Katharina Gaus

Rapid whole cell imaging reveals a calcium-APPL1-dynein nexus that regulates cohort trafficking of stimulated EGF receptors

Publisher: Springer Science and Business Media LLC

Date: 17-02-2021

DOI: 10.1038/S42003-021-01740-Y

Abstract: The endosomal system provides rich signal processing capabilities for responses elicited by growth factor receptors and their ligands. At the single cell level, endosomal trafficking becomes a critical component of signal processing, as exemplified by the epidermal growth factor (EGF) receptors. Activated EGFRs are trafficked to the phosphatase-enriched peri-nuclear region (PNR), where they are dephosphorylated and degraded. The details of the mechanisms that govern the movements of stimulated EGFRs towards the PNR, are not completely known. Here, exploiting the advantages of lattice light-sheet microscopy, we show that EGFR activation by EGF triggers a transient calcium increase causing a whole-cell level redistribution of Adaptor Protein, Phosphotyrosine Interacting with PH Domain And Leucine Zipper 1 (APPL1) from pre-existing endosomes within one minute, the rebinding of liberated APPL1 directly to EGFR, and the dynein-dependent translocation of APPL1-EGF-bearing endosomes to the PNR within ten minutes. The cell spanning, fast acting network that we reveal integrates a cascade of events dedicated to the cohort movement of activated EGF receptors. Our findings support the intriguing proposal that certain endosomal pathways have shed some of the stochastic strategies of traditional trafficking and have evolved processes that provide the temporal predictability that typify canonical signaling.

Oxidized lipoproteins and macrophages

Publisher: Elsevier BV

Date: 04-2002

DOI: 10.1016/S1537-1891(02)00174-X

Abstract: Macrophages are important participants in the development of atherosclerotic lesions, in cholesterol accumulation, as mediators of the immune response, and as sources of secreted enzymes and growth factors. Besides potentially contributing to local oxidation of lesion lipoproteins, many aspects of macrophage function can be affected by interaction with oxidized lipoproteins. Here we review macrophage responses to oxidized lipoproteins and provide novel data on the effects of a major oxidation product, 7-ketocholesterol, on high-density lipoprotein (HDL) function in cholesterol removal from macrophages.

The structure and luminescence properties of europium(iii) triflate doped self-assembled pyromellitamide gels

Publisher: Royal Society of Chemistry (RSC)

Date: 2011

DOI: 10.1039/C1NJ20085B

New Biological Frontiers Illuminated by Molecular Sensors and Actuators

Publisher: Elsevier BV

Date: 09-2016

DOI: 10.1016/J.BPJ.2016.08.012

Shiga toxin induces tubular membrane invaginations for its uptake into cells

Publisher: Springer Science and Business Media LLC

Date: 29-11-2007

DOI: 10.1038/NATURE05996

Abstract: Clathrin seems to be dispensable for some endocytic processes and, in several instances, no cytosolic coat protein complexes could be detected at sites of membrane invagination. Hence, new principles must in these cases be invoked to account for the mechanical force driving membrane shape changes. Here we show that the Gb3 (glycolipid)-binding B-subunit of bacterial Shiga toxin induces narrow tubular membrane invaginations in human and mouse cells and model membranes. In cells, tubule occurrence increases on energy depletion and inhibition of dynamin or actin functions. Our data thus demonstrate that active cellular processes are needed for tubule scission rather than tubule formation. We conclude that the B-subunit induces lipid reorganization that favours negative membrane curvature, which drives the formation of inward membrane tubules. Our findings support a model in which the lateral growth of B-subunit-Gb3 microdomains is limited by the invagination process, which itself is regulated by membrane tension. The physical principles underlying this basic cargo-induced membrane uptake may also be relevant to other internalization processes, creating a rationale for conceptualizing the perplexing ersity of endocytic routes.

Mesoporous silicon photonic crystal microparticles: Towards single-cell optical biosensors

Publisher: Royal Society of Chemistry (RSC)

Date: 2011

DOI: 10.1039/C005340F

Label-free dynamic volumetric imaging of deforming giant unilamellar vesicles under micro-flows

Publisher: OSA

Date: 2017

DOI: 10.1364/BODA.2017.BOS2A.2

Clus-DoC: a combined cluster detection and colocalization analysis for single-molecule localization microscopy data.

Publisher: American Society for Cell Biology (ASCB)

Date: 07-11-2016

DOI: 10.1091/MBC.E16-07-0478

Abstract: Advances in fluorescence microscopy are providing increasing evidence that the spatial organization of proteins in cell membranes may facilitate signal initiation and integration for appropriate cellular responses. Our understanding of how changes in spatial organization are linked to function has been h ered by the inability to directly measure signaling activity or protein association at the level of in idual proteins in intact cells. Here we solve this measurement challenge by developing Clus-DoC, an analysis strategy that quantifies both the spatial distribution of a protein and its colocalization status. We apply this approach to the triggering of the T-cell receptor during T-cell activation, as well as to the functionality of focal adhesions in fibroblasts, thereby demonstrating an experimental and analytical workflow that can be used to quantify signaling activity and protein colocalization at the level of in idual proteins.

Electrochemical behavior of gold colloidal alkyl modified silicon surfaces

Publisher: American Chemical Society (ACS)

Date: 21-10-2009

DOI: 10.1021/AM900427W

Abstract: Herein, we report on the production of nanoelectrode arrays by attaching colloidal gold on silicon-bound mixed self-assembled monolayers of TFA-protected alkenylthiol (C(11)-S-TFA) and undecylenic acid (acid). Effective modification of the surface, tethering of the nanoparticles, and the direct influence of the deprotected alkenylthiol (C(11)-SH) /acid ratio on the number of adherent particles were demonstrated using X-ray photoelectron spectroscopy, electrochemistry, and atomic force microscopy. Cyclic voltammetry showed that the enhancement of electron transfer to the silicon surface by the presence of nanoparticles is influenced by the number of tethered nanoparticles.

Assessment of the fifth ligand-binding repeat (LR5) of the LDL receptor as an analytical reagent for LDL binding

Publisher: Royal Society of Chemistry (RSC)

Date: 2001

DOI: 10.1039/B008725O

Abstract: The fifth ligand binding repeat (LR5) of the low density lipoprotein (LDL) receptor was assessed ex vivo as an 'analytical reagent' to distinguish LDL state, in atherosclerosis risk monitoring. LR5 was immobilized to mercaptoundecanoic acid modified gold surfaces via a glycine linker. Surface plasmon resonance (SPR) was used to monitor LDL binding. Unfolded LR5 was ineffectual as an affinity ligand for LDL but refolded LR5 showed a high affinity for native LDL but little affinity for oxidized LDL. LR5 refolded in the presence of calcium or EDTA gave the equivalent LDL binding capacity. However, EDTA-LR5 was less stable than Ca-LR5 at pH 5 and, from tryptophan fluorescence evidence, they appeared to involve different regions of LR5 and/or LDL in the binding. Involvement of amino acid residues of the calcium cage of LR5 was tested in LDL binding by monitoring calcium ion release with a calcium ionophore. The results were consistent with approximately 7-8 LR5 binding per LDL, of which only some induce calcium release (a maximum of approximately 25 mol% calcium, based on LR5, was released during LDL binding). For LDL binding to the LDL receptor in vivo more than one ligand-binding repeat is needed and this may be consistent with LR5 acting here also at binding sites which other LRs normally occupy in the LDL-LDL receptor complex. This initial study is encouraging for the use of a minimum peptide repeat array based on the conserved region of the LRs as an affinity surface for atherosclerosis risk monitoring.

Classification of lactic acid bacteria with UV‐resonance Raman spectroscopy

Publisher: Wiley

Date: 18-01-2006

DOI: 10.1002/BIP.20448

Abstract: UV-resonance Raman spectroscopy is applied as a method for the identification of lactic acid bacteria from yogurt. Eight different strains of bacteria from Lactobacillus acidophilus, L. delbrueckii ssp. bulgaricus, and Streptococcus thermophilus were investigated. At an excitation wavelength of 244 nm signals from nucleic acids and proteins are selectively enhanced. Classification was accomplished using different chemometric methods. In a first attempt, the unsupervised methods hierarchical cluster analysis and principal component analysis were applied to investigate natural grouping in the data. In a second step the spectra were analyzed using several supervised methods: K-nearest neighbor classifier, nearest mean classifier, linear discriminant analysis, and support vector machines.

Triton X-100 promotes a cholesterol-dependent condensation of the plasma membrane

Publisher: Portland Press Ltd.

Date: 27-05-2009

DOI: 10.1042/BJ20090051

Abstract: The molecular components of membrane rafts are frequently defined by their biochemical partitioning into detergent-resistant membranes. In the present study, we used a combination of epifluorescence and two-photon microscopy to visualize and quantify whether this insolubility in detergent reflects a pre-existing organization of the PM (plasma membrane). We found that the treatment of cells with cold TX (Triton X-100) promotes a profound remodelling of the PM, including a rapid rearrangement of the glycosphingolipid GM1 and cholesterol into newly formed structures, only partial solubilization of fluid domains and the formation of condensed domains that cover 51% of the remaining membrane. TX does not appear to induce the coalescence of pre-existing domains instead, the domains that remain after TX treatment seem to be newly formed with a higher degree of condensation than those observed in native membranes. However, when cholesterol was complexed physically by treatment with a second detergent, such as saponin, cholesterol did not separate into the newly formed structures, condensation of the domains was unaltered, and the relative area corresponding to ordered domains increased to occupy 62% of the remaining membrane. Our results suggest that detergent can be used to enrich ordered domains for biochemical analysis, but that TX treatment alone substantially alters the lateral organization of the PM.

Role of lipid rafts in agrin-elicited acetylcholine receptor clustering

Publisher: Elsevier BV

Date: 09-2008

DOI: 10.1016/J.CBI.2008.03.020

Abstract: Emerging concepts of membrane organization point to the compartmentalization of the plasma membrane into distinct lipid microdomains. This lateral segregation within cellular membranes is based on cholesterol-sphingolipid-enriched microdomains or lipid rafts which can move laterally and assemble into large-scale domains to create plasma membrane specialized cellular structures at specific cell locations. Such domains are likely involved in the genesis of the postsynaptic specialization at the neuromuscular junction, which requires the accumulation of acetylcholine receptors (AChRs), through activation of the muscle specific kinase MuSK by the neurotropic factor agrin and the reorganization of the actin cytoskeleton. We used C2C12 myotubes as a model system to investigate whether agrin-elicited AChR clustering correlated with lipid rafts. In a previous study, using two-photon Laurdan confocal imaging, we showed that agrin-induced AChR clusters corresponded to condensed membrane domains: the biophysical hallmark of lipid rafts [F. Stetzkowski-Marden, K. Gaus, M. Recouvreur, A. Cartaud, J. Cartaud, Agrin elicits membrane condensation at sites of acetylcholine receptor clusters in C2C12 myotubes, J. Lipid Res. 47 (2006) 2121-2133]. We further demonstrated that formation and stability of AChR clusters depend on cholesterol. We also reported that three different extraction procedures (Triton X-100, pH 11 or isotonic Ca++, Mg++ buffer) generated detergent resistant membranes (DRMs) with similar cholesterol/GM1 ganglioside content, which are enriched in several signalling postsynaptic components, notably AChR, the agrin receptor MuSK, rapsyn and syntrophin. Upon agrin engagement, actin and actin-nucleation factors such as Arp2/3 and N-WASP were transiently recovered within raft fractions suggesting that the activation by agrin can trigger actin polymerization. Taken together, the present data suggest that AChR clustering at the neuromuscular junction relies upon a mechanism of raft coalescence driven by agrin-elicited actin polymerization.

Clustering of CD3ζ is sufficient to initiate T cell receptor signaling

Publisher: Cold Spring Harbor Laboratory

Date: 19-02-2020

DOI: 10.1101/2020.02.17.953463

Abstract: T cell activation is initiated when ligand binding to the T cell receptor (TCR) triggers intracellular phosphorylation of the TCR-CD3 complex. However, it remains unknown how biophysical properties of TCR engagement result in biochemical phosphorylation events. Here, we constructed an optogenetic tool that induces spatial clustering of CD3ζ chains in a light controlled manner. We showed that spatial clustering of the CD3ζ intracellular tail alone was sufficient to initialize T cell triggering including phosphorylation of CD3ζ, Zap70, PLCγ, ERK and initiated Ca 2+ flux. In reconstituted COS-7 cells, only Lck expression was required to initiate CD3ζ phosphorylation upon CD3ζ clustering, which leads to the recruitment of tandem SH2 domain of Zap70 from cell cytosol to the newly formed CD3ζ clusters at the plasma membrane. Taken together, our data suggest that clustering of the TCR can initialize proximal TCR signaling and thus constitute a biophysical mechanism of TCR triggering.

The Constrained Amino Acid β‐Acc Confers Potency and Selectivity to Integrin Ligands

Publisher: Wiley

Date: 15-05-2007

DOI: 10.1002/ANIE.200605248

Optimized time-gated generalized polarization imaging of Laurdan and di-4-ANEPPDHQ for membrane order image contrast enhancement

Publisher: Wiley

Date: 2010

DOI: 10.1002/JEMT.20801

Abstract: The membrane dyes Laurdan and di-4-ANEPPDHQ can be used to image membrane order due to a spectral blue-shift in the fluorescence emission between the liquid-ordered and liquid-disordered phases. These images typically take the form of a normalized intensity ratio image known as a generalized polarization (GP) plot. Here, we exploit the known excited state photophysics and time-resolved data acquisition via time-correlated single-photon counting (TCSPC) to demonstrate GP contrast enhancement for these two probes of 7 and 31%, respectively. This improvement in image contrast enhancement will be invaluable when studying the role of lipid rafts in fixed and live cell systems.

Surface plasmon resonance sensor for heparin measurements in blood plasma

Publisher: Elsevier BV

Date: 12-1998

DOI: 10.1016/S0956-5663(98)00094-3

Abstract: Surface plasmon resonance (SPR) was used as an affinity biosensor to determine absolute heparin concentrations in human blood plasma s les. Protamine and polyethylene imine (PEI) were evaluated as heparin affinity surfaces. Heparin adsorption onto protamine in blood plasma was specific with a lowest detection limit of 0.2 U/ml and a linear window of 0.2-2 U/ml. Although heparin adsorption onto PEI in buffer solution had indicated superior sensitivity to that on protamine, in blood plasma it was not specific for heparin and adsorbed plasma species to a steady-state equilibrium. By reducing the incubation time and diluting the plasma s les with buffer to 50%, the non-specific adsorption of plasma could be controlled and a PEI pre-treated with blood plasma could be used successfully for heparin determination. Heparin adsorption in 50% plasma was linear between 0.05 and 1 U/ml so that heparin plasma levels of 0.1-2 U/ml could be determined within a relative error of 11% and an accuracy of 0.05 U/ml.

Identification and Characterization of Associated with Lipid Droplet Protein 1: A Novel Membrane‐Associated Protein That Resides on Hepatic Lipid Droplets

Publisher: Wiley

Date: 02-08-2006

DOI: 10.1111/J.1600-0854.2006.00465.X

Abstract: Alcoholic and nonalcoholic liver steatosis and steatohepatitis are characterized by the massive accumulation of lipid droplets (LDs) in the cytosol of hepatocytes. Although LDs are ubiquitous and dynamic organelles found in the cells of a wide range of organisms, little is known about the mechanisms and sites of LD biogenesis. To examine the participation of these organelles in the pathophysiological disorders of steatotic livers, we used a combination of mass spectrometry (matrix-assisted laser desorption ionization-time of flight and LC-MS electrospray) and Western blot analysis to study the composition of LDs purified from rat liver after a partial hepatectomy. Fifty proteins were identified. Adipose differentiation-related protein was the most abundant, but other proteins such as calreticulin, TIP47, Sar1, Rab GTPases, Rho and actin were also found. In addition, we identified protein associated with lipid droplets I ALDI (tentatively named Associated with LD protein 1), a novel protein widely expressed in liver and kidney corresponding to the product of 0610006F02Rik (GI:27229118). Our results show that, upon lipid loading of the cells, ALDI translocates from the endoplasmic reticulum into nascent LDs and indicate that ALDI may be targeted to the initial lipid deposits that eventually form these droplets. Moreover, we used ALDI expression studies to view other processes related to these droplets, such as LD biogenesis, and to analyze LD dynamics. In conclusion, here we report the composition of hepatic LDs and describe a novel bona fide LD-associated protein that may provide new insights into the mechanisms and sites of LD biogenesis.

Self-calibrated line-scan STED-FCS to quantify lipid dynamics in model and cell membranes

Publisher: Elsevier BV

Date: 02-2015

DOI: 10.1016/J.BPJ.2014.12.007

HIV taken by STORM: Super-resolution fluorescence microscopy of a viral infection

Publisher: Springer Science and Business Media LLC

Date: 02-05-2012

DOI: 10.1186/1743-422X-9-84

Abstract: The visualization of viral proteins has been hindered by the resolution limit of conventional fluorescent microscopes, as the dimension of any single fluorescent signal is often greater than most virion particles. Super-resolution microscopy has the potential to unveil the distribution of proteins at the resolution approaching electron microscopy without relying on morphological features of existing characteristics of the biological specimen that are needed in EM. Using direct stochastic optical reconstruction microscopy (dSTORM) to achieve a lateral resolution of 15–20 nm, we quantified the 2-D molecular distribution of the major structural proteins of the infectious human immunodeficiency virus type 1 (HIV-1) before and after infection of lymphoid cells. We determined that the HIV-1 matrix and capsid proteins undergo restructuring soon after HIV-1 infection. This study provides the proof-of-concept for the use of dSTORM to visualize the changes in the molecular distribution of viral proteins during an infection.

Towards single molecule biosensors using super-resolution fluorescence microscopy

Publisher: Elsevier BV

Date: 07-2016

DOI: 10.1016/J.BIOS.2016.10.048

Abstract: Conventional immunosensors require many binding events to give a single transducer output which represents the concentration of the analyte in the s le. Because of the requirements to selectively detect species in complex s les, immunosensing interfaces must allow immobilisation of antibodies while repelling nonspecific adsorption of other species. These requirements lead to quite sophisticated interfacial design, often with molecular level control, but we have no tools to characterise how well these interfaces work at the molecular level. The work reported herein is an initial feasibility study to show that antibody-antigen binding events can be monitored at the single molecule level using single molecule localisation microscopy (SMLM). The steps to achieve this first requires showing that indium tin oxide surfaces can be used for SMLM, then that these surfaces can be modified with self-assembled monolayers using organophosphonic acid derivatives, that the amount of antigens and antibodies on the surface can be controlled and monitored at the single molecule level and finally antibody binding to antigen modified surfaces can be monitored. The results show the amount of antibody that binds to an antigen modified surface is dependent on both the concentration of antigen on the surface and the concentration of antibody in solution. This study demonstrates the potential of SMLM for characterising biosensing interfaces and as the transducer in a massively parallel, wide field, single molecule detection scheme for quantitative analysis.

High speed multiphoton imaging

Publisher: SPIE

Date: 09-12-2016

DOI: 10.1117/12.2242951

Distinct surveillance pathway for immunopathology during acute infection via autophagy and SR-BI

Publisher: Springer Science and Business Media LLC

Date: 03-10-2016

DOI: 10.1038/SREP34440

Abstract: The mechanisms protecting from immunopathology during acute bacterial infections are incompletely known. We found that in response to apoptotic immune cells and live or dead Listeria monocytogenes scavenger receptor BI (SR-BI), an anti-atherogenic lipid exchange mediator, activated internalization mechanisms with characteristics of macropinocytosis and, assisted by Golgi fragmentation, initiated autophagic responses. This was supported by scavenger receptor-induced local increases in membrane cholesterol concentrations which generated lipid domains particularly in cell extensions and the Golgi. SR-BI was a key driver of beclin-1-dependent autophagy during acute bacterial infection of the liver and spleen. Autophagy regulated tissue infiltration of neutrophils, suppressed accumulation of Ly6C + (inflammatory) macrophages and prevented hepatocyte necrosis in the core of infectious foci. Perifocal levels of Ly6C + macrophages and Ly6C − macrophages were unaffected, indicating predominant regulation of the focus core. SR-BI-triggered autophagy promoted co-elimination of apoptotic immune cells and dead bacteria but barely influenced bacterial sequestration and survival or inflammasome activation, thus exclusively counteracting damage inflicted by immune responses. Hence, SR-BI- and autophagy promote a surveillance pathway that partially responds to products of antimicrobial defenses and selectively prevents immunity-induced damage during acute infection. Our findings suggest that control of infection-associated immunopathology can be based on a unified defense operation.

Colloidal silicon quantum dots: from preparation to the modification of self-assembled monolayers for bioimaging and sensing applications

Publisher: SPIE

Date: 22-02-2017

DOI: 10.1117/12.2249592

Ultralow- and Low-Background Surfaces for Single-Molecule Localization Microscopy of Multistep Biointerfaces for Single-Molecule Sensing

Publisher: American Chemical Society (ACS)

Date: 08-2018

DOI: 10.1021/ACS.LANGMUIR.8B01487

Abstract: Single-molecule localization microscopy (SMLM) has created the opportunity of pushing fluorescence microscopy from being a biological imaging tool to a surface characterization and possibly even a quantitative analytical tool. The latter could be achieved by molecular counting using pointillist SMLM data sets. However, SMLM is especially sensitive to background fluorescent signals, which influences any subsequent analysis. Therefore, fabricating sensing surfaces that resist nonspecific adsorption of proteins, even after multiple modification steps, has become paramount. Herein is reported two different ways to modify surfaces: dichlorodimethylsilane-biotinylated bovine serum albumin-Tween-20 (DbT20) and poly-l-lysine grafted polyethylene glycol (PLL-PEG) mixed with biotinylated PLL-PEG (PLL-PEG/PEGbiotin). The results show that the ability to resist nonspecific adsorption of DbT20 surfaces deteriorates with an increase in the number of modification steps required after the addition of the DbT20, which limits the applicability of this surface for SMLM. As such, a new surface for SMLM that employs PLL-PEG/PEGbiotin was developed that exhibits ultralow amounts of nonspecific protein adsorption even after many modification steps. The utility of the surface was demonstrated for human influenza hemagglutinin-tagged mEos2, which was directly pulled down from cell lysates onto the PLL-PEG/PEGbiotin surface. The results strongly indicated that the PLL-PEG/PEGbiotin surface satisfies the criteria of SMLM imaging of a negligible background signal and negligible nonspecific adsorption.

Telomere Loop Dynamics in Chromosome End Protection.

Publisher: Elsevier BV

Date: 08-2018

DOI: 10.1016/J.MOLCEL.2018.06.025

Abstract: Telomeres regulate DNA damage response (DDR) and DNA repair activity at chromosome ends. How telomere macromolecular structure contributes to ATM regulation and its potential dissociation from control over non-homologous end joining (NHEJ)-dependent telomere fusion is of central importance to telomere-dependent cell aging and tumor suppression. Using super-resolution microscopy, we identify that ATM activation at mammalian telomeres with reduced TRF2 or at human telomeres during mitotic arrest occurs specifically with a structural change from telomere loops (t-loops) to linearized telomeres. Additionally, we find the TRFH domain of TRF2 regulates t-loop formation while suppressing ATM activity. Notably, we demonstrate that ATM activation and telomere linearity occur separately from telomere fusion via NHEJ and that linear DDR-positive telomeres can remain resistant to fusion, even during an extended G1 arrest, when NHEJ is most active. Collectively, these results suggest t-loops act as conformational switches that specifically regulate ATM activation independent of telomere mechanisms to inhibit NHEJ.

Annexin A6 is an organizer of membrane microdomains to regulate receptor localization and signalling

Publisher: Wiley

Date: 12-10-2011

DOI: 10.1002/IUB.540

Abstract: Annexin A6 (AnxA6) belongs to the conserved annexin protein family--a group of Ca(2+) -dependent membrane binding proteins. It is the largest of all annexin proteins and upon activation, binds to negatively charged phospholipids in the plasma membrane and endosomes. In addition, AnxA6 associates with cholesterol-rich membrane microdomains termed lipid rafts. Membrane cholesterol triggers Ca(2+) -independent translocation of AnxA6 to membranes and AnxA6 levels determine the number of caveolae, a form of specialized rafts at the cell surface. AnxA6 also has an F-actin binding domain and interacts with cytoskeleton components. Taken together, this suggests that AnxA6 has a scaffold function to link membrane microdomains with the organization of the cytoskeleton. Such a link facilitates AnxA6 to participate in plasma membrane repair and it would also impact on receptor signalling at the cell surface, growth factor, and lipoprotein receptor trafficking, Ca(2+) -channel activity and T cell activation. Hence, the regulation of cell surface receptors by AnxA6 may be facilitated by its unique structure that allows recruitment of interaction partners and simultaneously bridging specialized membrane domains with cortical actin surrounding activated receptors.

Annexin A6‐Induced Alterations in Cholesterol Transport and Caveolin Export from the Golgi Complex

Publisher: Wiley

Date: 16-08-2007

DOI: 10.1111/J.1600-0854.2007.00640.X

Characterization of a New Series of Fluorescent Probes for Imaging Membrane Order

Publisher: Public Library of Science (PLoS)

Date: 04-02-2013

DOI: 10.1371/JOURNAL.PONE.0052960

Rapid Whole Cell Imaging Reveals A Calcium-APPL1-Dynein Nexus That Regulates Cohort Trafficking of Stimulated EGF Receptors

Publisher: Cold Spring Harbor Laboratory

Date: 29-11-2018

DOI: 10.1101/481796

Abstract: Multicellular life processes such as proliferation and differentiation depend on cell surface signaling receptors that bind ligands generally referred to as growth factors. Recently, it has emerged that the endosomal system provides rich signal processing capabilities for responses elicited by these factors [1-3]. At the single cell level, endosomal trafficking becomes a critical component of signal processing, as exemplified by the epidermal growth factor (EGF) receptors of the receptor tyrosine kinase family. EGFRs, once activated by EGF, are robustly trafficked to the phosphatase-enriched peri-nuclear region (PNR), where they are dephosphorylated [4-8]. However, the details of the mechanisms regulating the movements of stimulated EGFR in time and space, i.e., towards the PNR, are not known. What endosomal regulators provide specificity to EGFR? Do modifications to the receptor upon stimulation regulate its trafficking? To understand the events leading to EGFR translocation, and especially the early endosomal dynamics that immediately follow EGFR internalization, requires the real-time, long-term, whole-cell imaging of multiple elements. Here, exploiting the advantages of lattice light-sheet microscopy [9], we show that the binding of EGF by its receptor, EGFR, triggers a transient calcium increase that peaks by 30 s, causing the desorption of APPL1 from pre-existing endosomes within one minute, the rebinding of liberated APPL1 to EGFR within three minutes, and the dynein-dependent translocation of APPL1-EGF-bearing endosomes to the PNR within five minutes. The novel, cell spanning, fast acting network that we reveal integrates a cascade of events dedicated to the cohort movement of activated EGFR receptors. Our findings support the intriguing proposal that certain endosomal pathways have shed some of the stochastic strategies of traditional trafficking, and have evolved behaviors whose predictability is better suited to signaling [10, 11]. Work presented here demonstrates that our whole cell imaging approach can be a powerful tool in revealing critical transient interactions in key cellular processes such as receptor trafficking.

A mobile endocytic network connects clathrin-independent receptor endocytosis to recycling and promotes T cell activation

Publisher: Springer Science and Business Media LLC

Date: 23-04-2018

DOI: 10.1038/S41467-018-04088-W

Abstract: Endocytosis of surface receptors and their polarized recycling back to the plasma membrane are central to many cellular processes, such as cell migration, cytokinesis, basolateral polarity of epithelial cells and T cell activation. Little is known about the mechanisms that control the organization of recycling endosomes and how they connect to receptor endocytosis. Here, we follow the endocytic journey of the T cell receptor (TCR), from internalization at the plasma membrane to recycling back to the immunological synapse. We show that TCR triggering leads to its rapid uptake through a clathrin-independent pathway. Immediately after internalization, TCR is incorporated into a mobile and long-lived endocytic network demarked by the membrane-organizing proteins flotillins. Although flotillins are not required for TCR internalization, they are necessary for its recycling to the immunological synapse. We further show that flotillins are essential for T cell activation, supporting TCR nanoscale organization and signaling.

Evidence for why tri(ethylene oxide) functionalized Si-C linked monolayers on Si(111) have inferior protein antifouling properties relative to the equivalent alkanethiol monolayers assembled on gold

Publisher: CSIRO Publishing

Date: 2005

DOI: 10.1071/CH05121

Abstract: High quality methoxy-terminated monolayers containing a tri(ethylene oxide) moiety were formed on Si(111)–H surfaces in thermal hydrosilylation reactions. X-ray photoelectron spectroscopy, contact angle, and X-ray reflectivity measurements suggested that the suboptimal protein anti-fouling properties of these Si–C linked monolayers were due to a reduced lateral packing density of the chains resulting in a disordered layer with insufficient internal and external hydrophilicity.

Dextran-Catechin: An anticancer chemically-modified natural compound targeting copper that attenuates neuroblastoma growth

Publisher: Impact Journals, LLC

Date: 21-06-2016

DOI: 10.18632/ONCOTARGET.10201

Surface plasmon resonance measurement of the binding of low-density lipoprotein at a heparin surface

Publisher: Elsevier BV

Date: 09-1999

DOI: 10.1006/JCIS.1999.6333

Time-Resolved Laurdan Fluorescence Reveals Insights into Membrane Viscosity and Hydration Levels

Publisher: Elsevier BV

Date: 10-2018

DOI: 10.1016/J.BPJ.2018.08.041

Stoichiometric quantification of spatially dense assemblies with qPAINT

Publisher: Cold Spring Harbor Laboratory

Date: 21-01-2019

DOI: 10.1101/525345

Abstract: Quantitative PAINT (qPAINT) is a useful method for counting well-separated molecules within nanoscale assemblies. But whether cross-reactivity in densely-packed arrangements perturbs measurements is unknown. Here we establish that qPAINT measurements are robust even when target molecules are separated by as little as 3 nm, sufficiently close that single-stranded DNA binding sites can interact.

Functional Implications of Plasma Membrane Condensation for T Cell Activation

Publisher: Public Library of Science (PLoS)

Date: 28-05-2008

DOI: 10.1371/JOURNAL.PONE.0002262

PALM imaging and cluster analysis of protein heterogeneity at the cell surface

Publisher: Wiley

Date: 17-06-2010

DOI: 10.1002/JBIO.200900089

Abstract: The authors employed photoactivatable localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM) imaging and image analysis based on Ripley's K-function to quantify the distribution and heterogeneity of proteins at the cell plasma membrane. The membrane targeting sequence of the N-terminal region of the T cell receptor-pathway kinase Lck fused to the photo-convertible fluorescent protein tdEos (Lck(N10)-tdEos), clusters into sub-100 nm regions which cover approximately 7% of the cell surface. 2-channel PALM imaging of Lck(N10)-tdEos and the N-terminus of the kinase Src (Src(N15)-PS-CFP2) are demonstrated. Finally, T cell microclusters at the immune synapse are imaged at super-resolution using dSTORM, showing that conventional TIRF images contain unresolved, small clusters. These methods are generally applicable to other cell and fluorophore systems to quantify 2-D molecular clustering at nanometer scales.

Evidence for annexin A6‐dependent plasma membrane remodelling of lipid domains

Publisher: Wiley

Date: 20-01-2015

DOI: 10.1111/BPH.13022

Rod-shaped mesoporous silica nanoparticles for nanomedicine: recent progress and perspectives

Publisher: Informa UK Limited

Date: 02-09-2018

DOI: 10.1080/17425247.2018.1517748

Abstract: Interest in mesoporous silica nanoparticles for drug delivery has resulted in a good understanding of the impact of size and surface chemistry of these nanoparticles on their performance as drug carriers. Shape has emerged as an additional factor that can have a significant effect on delivery efficacy. Rod-shaped mesoporous silica nanoparticles show improvements in drug delivery relative to spherical mesoporous silica nanoparticles. This review summarises the synthesis methods for producing rod-shaped mesoporous silica nanoparticles for use in nanomedicine. The second part covers recent progress of mesoporous silica nanorods by comparing the impact of sphere and rod-shape on drug delivery efficiency. As hollow mesoporous silica nanorods are capable of higher drug loads than most other drug delivery vehicles, such particles will reduce the amount of mesoporous silica in the body for efficient therapy. However, the importance of nanoparticle shape on drug delivery efficiency is not well understood for mesoporous silica. Studies that visualize and quantify the uptake pathway of mesoporous silica nanorods in specific cell types and compare the cellular uptake to the well-studied nanospheres should be the focus of research to better understand the role of shape in uptake.

Functionalization of porous silicon based photonic crystals with organic monolayers

Publisher: IEEE

Date: 2005

DOI: 10.1109/COMMAD.2004.1577529

Smart tissue culture: in situ monitoring of the activity of protease enzymes secreted from live cells using nanostructured photonic crystals

Publisher: American Chemical Society (ACS)

Date: 21-04-2009

DOI: 10.1021/NL900283J

Abstract: Monitoring enzyme secretion in tissue culture has proved challenging because to date the activity cannot be continuously measured in situ. In this Letter, we present a solution using biopolymer loaded photonic crystals of anodized silicon. Shifts in the optical response by proteolytic degradation of the biopolymer provide label-free sensing with unprecedented low detection limits (1 pg) and calculation of kinetic parameters. The enhancement in sensitivity relative to previous photonic crystal sensors constitutes a change in the sensing paradigm because here the entire pore space is responsive to the secreted enzyme rather than just the pore walls. In situ monitoring is demonstrated by detecting secretion of matrix metalloprotease 9 from stimulated human macrophages.

The actin cytoskeleton and membrane organisation in T lymphocytes

Publisher: Humana Press

Date: 2012

DOI: 10.1007/978-1-61779-788-0_5

Tropomyosin isoforms support actomyosin biogenesis to generate contractile tension at the epithelial zonula adherens

Publisher: Wiley

Date: 12-2014

DOI: 10.1002/CM.21202

Abstract: Epithelial cells generate contractile forces at their cell-cell contacts. These are concentrated at the specialized apical junction of the zonula adherens (ZA), where a ring of stabilized E-cadherin lies adjacent to prominent actomyosin bundles. Coupling of adhesion and actomyosin contractility yields tension in the junction. The biogenesis of junctional contractility requires actin assembly at the ZA as well as the recruitment of nonmuscle myosin II, but the molecular regulators of these processes are not yet fully understood. We now report a role for tropomyosins 5NM1 (Tm5NM1) and 5NM2 (Tm5NM2) in their generation. Both these tropomyosin isoforms were found at the ZA and their depletion by RNAi or pharmacological inhibition reduced both F-actin and myosin II content at the junction. Photoactivation analysis revealed that the loss of F-actin was attributable to a decrease in filament stability. These changes were accompanied by a decrease in E-cadherin content at junctions. Ultimately, both long-term depletion of Tm5NM1/2 and acute inhibition with drugs caused junctional tension to be reduced. Thus these tropomyosin isoforms are novel contributors to junctional contractility and integrity.

Imaging lipid domains in cell membranes: The advent of super-resolution fluorescence microscopy

Publisher: Frontiers Media SA

Date: 2013

DOI: 10.3389/FPLS.2013.00503

Hybrid lipid bilayers in nanostructured silicon: A biomimetic mesoporous scaffold for optical detection of cholera toxin

Publisher: Royal Society of Chemistry (RSC)

Date: 2007

DOI: 10.1039/B702762A

Abstract: Cholera toxin levels are optically detected by affinity capture within hybrid lipid bilayer membranes formed in the nanostructures of porous silicon photonic crystals.

Porous silicon based narrow line-width rugate filters

Publisher: Elsevier BV

Date: 02-2007

DOI: 10.1016/J.OPTMAT.2005.10.012

Ultrasensitive and Specific Measurement of Protease Activity Using Functionalized Photonic Crystals

Publisher: American Chemical Society (ACS)

Date: 08-09-2015

DOI: 10.1021/ACS.ANALCHEM.5B02529

Abstract: Herein is presented a microsensor technology as a diagnostic tool for detecting specific matrix metalloproteinases (MMPs) at very low concentrations. MMP-2 and MMP-9 are detected using label free porous silicon (PSi) photonic crystals that have been made selective for a given MMP by filling the nanopores with synthetic polymeric substrates containing a peptide sequence for that MMP. Proteolytic cleavage of the peptide sequence results in a shift in wavelength of the main peak in the reflectivity spectrum of the PSi device, which is dependent on the amount of MMP present. The ability to detect picogram amounts of MMP-2 and MMP-9 released by primary retinal pigment epithelial (RPE) cells and iris pigment epithelial (IPE) cells stimulated with lipopolysaccharide (LPS) is demonstrated. It was found that both cell types secrete higher amounts of MMP-2 than MMP-9 in their stimulated state, with RPE cells producing higher amounts of MMPs than IPE cells. The microsensor performance was compared to conventional protease detection systems, including gelatin zymography and enzyme linked immunosorbent assay (ELISA). It was found that the PSi microsensors were more sensitive than gelatin zymography PSi microsensors detected the presence of both MMP-2 and MMP-9 while zymography could only detect MMP-2. The MMP-2 and MMP-9 quantification correlated well with the ELISA. This new method of detecting protease activity shows superior performance to conventional protease assays and has the potential for translation to high-throughput multiplexed analysis.

Low density lipoprotein interaction with amino acid-modified self assembled monolayers on surface plasmon resonance surfaces

Publisher: Elsevier BV

Date: 10-2002

DOI: 10.1016/S0003-2670(02)00256-8

Mechanisms of protein nanoscale clustering

Publisher: Elsevier BV

Date: 02-2017

DOI: 10.1016/J.CEB.2016.09.004

Abstract: Due to recent technical developments in microscopy, huge advances have been made in our understanding of the architecture of the cell membrane. It is now well appreciated that nanoscale clustering is a common feature of membrane proteins. Many of these clusters have been implicated in signal initiation and integration platforms. However, the mechanisms that mediate the dynamic nanoscale arrangement of membrane proteins are not fully understood and could involve lipid domains, electrostatic interactions between proteins and lipid, protein scaffolding as well as purely mechanical processes. In this review we summarise these mechanisms giving rise to dynamic nanoscale protein reorganisation in the plasma membrane with reference to recent ex les of immune receptor clustering to illustrate general principles.

Pair correlation microscopy reveals the role of nanoparticle shape in intracellular transport and site of drug release

Publisher: Springer Science and Business Media LLC

Date: 12-09-2017

DOI: 10.1038/NNANO.2016.160

Abstract: Nanoparticle size, surface charge and material composition are known to affect the uptake of nanoparticles by cells. However, whether nanoparticle shape affects transport across various barriers inside the cell remains unclear. Here we used pair correlation microscopy to show that polymeric nanoparticles with different shapes but identical surface chemistries moved across the various cellular barriers at different rates, ultimately defining the site of drug release. We measured how micelles, vesicles, rods and worms entered the cell and whether they escaped from the endosomal system and had access to the nucleus via the nuclear pore complex. Rods and worms, but not micelles and vesicles, entered the nucleus by passive diffusion. Improving nuclear access, for ex le with a nuclear localization signal, resulted in more doxorubicin release inside the nucleus and correlated with greater cytotoxicity. Our results therefore demonstrate that drug delivery across the major cellular barrier, the nuclear envelope, is important for doxorubicin efficiency and can be achieved with appropriately shaped nanoparticles.

Peptide-modified optical filters for detecting protease activity

Publisher: American Chemical Society (ACS)

Date: 31-10-2007

DOI: 10.1021/NN700141N

Abstract: The organic derivatization of silicon-based nanoporous photonic crystals is presented as a method to immobilize peptides for the detection of protease enzymes in solution. A narrow-line-width rugate filter, a one-dimensional photonic crystal, is fabricated that exhibits a high-reflectivity optical resonance that is sensitive to small changes in the refractive index at the pore walls. To immobilize peptide in the pore of the photonic crystal, the hydrogen-terminated silicon surface was first modified with the alkene 10-succinimidyl undecenoate via hydrosilylation. The monolayer with the succinimide ester moiety at the distal end served the dual function of protecting the underlying silicon from oxidation as well as providing a surface suitable for subsequent derivatization with amines. The surface was further modified with 1-aminohexa(ethylene glycol) (EG(6)) to resist nonspecific adsorption of proteins common in complex biological s les. The distal hydroxyl of the EG(6) is activated using the solid-phase coupling reagent disuccinimidyl carbonate for selective immobilization of peptides as protease recognition elements. X-ray photoelectron spectroscopy analysis reveals high activation and coupling efficiency at each stage of the functionalization. Exposure of the peptide-modified crystals to the protease subtilisin in solution causes a change in the refractive index, resulting in a shift of the resonance to shorter wavelengths, indicating cleavage of organic material within the pores. The lowest detected concentration of enzyme was 37 nM (7.4 pmol in 200 microL).

Fluorescence localization microscopy

Publisher: Informa UK Limited

Date: 07-2012

DOI: 10.4161/CIB.20348

Apolipoprotein A-I-stimulated Apolipoprotein E Secretion from Human Macrophages Is Independent of Cholesterol Efflux

Publisher: Elsevier BV

Date: 06-2004

DOI: 10.1074/JBC.M401177200

Quantitative imaging of membrane lipid order in cells and organisms

Publisher: Springer Science and Business Media LLC

Date: 08-12-2012

DOI: 10.1038/NPROT.2011.419

Abstract: It is now recognized that lipids and proteins in cellular membranes are not homogenously distributed. A high degree of membrane order is the biophysical hallmark of cholesterol-enriched lipid rafts, which may induce the lateral sorting of proteins within the membrane. Here we describe a quantitative fluorescence microscopy technique for imaging localized lipid environments and measuring membrane lipid order in live and fixed cells, as well as in intact tissues. The method is based on the spectral ratiometric imaging of the polarity-sensitive membrane dyes Laurdan and di-4-ANEPPDHQ. Laurdan typically requires multiphoton excitation, making it suitable for the imaging of tissues such as whole, living zebrafish embryos, whereas di-4-ANEPPDHQ imaging can be achieved with standard confocal microscopes. This approach, which takes around 4 h, directly examines the organization of cellular membranes and is distinct from alternative approaches that infer membrane order by measuring probe partitioning or dynamics.

tagPAINT: covalent labelling of genetically encoded protein tags for DNA-PAINT imaging

Publisher: Cold Spring Harbor Laboratory

Date: 10-04-2019

DOI: 10.1101/604462

Abstract: Recently, DNA-PAINT single molecule localisation microscopy (SMLM) has shown great promise for quantitative imaging. However, labelling strategies so far have relied on approaches that are multivalent or affinity-based. Here, we demonstrate tagPAINT - the covalent labelling of expressed protein tags (SNAP tag and Halo tag) with single DNA docking strands for single molecule localisation microscopy via DNA-PAINT. We utilised tagPAINT for T-cell receptor signalling proteins at the immune synapse as a proof of principle.

... yet even flawed films raise interest in research [3]

Publisher: Springer Science and Business Media LLC

Date: 09-2004

DOI: 10.1038/431244C

Imaging galectin-3 dependent endocytosis with lattice light-sheet microscopy

Publisher: SPIE

Date: 29-04-2017

DOI: 10.1117/12.2275706

High contrast imaging and flexible photomanipulation for quantitative in vivo multiphoton imaging with polygon scanning microscope.

Publisher: Wiley

Date: 10-04-2018

DOI: 10.1002/JBIO.201700341

Abstract: In this study, we introduce two key improvements that overcome limitations of existing polygon scanning microscopes while maintaining high spatial and temporal imaging resolution over large field of view (FOV). First, we proposed a simple and straightforward means to control the scanning angle of the polygon mirror to carry out photomanipulation without resorting to high speed optical modulators. Second, we devised a flexible data s ling method directly leading to higher image contrast by over 2-fold and digital images with 100 megapixels (10 240 × 10 240) per frame at 0.25 Hz. This generates sub-diffraction limited pixels (60 nm per pixels over the FOV of 512 μm) which increases the degrees of freedom to extract signals computationally. The unique combined optical and digital control recorded fine fluorescence recovery after localized photobleaching (r ~10 μm) within fluorescent giant unilamellar vesicles and micro-vascular dynamics after laser-induced injury during thrombus formation in vivo. These new improvements expand the quantitative biological-imaging capacity of any polygon scanning microscope system.

The platelet glycoprotein Ib-IX-V complex anchors lipid rafts to the membrane skeleton: Implications for activation-dependent cytoskeletal translocation of signaling molecules

Publisher: Elsevier BV

Date: 2010

DOI: 10.1111/J.1538-7836.2009.03656.X

Abstract: The glycoprotein (GP) Ib-IX-V complex attaches platelets to areas of endothelial damage by binding von Willebrand factor (VWF), an interaction that transmits intracellular activation signals. These signals require that the complex associates with both lipid rafts and the membrane cytoskeleton, but it is not clear whether the same GPIb-IX-V subpopulation associates with both structures. To determine which subpopulation of GPIb-IX-V associates with lipid rafts, and the consequences of that interaction. We analyzed the content of proteins (particularly the GPIb-IX-V complex) and lipids in rafts from detergent lysates of platelets before and after removal of the actin cytoskeleton alone or both the actin cytoskeleton and membrane skeleton (by successive centrifugations of 15,800 x g and 100,000 x g). In unstimulated platelets, little raft-associated GPIb-IX-V sedimented with the actin skeleton most was removed by sedimentation of the membrane skeleton. The Src family kinase Lyn followed the same pattern. In VWF-activated platelets, almost all of the GPIb-IX-V complex and Lyn in rafts sedimented with the actin cytoskeleton, consistent with a previously described crosslinking of the membrane and actin skeletal structures following platelet activation. Disruption of the GPIbalpha-filamin linkage with N-ethylmaleimide prevented depletion of raft-associated GPIb-IX-V by skeletal sedimentation. Not all raft-associated proteins and lipids followed this pattern. These results suggest that the raft association and cytoskeletal linkage of the GPIb-IX-V complex are interrelated, and both are required for optimal receptor function, perhaps because raft association attracts signaling proteins and membrane skeletal association allows these proteins to move en masse to new locations.

Imaging membrane lipid order in whole, living vertebrate organisms

Publisher: Elsevier BV

Date: 07-2010

DOI: 10.1016/J.BPJ.2010.04.022

Fluctuation-based imaging of nuclear Rac1 activation by protein oligomerisation

Publisher: Springer Science and Business Media LLC

Date: 27-02-2014

DOI: 10.1038/SREP04219

Abstract: Here we describe a fluctuation-based method to quantify how protein oligomerisation modulates signalling activity of a multifunctional protein. By recording fluorescence lifetime imaging microscopy (FLIM) data of a FRET biosensor in a format that enables concomitant phasor and cross Number and Brightness (cN& B) analysis, we measure the nuclear dynamics of a Rac1 FRET biosensor and assess how Rac1 homo-oligomers (N& B) regulate Rac1 activity (hetero-oligomerisation with the biosensor affinity reagent, PBD, by FLIM-FRET) or interaction with an unknown binding partner (cN& B). The high spatiotemporal resolution of this method allowed us to discover that upon DNA damage monomeric and active Rac1 in the nucleus is segregated from dimeric and inactive Rac1 in the cytoplasm. This reorganisation requires Rac1 GTPase activity and is associated with an importin-α2 redistribution. Only with this multiplexed approach can we assess the oligomeric state a molecular complex must form in order to regulate a complex signalling network.

Do mechanical forces contribute to nanoscale membrane organisation in T cells?

Publisher: Elsevier BV

Date: 04-2015

DOI: 10.1016/J.BBAMCR.2014.10.025

Abstract: Mechanotransduction describes how a cell senses and interacts with its environment. The concept originated in adhesion biology where adhesion receptors, integrins, facilitate force transmission between the extracellular matrix and the intracellular actin cytoskeleton. Indeed, during any adhesive contacts, cells do exert mechanical force. Hence, the probing of the local environment by cells results in mechanical cues that contribute to cellular functions and cell fate decisions such as migration, proliferation, differentiation and apoptosis. On the molecular level, mechanical forces can rearrange proteins laterally within the membrane, regulate their activity by inducing conformational changes and probe the mechanical properties and bond strength of receptor-ligands. From this point of view, it appears surprising that molecular forces have been largely overlooked in membrane organisation and ligand discrimination processes in lymphocytes. During T cell activation, the T cell receptor recognises and distinguishes antigenic from benign endogenous peptides to initiate the reorganisation of membrane proteins into signalling clusters within the immunological synapse. In this review, we asked whether characteristics of fibroblast force sensing could be applied to immune cell antigen recognition and signalling, and outline state-of-the-art experimental strategies for studying forces in the context of membrane organisation. This article is part of a Special Issue entitled: Nanoscale membrane orgainisation and signalling.

Evaluation of surface plasmon resonance (SPR) for heparin assay II. Polyethylene imine (PEI) as an affinity surface

Publisher: Elsevier BV

Date: 10-1997

DOI: 10.1006/JCIS.1997.5098

Abstract: Heparin adsorption onto polyethylene imine (PEI) films was investigated. The adsorption was followed with time-dependent surface plasmon resonance (SPR) for PEI cast on gold films, and the thickness of the adsorbed heparin was determined by fitting the angular reflection curves to Fresnel's equations. The average thickness of the PEI determined the heparin adsorption range and it was found that both heparin binding capacity and sensitivity increased with PEI thickness. At an appropriate thickness of 10 &plusmn 2 nm, adsorption of heparin in the clinically applicable range of 0-2 U/ml caused a linear concentration dependent change in the SPR reflectivity at a fixed angle, with excellent sensitivity (0.05 U/ml). The results are compared to heparin adsorption on immobilized protamine and the surface coverage and probable heparin distribution discussed. Copyright 1997 Academic Press. Copyright 1997Academic Press

Evaluation of surface plasmon resonance (SPR) for heparin assay

Publisher: Elsevier BV

Date: 10-1997

DOI: 10.1006/JCIS.1997.5097

Abstract: The concentration of heparin, an anticoagulant in blood, is usually inferred from clotting type assay, which determines a parameter related to the heparin activity. Because of the heterogeneity of heparin, however, it is desirable to monitor the absolute concentration of heparin directly in the clinical range of 0-2 U/ml. Surface plasmon resonance (SPR) provides a optical direct method of monitoring binding events. Gold films, as required for SPR, were modified with protamine the immoblized protamine interacts electrostatically with heparin so that the heparin adsorption is dependent on the absolute concentration. The "thickness" of the immobilized protamine layer determined the linear range of the sensor's response and the sensitivity. Less densely packed layers of protamine showed a lower detection limit for heparin, suggesting a mixing of the heparin into the incomplete protamine layer. On the other hand, thicker, denser protamine layers did not show a low concentration sensitivity to heparin although their maximum heparin binding capacity was increased. It was shown that the linear response range of the protamine modified SPR device to heparin could be modulated by altering both the protamine loading and its method of immobilization. Copyright 1997 Academic Press. Copyright 1997Academic Press

NicoLase - An open-source diode laser combiner, fiber launch, and sequencing controller for fluorescence microscopy

Publisher: Public Library of Science (PLoS)

Date: 16-03-2017

DOI: 10.1371/JOURNAL.PONE.0173879

Visualizing membrane microdomains by Laurdan 2-photon microscopy (Review)

Publisher: Informa UK Limited

Date: 2006

DOI: 10.1080/09687860500466857

Abstract: Lateral segregation of cell membrane components gives rise to microdomains with a different structure within the membrane. Most prominently, lipid rafts are defined as domains in liquid ordered phase whereas surrounding membranes are more fluid. Here we review a 2-photon fluorescence microscopy approach, which allows the visualization of membrane fluidity. The fluorescent probe Laurdan exhibits a blue shift in emission with increasing membrane condensation caused by an alteration in the dipole moment of the probe as a consequence of exclusion of water molecules from the lipid bilayer. The quantification of membrane order is achieved by the Generalized Polarization (GP) values, which are defined as normalized intensity ratios of two emission channels. GP images are therefore not biased by probe concentrations and membrane ruffles. Furthermore, Laurdan reports membrane structure independently from the lipid and protein cargo of the membrane domains. We give ex les where Laurdan microscopy was instrumental in quantifying the formation of condensed membrane domains and their cellular requirements. Moreover we discuss how microdomains identified by Laurdan microscopy are consistent with domains identified by other methodologies and put GP images in the context of current raft hypotheses.

Binding of transcription factor GabR to DNA requires recognition of DNA shape at a location distinct from its cognate binding site

Publisher: Oxford University Press (OUP)

Date: 17-12-2016

DOI: 10.1093/NAR/GKV1466

The lipid raft hypothesis revisited – New insights on raft composition and function from super‐resolution fluorescence microscopy

Publisher: Wiley

Date: 14-06-2012

DOI: 10.1002/BIES.201200044

Abstract: Recently developed super-resolution microscopy techniques are changing our understanding of lipid rafts and membrane organisation in general. The lipid raft hypothesis postulates that cholesterol can drive the formation of ordered domains within the plasma membrane of cells, which may serve as platforms for cell signalling and membrane trafficking. There is now a wealth of evidence for these domains. However, their study has hitherto been h ered by the resolution limit of optical microscopy, making the definition of their properties problematic and contentious. New microscopy techniques circumvent the resolution limit and, for the first time, allow the fluorescence imaging of structures on length scales below 200 nm. This review describes such techniques, particularly as applied to the study of membrane organisation, synthesising newly emerging facets of lipid raft biology into a state-of-the art model.

A FRET sensor enables quantitative measurements of membrane charges in live cells

Publisher: Springer Science and Business Media LLC

Date: 13-03-2017

DOI: 10.1038/NBT.3828

Abstract: Membrane charge has a critical role in protein trafficking and signaling. However, quantification of the effective electrostatic potential of cellular membranes has remained challenging. We developed a fluorescence membrane charge sensor (MCS) that reports changes in the membrane charge of live cells via Förster resonance energy transfer (FRET). MCS is permanently attached to the inner leaflet of the plasma membrane and shows a linear, reversible and fast response to changes of the electrostatic potential. The sensor can monitor a wide range of cellular treatments that alter the electrostatic potential, such as incorporation and redistribution of charged lipids and alterations in cytosolic ion concentration. Applying the sensor to T cell biology, we used it to identify charged membrane domains in the immunological synapse. Further, we found that electrostatic interactions prevented spontaneous phosphorylation of the T cell receptor and contributed to the formation of signaling clusters in T cells.

Dynamic control of β1 integrin adhesion by the plexinD1-sema3E axis

Publisher: Proceedings of the National Academy of Sciences

Date: 16-12-2013

DOI: 10.1073/PNAS.1314209111

Abstract: Cell-expressed integrins mediate adhesion with other cells and with extracellular matrix and are essential for embryonic development and for controlling leukocyte migration in later life. Integrin adhesion depends on conformational change leading to activation, although it remains unknown exactly how integrins alter their conformational state and adhesion in response to guidance cues. We show that the guidance molecule plexinD1 controls clustering of integrins in patches on the cell membrane and that the activation state of in idual integrins in these patches can be switched off by binding of sema3E to plexinD1. Disruption of this pathway causes abnormal thymocyte adhesion regulation and migration during development, leading to autoimmune phenomena.

Enhancing Quantum Dots for Bioimaging using Advanced Surface Chemistry and Advanced Optical Microscopy: Application to Silicon Quantum Dots (SiQDs)

Publisher: Wiley

Date: 31-08-2015

DOI: 10.1002/ADMA.201503223

Abstract: Fluorescence lifetime imaging microscopy is successfully demonstrated in both one- and two-photon cases with surface modified, nanocrystalline silicon quantum dots in the context of bioimaging. The technique is further demonstrated in combination with Förster resonance energy transfer studies where the color of the nanoparticles is tuned by using organic dye acceptors directly conjugated onto the nanoparticle surface.

The Synthesis of Ketone‐Derived Enamides by Elimination of HCN from Cyanoamides

Publisher: Wiley

Date: 29-07-2016

DOI: 10.1002/EJOC.201600638

Method for co-cluster analysis in multichannel single-molecule localisation data

Publisher: Springer Science and Business Media LLC

Date: 19-03-2014

DOI: 10.1007/S00418-014-1208-Z

Abstract: We demonstrate a combined univariate and bivariate Getis and Franklin's local point pattern analysis method to investigate the co-clustering of membrane proteins in two-dimensional single-molecule localisation data. This method assesses the degree of clustering of each molecule relative to its own species and relative to a second species. Using simulated data, we show that this approach can quantify the degree of cluster overlap in multichannel point patterns. The method is validated using photo-activated localisation microscopy and direct stochastic optical reconstruction microscopy data of the proteins Lck and CD45 at the T cell immunological synapse. Analysing co-clustering in this manner is generalizable to higher numbers of fluorescent species and to three-dimensional or live cell data sets.

Tropomyosin Tm5NM1 spatially restricts Src kinase activity through perturbation of Rab11 vesicle trafficking

Publisher: Informa UK Limited

Date: 12-2014

DOI: 10.1128/MCB.00796-14

Super-resolution microscopy of the immunological synapse

Publisher: Elsevier BV

Date: 06-2013

DOI: 10.1016/J.COI.2013.04.002

Abstract: Deciphering the spatial organisation of signalling proteins is the key to understanding the mechanisms underlying immune cell activation. Every advance in imaging technology has led to major breakthroughs in unravelling how receptor and signalling proteins are distributed within the plasma membrane and how membrane signalling is integrated with endosomes and vesicular trafficking. Recently, super-resolution fluorescence microscopy has been applied to immunological synapses, gaining new insights into the nanoscale organisation of signalling processes. Here, we review the advantages and potential of super-resolution microscopy for elucidating the regulation of many aspects of immune signalling.

Substrate independent assembly of optical structures guided by biomolecular interactions

Publisher: American Chemical Society (ACS)

Date: 05-11-2010

DOI: 10.1021/AM1007084

Abstract: The chip-scale integration of optical components is crucial for technologies as erse as optical communications, optoelectronics displays, and photovoltaics. However, the realization of integrated optical devices from discrete components is often h ered by the lack of a universal substrate for achieving monolithic integration and by incompatibilities between materials. Emergent technologies such as chip-scale biophotonics, organic optoelectronics, and optofluidics present a host of new challenges for optical device integration, which cannot be solved with existing bonding techniques. Here, we report a new method for substrate independent integration of dissimilar optical components by way of biological recognition-directed assembly. Bonding in this scheme is achieved by locally modifying the substrate with a protein receptor and the optical component with a biomolecular ligand or vice versa. The key features of this new technology include substrate independent assembly, cross-platform vertical scale integration, and selective registration of components based on complementary biomolecular interactions.

Membrane Domains as Signaling Centers in Macrophages and T-Cells: From Concepts to Experiments

Publisher: Bentham Science Publishers Ltd.

Date: 12-2008

DOI: 10.2174/187152208787169152

Tracking molecular dynamics without tracking: image correlation of photo-activation microscopy

Publisher: IOP Publishing

Date: 09-03-2015

DOI: 10.1088/2050-6120/3/1/014006

Abstract: Measuring protein dynamics in the plasma membrane can provide insights into the mechanisms of receptor signaling and other cellular functions. To quantify protein dynamics on the single molecule level over the entire cell surface, sophisticated approaches such as single particle tracking (SPT), photo-activation localization microscopy (PALM) and fluctuation-based analysis have been developed. However, analyzing molecular dynamics of fluorescent particles with intermittent excitation and low signal-to-noise ratio present at high densities has remained a challenge. We overcame this problem by applying spatio-temporal image correlation spectroscopy (STICS) analysis to photo-activated (PA) microscopy time series. In order to determine under which imaging conditions this approach is valid, we simulated PA images of diffusing particles in a homogeneous environment and varied photo-activation, reversible blinking and irreversible photo-bleaching rates. Further, we simulated data with high particle densities that populated mobile objects (such as adhesions and vesicles) that often interfere with STICS and fluctuation-based analysis. We demonstrated in experimental measurements that the diffusion coefficient of the epidermal growth factor receptor (EGFR) fused to PAGFP in live COS-7 cells can be determined in the plasma membrane and revealed differences in the time-dependent diffusion maps between wild-type and mutant Lck in activated T cells. In summary, we have developed a new analysis approach for live cell photo-activation microscopy data based on image correlation spectroscopy to quantify the spatio-temporal dynamics of single proteins.

A Kinetic Model to Evaluate Cholesterol Efflux from THP-1 Macrophages to Apolipoprotein A-1

Publisher: American Chemical Society (ACS)

Date: 31-07-2001

DOI: 10.1021/BI010323N

Abstract: The kinetics (0 to 3 h) of cholesterol efflux to delipidated apolipoprotein A-1 were investigated, and the experimental data were best fitted to a mathematical model that involves two independent pathways of cholesterol efflux. The first pathway with a rate constant of 4.6 h(-1) is fast but removes only 3-5% of total cholesterol. After preconditioning apoA-1, it was found that this pathway remains, and hence it is a property of the cholesterol-loaded cells rather than due to modification on the apolipoprotein. This fast initial efflux does not seem to contribute to cholesterol efflux at later stages (>1 h) where a second pathway predominates. However, the fast initial efflux pool can be restored if apoA-1 is withdrawn. The second slower pathway (k(membrane--media) = 0.79 h(-1)) is associated with cholesterol ester hydrolysis whose rate constant could be experimentally verified (k(cal) = 0.43, k(exp) = 0.38 +/- 0.05). The model suggests that two different plasma membrane domains are involved in the two pathways. Loading of the cells with an oxysterol, 7-ketocholesterol (7K), inhibits efflux from both pathways. The model predicts that 7K decreases the initial efflux by decreasing the available cholesterol (by possibly affecting lipid packing), while all rate constants in the second pathway are decreased. In conclusion, the kinetic model suggests that cholesterol efflux to apoA-1 is a two-step process. In the first step, some of the plasma membrane cholesterol contributes to a fast initial efflux (possibly from lipid rafts) and leads to a second pathway that mobilizes intracellular cholesterol mobilization.

Biofunctionalization of free-standing porous silicon films for self-assembly of photonic devices

Publisher: Royal Society of Chemistry (RSC)

Date: 2012

DOI: 10.1039/C1SM06651J

Plasma membrane polarization during mating in yeast cells

Publisher: Rockefeller University Press

Date: 12-06-2006

DOI: 10.1083/JCB.200602007

Abstract: The yeast mating cell provides a simple paradigm for analyzing mechanisms underlying the generation of surface polarity. Endocytic recycling and slow diffusion on the plasma membrane were shown to facilitate polarized surface distribution of Snc1p (Valdez-Taubas, J., and H.R. Pelham. 2003. Curr. Biol. 13:1636–1640). Here, we found that polarization of Fus1p, a raft-associated type I transmembrane protein involved in cell fusion, does not depend on endocytosis. Instead, Fus1p localization to the tip of the mating projection was determined by its cytosolic domain, which binds to peripheral proteins involved in mating tip polarization. Furthermore, we provide evidence that the lipid bilayer at the mating projection is more condensed than the plasma membrane enclosing the cell body, and that sphingolipids are required for this lipid organization.

Observing the Reversible Single Molecule Electrochemistry of Alexa Fluor 647 Dyes by Total Internal Reflection Fluorescence Microscopy

Publisher: Wiley

Date: 30-08-2019

DOI: 10.1002/ANIE.201907298

Abstract: Alexa Fluor 647 is a widely used fluorescent probe for cell bioimaging and super-resolution microscopy. Herein, the reversible fluorescence switching of Alexa Fluor 647 conjugated to bovine serum albumin (BSA) and adsorbed onto indium tin oxide (ITO) electrodes under electrochemical potential control at the level of single protein molecules is reported. The modulation of the fluorescence as a function of potential was observed using total internal reflectance fluorescence (TIRF) microscopy. The fluorescence intensity of the Alexa Fluor 647 decreased, or reached background levels, at reducing potentials but returned to normal levels at oxidizing potentials. These electrochemically induced changes in fluorescence were sensitive to pH despite that BSA-Alexa Fluor 647 fluorescence without applied potential is insensitive to pH between values of 4-10. The observed pH dependence indicated the involvement of electron and proton transfer in the fluorescence switching mechanism.

Actin Dynamics Drive Membrane Reorganization and Scission in Clathrin-Independent Endocytosis

Publisher: Elsevier BV

Date: 02-2010

DOI: 10.1016/J.CELL.2010.01.010

Abstract: Nascent transport intermediates detach from donor membranes by scission. This process can take place in the absence of dynamin, notably in clathrin-independent endocytosis, by mechanisms that are yet poorly defined. We show here that in cells scission of Shiga toxin-induced tubular endocytic membrane invaginations is preceded by cholesterol-dependent membrane reorganization and correlates with the formation of membrane domains on model membranes, suggesting that domain boundary forces are driving tubule membrane constriction. Actin triggers scission by inducing such membrane reorganization process. Tubule occurrence is indeed increased upon cellular depletion of the actin nucleator component Arp2, and the formation of a cortical actin shell in liposomes is sufficient to trigger the scission of Shiga toxin-induced tubules in a cholesterol-dependent but dynamin-independent manner. Our study suggests that membranes in tubular Shiga toxin-induced invaginations are poised to undergo actin-triggered reorganization leading to scission by a physical mechanism that may function independently from or in synergy with pinchase activity.

Single molecule localization microscopy with autonomous feedback loops for ultrahigh precision

Publisher: Cold Spring Harbor Laboratory

Date: 05-12-2018

DOI: 10.1101/487728

Abstract: Single-molecule localization microscopy (SMLM) promises to provide truly molecular scale images of biological specimens 1–5 . However, mechanical instabilities in the instrument, readout errors and s le drift constitute significant challenges and severely limit both the useable data acquisition length and the localization accuracy of single molecule emitters 6 . Here, we developed an actively stabilized total internal fluorescence (TIRF) microscope that performs 3D real-time drift corrections and achieves a stability of ≤1 nm. Self-alignment of the emission light path and corrections of readout errors of the camera automate channel alignment and ensure localization precisions of 1-4 nm in DNA origami structures and cells for different labels. We used Feedback SMLM to measure the separation distance of signaling receptors and phosphatases in T cells. Thus, an improved SMLM enables direct distance measurements between molecules in intact cells on the scale between 1-20 nm, potentially replacing Förster resonance energy transfer (FRET) to quantify molecular interactions 7 . In summary, by overcoming the major bottlenecks in SMLM imaging, it is possible to generate molecular images with nanometer accuracy and conduct distance measurements on the biological relevant length scales.

The aPKC/Par3/Par6 Polarity Complex and Membrane Order Are Functionally Interdependent in Epithelia During Vertebrate Organogenesis

Publisher: Wiley

Date: 23-11-2016

DOI: 10.1111/TRA.12339

Abstract: The differential distribution of lipids between apical and basolateral membranes is necessary for many epithelial cell functions, but how this characteristic membrane organization is integrated within the polarity network during ductal organ development is poorly understood. Here we quantified membrane order in the gut, kidney and liver ductal epithelia in zebrafish larvae at 3-11 days post fertilization (dpf) with Laurdan 2-photon microscopy. We then applied a combination of Laurdan imaging, antisense knock-down and analysis of polarity markers to understand the relationship between membrane order and apical-basal polarity. We found a reciprocal relationship between membrane order and the cell polarity network. Reducing membrane condensation by exogenously added oxysterol or depletion of cholesterol reduced apical targeting of the polarity protein, aPKC. Conversely, using morpholino knock down in zebrafish, we found that membrane order was dependent upon the Crb3 and Par3 polarity protein expression in ductal epithelia. Hence our data suggest that the biophysical property of membrane lipid packing is a regulatory element in apical basal polarity.

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

Publisher: Elsevier BV

Date: 10-2006

DOI: 10.1194/JLR.M600182-JLR200

Detection of oxidized low-density lipoproteins using surface plasmon resonance

Publisher: American Chemical Society (ACS)

Date: 15-05-1999

DOI: 10.1021/AC981219N

Abstract: Management of atherosclerosis is a high priority target. If this is to be achieved, the early detection of risk and risk factors are paramount and integrated with this is a need for the detection of the oxidation state of a patient's low density lipoprotein (LDL). Presently no readily usable technique exists for their rapid determination and in order to develop such a technique a monitoring system must be devised which distinguishes a parameter which changes on oxidation and distinguishes critical and noncritical oxidation products. The strategy which is investigated here is based on the use of a heparin-modified Au-surface plasmon resonance (SPR) device as a modulator of LDL binding, according to its oxidation state. Heparin is strongly negatively charged and seven binding sites for heparin have been identified on the LDL apoprotein consisting of arginine and lysine clusters these are regarded as identical to the LDL receptor binding sites. The heparin-modified surface was calibrated for LDL and a calibration factor of 1.84 × 10(9) particles mm(-)(2) Δ(o)(-)(1) SPR and instrumental resolution of 9 × 10(6) particles mm(-)(2) obtained which gives sufficient scope to distinguish LDL dependent binding. LDL oxidation could involve the protein and/or lipoprotein, the latter being of interest for athersclerosis risk and the LDL binding to heparin was shown to decrease with degree of protein oxidation as determined by the free amino groups (fluorescamine assay), but was not influenced by lipid oxidation (determined by thiobarbituric acid reactive substances assay, TBARS). The SPR based assay was tested for LDL in plasma and the calibration found to follow that obtained in buffer, although the scatter was higher, probably due to interference from other plasma species. Nevertheless, in the context of the normal distribution of LDL in healthy patients, the assay would almost certainly be able to determine Ox-LDL in atherosclerotic patients.

Myelin basic protein-dependent plasma membrane reorganization in the formation of myelin

Publisher: Wiley

Date: 12-10-2006

DOI: 10.1038/SJ.EMBOJ.7601376

Enhanced flux in direct contact membrane distillation using superhydrophobic PVDF nanofibre membranes embedded with organically modified SiO2 nanoparticles

Publisher: Wiley

Date: 05-07-2019

DOI: 10.1002/JCTB.6104

3D super-resolution imaging by localization microscopy

Publisher: Springer New York

Date: 22-09-2014

DOI: 10.1007/978-1-4939-1752-5_11

Abstract: Fluorescence microscopy is an important tool in all fields of biology to visualize structures and monitor dynamic processes and distributions. Contrary to conventional microscopy techniques such as confocal microscopy, which are limited by their spatial resolution, super-resolution techniques such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) have made it possible to observe and quantify structure and processes on the single molecule level. Here, we describe a method to image and quantify the molecular distribution of membrane-associated proteins in two and three dimensions with nanometer resolution.

Observing the Reversible Single Molecule Electrochemistry of Alexa Fluor 647 Dyes by Total Internal Reflection Fluorescence Microscopy

Publisher: Wiley

Date: 30-08-2019

DOI: 10.1002/ANGE.201907298

Single-Molecule Sensors: Challenges and Opportunities for Quantitative Analysis

Publisher: Wiley

Date: 22-07-2016

DOI: 10.1002/ANIE.201600495

Abstract: Measurement science has been converging to smaller and smaller s les, such that it is now possible to detect single molecules. This Review focuses on the next generation of analytical tools that combine single-molecule detection with the ability to measure many single molecules simultaneously and/or process larger and more complex s les. Such single-molecule sensors constitute a new type of quantitative analytical tool, as they perform analysis by molecular counting and thus potentially capture the heterogeneity of the s le. This Review outlines the advantages and potential of these new, quantitative single-molecule sensors, the measurement challenges in making single-molecule devices suitable for analysis, the inspiration biology provides for overcoming these challenges, and some of the solutions currently being explored.

Flotillins Are Involved in the Polarization of Primitive and Mature Hematopoietic Cells

Publisher: Public Library of Science (PLoS)

Date: 22-12-2009

DOI: 10.1371/JOURNAL.PONE.0008290

Flexible polygon‐mirror based laser scanning microscope platform for multiphoton in‐vivo imaging

Publisher: Wiley

Date: 06-02-2017

DOI: 10.1002/JBIO.201600289

Abstract: Commercial microscopy systems make use of tandem scanning i.e. either slow or fast scanning. We constructed, for the first time, an advanced control system capable of delivering a dynamic line scanning speed ranging from 2.7 kHz to 27 kHz and achieve variable frame rates from 5 Hz to 50 Hz (512 × 512). The dynamic scanning ability is digitally controlled by a new customized open-source software named PScan1.0. This permits manipulation of scanning rates either to gain higher fluorescence signal at slow frame rate without increasing laser power or increase frame rates to capture high speed events. By adjusting imaging speed from 40 Hz to 160 Hz, we capture a range of calcium waves and transient peaks from soma and dendrite of single fluorescence neuron (CAL-520AM). Motion artifacts arising from respiratory and cardiac motion in small animal imaging reduce quality of real-time images of single cells in-vivo. An image registration algorithm, integrated with PScan1.0, was shown to perform both real time and post-processed motion correction. The improvement is verified by quantification of blood flow rates. This work describes all the steps necessary to develop a high performance and flexible polygon-mirror based multiphoton microscope system for in-vivo biological imaging.

Si-C linked oligo(ethylene glycol) layers in silicon-based photonic crystals: Optimization for implantable optical materials

Publisher: Elsevier BV

Date: 07-2007

DOI: 10.1016/J.BIOMATERIALS.2007.03.014

Abstract: Porous silicon has shown potential for various applications in biology and medicine, which require that the material (1) remain stable for the length of the intended application and (2) resist non-specific adsorption of proteins. Here we explore the efficacy of short oligo(ethylene glycol) moieties incorporated into organic layers via two separate strategies in achieving these aims. In the first strategy the porous silicon structure was modified in a single step via hydrosilylation of alpha-oligo(ethylene glycol)-omega-alkenes containing three or six ethylene glycol units. The second strategy employs two steps: (1) hydrosilylation of succinimidyl-10-undecenoate and (2) coupling of an amino hexa(ethylene glycol) species. The porous silicon photonic crystals modified by the two-step strategy displayed greater stability relative to the single step procedure when exposed to conditions of physiological temperature and pH. Both strategies produced layers that resist non-specific adsorption of proteins as determined with fluorescently labelled bovine serum albumin. The antifouling behaviour and greater stability to physiological conditions provided by this chemistry enhances the suitability of porous silicon for biomaterials applications.

How does the kinase Lck phosphorylate the T cell receptor? Spatial organization as a regulatory mechanism

Publisher: Frontiers Media SA

Date: 2012

DOI: 10.3389/FIMMU.2012.00167

Super-Resolution Imaging by Localization Microscopy

Publisher: Humana Press

Date: 18-09-2012

DOI: 10.1007/978-1-62703-137-0_6

Abstract: Photoactivated localization microscopy (PALM) and the related technique of Stochastic optical reconstruction microscopy (STORM) are super-resolution imaging methods based on the precise localization of single molecules. Instruments based on these techniques are now commercially available and are capable of generating images with lateral resolutions in the tens of nanometers range. Here, we give an overview of the current state of this technology including live-cell and 3D PALM and provide an in-depth protocol for performing PALM experiments in a fixed cell monolayer. This includes both the instrumentation/acquisition aspects and the data analysis required for generating quantitative, super-resolution data of molecular distributions. In this ex le, the system under investigation will be fixed HeLa cells transfected with the photo-switchable fluorescent protein PS-CFP2 targeted to the plasma membrane by fusion to the N-terminus of the protein kinase Lck.

The organisation of the cell membrane: do proteins rule lipids?

Publisher: Elsevier BV

Date: 06-2014

DOI: 10.1016/J.CBPA.2014.04.009

Abstract: Cell membranes are a complex adaptive system: they are constantly re-organised in response to extra- and intracellular inputs and their local and global structure ultimately determines how, where and when these inputs are processed. This requires a tight coupling of signalling and membranes in localised and specialised compartments. While lipids are essential components of cell membranes, they mostly lack a direct link to the input signals. Here we review how proteins can deform locally membranes, modify and reorganise lipids to form membrane domains and regulate properties like membrane charges and diffusion. From this point-of-view, it appears that proteins play a central role in regulating membrane organisation.

Turning single-molecule localization microscopy into a quantitative bioanalytical tool

Publisher: Springer Science and Business Media LLC

Date: 02-02-2017

DOI: 10.1038/NPROT.2016.166

Abstract: Single-molecule localization microscopy (SMLM) generates super-resolution images by serially detecting in idual fluorescent molecules. The power of SMLM, however, goes beyond images: biologically relevant information can be extracted from the mathematical relationships between the positions of the fluorophores in space and time. Here we review the history of SMLM and how recent progress in methods for spatial point analysis has enabled quantitative measurement of SMLM data, providing insights into biomolecule patterning, clustering and oligomerization in biological systems.

Domain-specific lipid distribution in macrophage plasma membranes

Publisher: Elsevier BV

Date: 07-2005

DOI: 10.1194/JLR.M500103-JLR200

Conformational states control Lck switching between free and confined diffusion modes in T cells

Publisher: Cold Spring Harbor Laboratory

Date: 18-10-2018

DOI: 10.1101/446732

Abstract: T cell receptor (TCR) phosphorylation by Lck is an essential step in T cell activation. It is known the conformational states of Lck control enzymatic activity however, the underlying principles of how Lck finds its substrate in the plasma membrane remain elusive. Here, single-particle tracking is paired with photoactivatable localization microscopy (sptPALM) to observe the diffusive modes of Lck in the plasma membrane. In idual Lck molecules switched between free and confined diffusion in resting and stimulated T cells. Conformational state, but not partitioning into membrane domains, caused Lck confinement as open conformation Lck was more confined than closed. Further confinement of kinase-dead versions of Lck suggests that Lck interacts with open active Lck to cause confinement, irrespectively of kinase activity. Our data supports a model that confined diffusion of open Lck results in high local phosphorylation rates and closed Lck diffuses freely to enable wide-range scanning of the plasma membrane.

Galectin-3 drives glycosphingolipid-dependent biogenesis of clathrin-independent carriers

Publisher: Springer Science and Business Media LLC

Date: 18-05-2014

DOI: 10.1038/NCB2970

Abstract: Several cell surface molecules including signalling receptors are internalized by clathrin-independent endocytosis. How this process is initiated, how cargo proteins are sorted and membranes are bent remains unknown. Here, we found that a carbohydrate-binding protein, galectin-3 (Gal3), triggered the glycosphingolipid (GSL)-dependent biogenesis of a morphologically distinct class of endocytic structures, termed clathrin-independent carriers (CLICs). Super-resolution and reconstitution studies showed that Gal3 required GSLs for clustering and membrane bending. Gal3 interacted with a defined set of cargo proteins. Cellular uptake of the CLIC cargo CD44 was dependent on Gal3, GSLs and branched N-glycosylation. Endocytosis of β1-integrin was also reliant on Gal3. Analysis of different galectins revealed a distinct profile of cargoes and uptake structures, suggesting the existence of different CLIC populations. We conclude that Gal3 functionally integrates carbohydrate specificity on cargo proteins with the capacity of GSLs to drive clathrin-independent plasma membrane bending as a first step of CLIC biogenesis.

Electrochemically controlled blinking of fluorophores to enable quantitative stochastic optical reconstruction microscopy (STORM) imaging

Publisher: Cold Spring Harbor Laboratory

Date: 13-09-2023

DOI: 10.1101/2023.09.13.557504

High F-Content Perfluoropolyether-Based Nanoparticles for Targeted Detection of Breast Cancer by 19F Magnetic Resonance and Optical Imaging

Publisher: American Chemical Society (ACS)

Date: 17-08-2018

DOI: 10.1021/ACSNANO.8B03726

Abstract: Two important challenges in the field of

Different functionalization of the internal and external surfaces in mesoporous materials for biosensing applications using click chemistry

Publisher: American Chemical Society (ACS)

Date: 09-12-2011

DOI: 10.1021/LA102599M

Cyclodextrins differentially mobilize free and esterified cholesterol from primary human foam cell macrophages

Publisher: Elsevier BV

Date: 06-2003

DOI: 10.1194/JLR.M200464-JLR200

A photoelectrochemical platform for the capture and release of rare single cells

Publisher: Springer Science and Business Media LLC

Date: 12-06-2018

DOI: 10.1038/S41467-018-04701-Y

Abstract: For many normal and aberrant cell behaviours, it is important to understand the origin of cellular heterogeneity. Although powerful methods for studying cell heterogeneity have emerged, they are more suitable for common rather than rare cells. Exploring the heterogeneity of rare single cells is challenging because these rare cells must be first pre-concentrated and undergo analysis prior to classification and expansion. Here, a versatile capture & release platform consisting of an antibody-modified and electrochemically cleavable semiconducting silicon surface for release of in idual cells of interest is presented. The captured cells can be interrogated microscopically and tested for drug responsiveness prior to release and recovery. The capture & release strategy was applied to identify rare tumour cells from whole blood, monitor the uptake of, and response to, doxorubicin and subsequently select cells for single-cell gene expression based on their response to the doxorubicin.

Activation of Endothelial Nitric Oxide (eNOS) Occurs through Different Membrane Domains in Endothelial Cells

Publisher: Public Library of Science (PLoS)

Date: 15-03-2016

DOI: 10.1371/JOURNAL.PONE.0151556

Disruption of Serinc1, which facilitates serine-derived lipid synthesis, fails to alter macrophage function, lymphocyte proliferation or autoimmune disease susceptibility

Publisher: Elsevier BV

Date: 02-2017

DOI: 10.1016/J.MOLIMM.2016.12.007

Abstract: During immune cell activation, serine-derived lipids such as phosphatidylserine and sphingolipids contribute to the formation of protein signaling complexes within the plasma membrane. Altering lipid composition in the cell membrane can subsequently affect immune cell function and the development of autoimmune disease. Serine incorporator 1 (SERINC1) is a putative carrier protein that facilitates synthesis of serine-derived lipids. To determine if SERINC1 has a role in immune cell function and the development of autoimmunity, we characterized a mouse strain in which a retroviral insertion abolishes expression of the Serinc1 transcript. Expression analyses indicated that the Serinc1 transcript is readily detectable and expressed at relatively high levels in wildtype macrophages and lymphocytes. The ablation of Serinc1 expression in these immune cells, however, did not significantly alter serine-derived lipid composition or affect macrophage function and lymphocyte proliferation. Analyses of Serinc1-deficient mice also indicated that systemic ablation of Serinc1 expression did not affect viability, fertility or autoimmune disease susceptibility. These results suggest that Serinc1 is dispensable for certain immune cell functions and does not contribute to previously reported links between lipid composition in immune cells and autoimmunity.

Modifying Porous Silicon with Self-Assembled Monolayers for Biomedical Applications: The Influence of Surface Coverage on Stability and Biomolecule Coupling

Publisher: Wiley

Date: 08-12-2008

DOI: 10.1002/ADFM.200800640

Caveolin-1-dependent and -independent membrane domains

Publisher: Elsevier BV

Date: 08-2009

DOI: 10.1194/JLR.M800601-JLR200

Real-Time Bioimpedance Sensing of Antifibrotic Drug Action in Primary Human Cells

Publisher: American Chemical Society (ACS)

Date: 19-09-2017

DOI: 10.1021/ACSSENSORS.7B00442

Abstract: Fibrotic diseases are among the most serious health issues with severe burdens due to their chronic nature and a large number of patients suffering from the debilitating effects and long-term sequelae. Collagenase treatment is a nonsurgical option but has limited results. To date, there is no potent noninvasive solution for fibrosis. Part of the reason for this is the lack of appropriate in vitro live cell screening tools to assess the efficacy of new therapeutical agents. Here, we demonstrate the utility of a cell-based electrochemical impedance biosensor platform to screen the efficacy of potential antifibrotic compounds. The platform employs a label-free and noninvasive strategy to detect the progression of fibrosis and the potency of the antifibrotic molecules in real-time. The fundamental principle that governs this novel system is that dynamic changes in cell shape and adhesion during fibrosis can be measured accurately by monitoring the changes in the impedance. This is achieved by growing the cells on a transparent interdigitated indium tin oxide (ITO) electrodes. It was demonstrated by monitoring the efficacy of a model antifibrotic compound, PXS64, on cells collected from patients with Dupuytren's contracture. We confirmed the validity of the developed biochemical impedance biosensor as an tool for in vitro screening of antifibrotic compounds and provided quantitative information on subcellular influences of the examined chemical molecules using correlative microscopy analyses that monitor the average cell area, cell morphology, and the amount and directionality of the deposited extracellular matrix protein collagen and measurement of cytosolic Ca

Single-step DNA immobilization on antifouling self-assembled monolayers covalently bound to silicon (111)

Publisher: American Chemical Society (ACS)

Date: 14-03-2006

DOI: 10.1021/LA060331A

Abstract: Hydrosilylation of alkenes with epoxide-terminated tri(ethylene oxide) moieties on Si-H surfaces yields homogeneous monolayers for the efficient coupling of biomolecules. The wetting properties of the epoxide-functionalized surface allow for the spotting of solutions of biomolecules, making the surface amenable to microarraying. Immobilization of thiolated DNA was achieved in a single step to fabricate biorecognition interfaces showing the hybridization of complementary DNA at low concentrations and negligible binding of noncomplementary DNA.

Efficient synthesis of fused bicyclic ethers and their application in herbicide chemistry

Publisher: Elsevier BV

Date: 10-2014

DOI: 10.1016/J.BMCL.2014.08.043

Abstract: A series of triketones 2 and 3 featuring novel fused bicyclic aryl ethers have been prepared. The syntheses utilized ring-closing olefin metathesis (compounds 2), or oxidative cyclization of allylphenols as the key steps. The herbicidal activity of the targeted triketones 2 and 3 on various grasses and broad-leaved weeds was determined and compared with compound 1. The strength of the novel compounds 2 and 3 is their good herbicidal activity on broad-leaved weeds in post-emergent applications, whereas activity on grasses was inferior compared to 1. In addition, computational methods have been applied to provide a deeper understanding of the SAR found for compounds 2 and 3.

Direct-laser writing for subnanometer focusing and single-molecule imaging

Publisher: Springer Science and Business Media LLC

Date: 03-02-2022

DOI: 10.1038/S41467-022-28219-6

Abstract: Two-photon direct laser writing is an additive fabrication process that utilizes two-photon absorption of tightly focused femtosecond laser pulses to implement spatially controlled polymerization of a liquid-phase photoresist. Two-photon direct laser writing is capable of nanofabricating arbitrary three-dimensional structures with nanometer accuracy. Here, we explore direct laser writing for high-resolution optical microscopy by fabricating unique 3D optical fiducials for single-molecule tracking and 3D single-molecule localization microscopy. By having control over the position and three-dimensional architecture of the fiducials, we improve axial discrimination and demonstrate isotropic subnanometer 3D focusing ( .8 nm) over tens of micrometers using a standard inverted microscope. We perform 3D single-molecule acquisitions over cellular volumes, unsupervised data acquisition and live-cell single-particle tracking with nanometer accuracy.

The myelin proteolipid plasmolipin forms oligomers and induces liquid-ordered membranes in the Golgi complex

Publisher: The Company of Biologists

Date: 07-2015

DOI: 10.1242/JCS.166249

Abstract: Myelin comprises a compactly stacked massive surface area of protein-poor thick membrane that insulates axons to allow fast signal propagation. Increasing levels of the myelin protein plasmolipin (PLLP) were correlated with post-natal myelination however, its function is unknown. Here, the intracellular localization and dynamics of PLLP were characterized in primary glial and cultured cells using fluorescently labeled PLLP and antibodies against PLLP. PLLP localized to and recycled between the plasma membrane and the Golgi complex. In the Golgi complex, PLLP forms oligomers based on fluorescence resonance energy transfer (FRET) analyses. PLLP oligomers blocked Golgi to plasma membrane transport of the secretory protein vesicular stomatitis virus G protein (VSVG), but not of a VSVG mutant with an elongated transmembrane domain. Laurdan staining analysis showed that this block is associated with PLLP-induced proliferation of liquid-ordered membranes. These findings show the capacity of PLLP to assemble potential myelin membrane precursor domains at the Golgi complex through its oligomerization and ability to attract liquid-ordered lipids. These data support a model in which PLLP functions in myelin biogenesis through organization of myelin liquid-ordered membranes in the Golgi complex.

Monolayer surface chemistry enables 2-colour single molecule localisation microscopy of adhesive ligands and adhesion proteins

Publisher: Springer Science and Business Media LLC

Date: 20-08-2018

DOI: 10.1038/S41467-018-05837-7

Abstract: Nanofabricated and nanopatterned surfaces have revealed the sensitivity of cell adhesion to nanoscale variations in the spacing of adhesive ligands such as the tripeptide arginine-glycine-aspartic acid (RGD). To date, surface characterisation and cell adhesion are often examined in two separate experiments so that the localisation of ligands and adhesion proteins cannot be combined in the same image. Here we developed self-assembled monolayer chemistry for indium tin oxide (ITO) surfaces for single molecule localisation microscopy (SMLM). Cell adhesion and spreading were sensitive to average RGD spacing. At low average RGD spacing, a threshold exists of 0.8 RGD peptides per µm 2 that tether cells to the substratum but this does not enable formation of focal adhesions. These findings suggest that cells can sense and engage single adhesive ligands but ligand clustering is required for cell spreading. Thus, our data reveal subtle differences in adhesion biology that may be obscured in ensemble measurements.

Visualizing lipid structure and raft domains in living cells with two-photon microscopy

Publisher: Proceedings of the National Academy of Sciences

Date: 12-12-2003

DOI: 10.1073/PNAS.2534386100

Abstract: The lateral organization of cellular membranes is formed by the clustering of specific lipids, such as cholesterol and sphingolipids, into highly condensed domains (termed lipid rafts). Hence such domains are distinct from the remaining membrane by their lipid structure (liquid-ordered vs. -disordered domains). Here, we directly visualize membrane lipid structure of living cells by using two-photon microscopy. In macrophages, liquid-ordered domains are particularly enriched on membrane protrusions (filopodia), adhesion points and cell–cell contacts and cover 10–15% of the cell surface at 37°C. By deconvoluting the images, we demonstrate the existence of phase separation in vivo . We compare the properties of microscopically visible domains ( μm 2 ), with those of isolated detergent-resistant membranes and provide evidence that membrane coverage by lipid rafts and their fluidity are principally governed by cholesterol content, thereby providing strong support for the lipid raft hypothesis.

Probing DNA–peptide interaction forces at the single‐molecule level

Publisher: Wiley

Date: 27-11-2006

DOI: 10.1002/PSC.820

Abstract: The versatility of chemical peptide synthesis combined with the high sensitivity of AFM single-molecule force spectroscopy allows us to investigate, quantify, and control molecular recognition processes (molecular nanotechnology), offering a tremendous potential in chemical biology.Single-molecule force spectroscopy experiments are able to detect fast intermediate transition states, details of the energy landscape, and structural changes, while avoiding multiple binding events that can occur under ensemble conditions. Dynamic force spectroscopy (DFS) is even able to provide data on the complex lifetime. This minireview outlines the biophysical methodology, discusses different experimental set-ups, and presents representative results in the form of two case studies, both dealing with DNA-binding peptides. They may serve as model systems, e.g., for transcription factors or gene transfection agents.

Simultaneous impedance spectroscopy and fluorescence microscopy for the real-time monitoring of the response of cells to drugs

Publisher: Royal Society of Chemistry (RSC)

Date: 2017

DOI: 10.1039/C6SC05159F

Abstract: A dual fluorescence microscopy and electrochemical strategy to investigate how cell–surface interactions influence the cellular responses to cues for the cell-based biosensing of drug efficacy is reported herein.

Introducing membrane charge and membrane potential to T cell signaling

Publisher: Frontiers Media SA

Date: 09-11-2017

DOI: 10.3389/FIMMU.2017.01513

The MARVEL transmembrane motif of occludin mediates oligomerization and targeting to the basolateral surface in epithelia

Publisher: The Company of Biologists

Date: 08-2012

DOI: 10.1242/JCS.100289

Abstract: Occludin (Ocln), a MARVEL-motif-containing protein, is found in all tight junctions. MARVEL motifs are comprised of four transmembrane helices associated with the localization to or formation of erse membrane subdomains by interacting with the proximal lipid environment. The functions of the Ocln MARVEL motif are unknown. Bioinformatics sequence- and structure-based analyses demonstrated that the MARVEL domain of Ocln family proteins has distinct evolutionarily conserved sequence features that are consistent with its basolateral membrane localization. Live-cell microscopy, fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) were used to analyze the intracellular distribution and self-association of fluorescent-protein-tagged full-length human Ocln or the Ocln MARVEL motif excluding the cytosolic C- and N-termini (amino acids 60–269, FP-MARVEL-Ocln). FP-MARVEL-Ocln efficiently arrived at the plasma membrane (PM) and was sorted to the basolateral PM in filter-grown polarized MDCK cells. A series of conserved aromatic amino acids within the MARVEL domain were found to be associated with Ocln dimerization using BiFC. FP-MARVEL-Ocln inhibited membrane pore growth during Triton-X-100-induced solubilization and was shown to increase the membrane-ordered state using Laurdan, a lipid dye. These data demonstrate that the Ocln MARVEL domain mediates self-association and correct sorting to the basolateral membrane.

HIV-1 Nef mobilizes lipid rafts in macrophages through a pathway that competes with ABCA1-dependent cholesterol efflux

Publisher: Elsevier BV

Date: 04-2012

DOI: 10.1194/JLR.M023119

The ATP binding cassette transporter, ABCG1, localizes to cortical actin filaments

Publisher: Springer Science and Business Media LLC

Date: 06-02-2017

DOI: 10.1038/SREP42025

Abstract: The ATP-binding cassette sub-family G member 1 (ABCG1) exports cellular cholesterol to high-density lipoproteins (HDL). However, a number of recent studies have suggested ABCG1 is predominantly localised to intracellular membranes. In this study, we found that ABCG1 was organized into two distinct cellular pools: one at the plasma membrane and the other associated with the endoplasmic reticulum (ER). The plasma membrane fraction was organized into filamentous structures that were associated with cortical actin filaments. Inhibition of actin polymerization resulted in complete disruption of ABCG1 filaments. Cholesterol loading of the cells increased the formation of the filamentous ABCG1, the proximity of filamentous ABCG1 to actin filaments and the diffusion rate of membrane associated ABCG1. Our findings suggest that the actin cytoskeleton plays a critical role in the plasma membrane localization of ABCG1.

Spacing of integrin ligands influences signal transduction in endothelial cells

Publisher: Elsevier BV

Date: 08-2011

DOI: 10.1016/J.BPJ.2011.06.064

Precise, high-throughput production of multicellular spheroids with a bespoke 3D bioprinter

Publisher: Cold Spring Harbor Laboratory

Date: 07-04-2020

DOI: 10.1101/2020.04.06.028548

Abstract: 3D in vitro cancer models are important therapeutic and biological discovery tools, yet formation of multicellular spheroids in a throughput and highly controlled manner to achieve robust and statistically relevant data, remains challenging. Here, we developed an enabling technology consisting of a bespoke drop-on-demand 3D bioprinter capable of high-throughput printing of 96-well plates of spheroids. 3D-multicellular spheroids are embedded inside a tissue-like matrix with precise control over size and cell number. Application of 3D bioprinting for high-throughput drug screening was demonstrated with doxorubicin. Measurements showed that IC 50 values were sensitive to spheroid size, embedding and how spheroids conform to the embedding, revealing parameters shaping biological responses in these models. Our study demonstrates the potential of 3D bioprinting as a robust high-throughput platform to screen biological and therapeutic parameters. In vitro 3D cell cultures serve as more realistic models, compared to 2D cell culture, for understanding erse biology and for drug discovery. Preparing 3D cell cultures with defined parameters is challenging, with significant failure rates when embedding 3D multicellular spheroids into extracellular mimics. Here, we report a new 3D bioprinter we developed in conjunction with bioinks to allow 3D-multicellular spheroids to be produced in a high-throughput manner. High-throughput production of embedded multicellular spheroids allowed entire drug-dose responses to be performed in 96-well plate format with statistically relevant numbers of data points. We have deconvoluted important parameters in drug responses including the impact of spheroid size and embedding in an extracellular matrix mimic on IC 50 values.

Prolonged Intake of Dietary Lipids Alters Membrane Structure and T Cell Responses in LDLr−/− Mice

Publisher: The American Association of Immunologists

Date: 15-05-2016

DOI: 10.4049/JIMMUNOL.1501261

Abstract: Although it is recognized that lipids and membrane organization in T cells affect signaling and T cell activation, to what extent dietary lipids alter T cell responsiveness in the absence of obesity and inflammation is not known. In this study, we fed low-density lipoprotein receptor knockout mice a Western high-fat diet for 1 or 9 wk and examined T cell responses in vivo along with T cell lipid composition, membrane order, and activation ex vivo. Our data showed that high levels of circulating lipids for a prolonged period elevated CD4+ and CD8+ T cell proliferation and resulted in an increased proportion of CD4+ central-memory T cells within the draining lymph nodes following induction of contact hypersensitivity. In addition, the 9-wk Western high-fat diet elevated the total phospholipid content and monounsaturated fatty acid level, but decreased saturated phosphatidylcholine and sphingomyelin within the T cells. The altered lipid composition in the circulation, and of T cells, was also reflected by enhanced membrane order at the activation site of ex vivo activated T cells that corresponded to increased IL-2 mRNA levels. In conclusion, dietary lipids can modulate T cell lipid composition and responses in lipoprotein receptor knockout mice even in the absence of excess weight gain and a proinflammatory environment.

tagPAINT: covalent labelling of genetically encoded protein tags for DNA-PAINT imaging

Publisher: The Royal Society

Date: 12-2019

DOI: 10.1098/RSOS.191268

Abstract: Recently, DNA-PAINT single-molecule localization microscopy (SMLM) has shown great promise for quantitative imaging however, labelling strategies thus far have relied on multivalent and affinity-based approaches. Here, the covalent labelling of expressed protein tags (SNAP tag and Halo tag) with single DNA-docking strands and application of SMLM via DNA-PAINT is demonstrated. tagPAINT is then used for T-cell receptor signalling proteins at the immune synapse as a proof of principle.

The relative importance of topography and RGD ligand density for endothelial cell adhesion

Publisher: Public Library of Science (PLoS)

Date: 11-07-2011

DOI: 10.1371/JOURNAL.PONE.0021869

An RPTPα/Src family kinase/Rap1 signaling module recruits myosin IIB to support contractile tension at apical E-cadherin junctions

Publisher: American Society for Cell Biology (ASCB)

Date: 04-2015

DOI: 10.1091/MBC.E14-07-1223

Abstract: Cell–cell adhesion couples the contractile cortices of epithelial cells together, generating tension to support a range of morphogenetic processes. E-cadherin adhesion plays an active role in generating junctional tension by promoting actin assembly and cortical signaling pathways that regulate myosin II. Multiple myosin II paralogues accumulate at mammalian epithelial cell–cell junctions. Earlier, we found that myosin IIA responds to Rho-ROCK signaling to support junctional tension in MCF-7 cells. Although myosin IIB is also found at the zonula adherens (ZA) in these cells, its role in junctional contractility and its mode of regulation are less well understood. We now demonstrate that myosin IIB contributes to tension at the epithelial ZA. Further, we identify a receptor type-protein tyrosine phosphatase alpha–Src family kinase–Rap1 pathway as responsible for recruiting myosin IIB to the ZA and supporting contractile tension. Overall these findings reinforce the concept that orthogonal E-cadherin–based signaling pathways recruit distinct myosin II paralogues to generate the contractile apparatus at apical epithelial junctions.

Insights into adhesion biology using single-molecule localization microscopy

Publisher: Wiley

Date: 04-02-2014

DOI: 10.1002/CPHC.201301041

Abstract: Focal adhesions are complex multi-protein structures that mediate cell adhesion and cell migration in multicellular organisms. Most of the protein components involved in focal adhesion formation have been identified, but a major challenge remains: determination of the spatial and temporal dynamics of adhesion proteins in order to understand the molecular mechanisms of adhesion assembly, maturation, signal regulation, and disassembly. Progress in this field has been h ered by the limited resolution of fluorescence microscopy. Recent advances have led to the development of super-resolution techniques including single-molecule localization microscopy (SMLM). Here, we discuss how the application of these techniques has revealed important new insights into focal adhesion structure and dynamics, including the first description of the three-dimensional nano-architecture of focal adhesions and of the dynamic exchange of integrins in focal adhesions. Hence, SMLM has contributed to the refinement of existing models of adhesions as well as the establishment of novel models, thereby opening new research directions. With current improvements in SMLM instrumentation and analysis, it has become possible to study cellular adhesions at the single-molecule level.

Electrochemical switching of Si(100) modular assemblies

Publisher: American Chemical Society (ACS)

Date: 22-12-2012

DOI: 10.1021/JA210048X

Abstract: We report on a modular approach for producing well-defined and electrochemically switchable surfaces on Si(100). The switching of these surfaces is shown to change a Si(100) surface from resistant to cell adsorption to promoting cell adhesion. The electrochemical conversion of the modified electrode surface is demonstrated by X-ray photoelectron spectroscopy, X-ray reflectometry, contact angle and cell adhesion studies.

Discreet and distinct clustering of five model membrane proteins revealed by single molecule localization microscopy

Publisher: Informa UK Limited

Date: 02-01-2015

DOI: 10.3109/09687688.2014.990997

Abstract: Compartmentalization is a functionally important property of the plasma membrane, yet the underlying principles that organize membrane proteins into distinct domains are not well understood. Using single molecule localization microscopy, we assessed the clustering of five model membrane proteins in the plasma membrane of HeLa cells. All five proteins formed discrete and distinct nano-scaled clusters. The extent of clustering of the five proteins, independent of their membrane anchors, increased significantly when the fluorescent protein mEOS2 was employed, suggesting that protein-protein interactions are a key driver for clustering. Further, actin depolymerization or reduction of membrane order had a greater, and in some instances opposing effects on the clustering of membrane proteins fused to mEOS2 compared to PS-CFP2-fusion proteins. The data propose that protein interactions can override the lateral organization imposed by membrane anchors to provide an exquisite regulation of the mosaic-like compartmentalization of the plasma membrane.

Caveolin-1-Mediated Apolipoprotein A-I Membrane Binding Sites Are Not Required for Cholesterol Efflux

Publisher: Public Library of Science (PLoS)

Date: 12-08-2011

DOI: 10.1371/JOURNAL.PONE.0023353

A 3D Bioprinter Specifically Designed for the High-Throughput Production of Matrix-Embedded Multicellular Spheroids

Publisher: Elsevier BV

Date: 10-2020

DOI: 10.1016/J.ISCI.2020.101621

Endocytic crosstalk: cavins, caveolins, and caveolae regulate clathrin-independent endocytosis.

Publisher: Public Library of Science (PLoS)

Date: 08-04-2014

DOI: 10.1371/JOURNAL.PBIO.1001832

Microscopy approaches to investigate protein dynamics and lipid organization

Publisher: Informa UK Limited

Date: 21-07-2014

DOI: 10.3109/09687688.2014.937469

Abstract: The structure of cell membranes has been intensively investigated and many models and concepts have been proposed for the lateral organization of the plasma membrane. While proteomics and lipidomics have identified many if not all membrane components, how lipids and proteins interactions are coordinated in a specific cell function remains poorly understood. It is generally accepted that the organization of the plasma membrane is likely to play a critical role in the regulation of cell function such as receptor signalling by governing molecular interactions and dynamics. In this review we present different plasma membrane models and discuss microscopy approaches used for investigating protein behaviour, distribution and lipid organization.

Short peptide receptor mimics for atherosclerosis risk assessment of LDL

Publisher: Elsevier BV

Date: 03-2002

DOI: 10.1016/S0956-5663(02)00168-9

Abstract: Short peptides sequences were selected that showed binding selectivity towards healthy or oxidised (unhealthy) low density lipoprotein (LDL), respectively. These were investigated for application in atherosclerosis risk monitoring. Comparison was also made with the LDL receptor ligand repeat peptide (LR5). The peptides were immobilised on a gold surface plasmon resonance surface and LDL binding detected as a shift in the resonance. 3.7x10(7) (+/-5.6x10(6)) LDL/mm(2)/microg/ml solution LDL were bound on GlySerAspGlu-OH and 6.8x10(7) (+/-9.2x10(6)) LDL/mm(2)/microg/ml on GlyCystineSerAspGlu, compared with approximately 10(8) LDL/mm(2)/microg/ml on LR5. In this first group, binding of LDL decreased with oxidation level and a good correlation was found between LDL binding and residual amino groups on the apoprotein of the LDL following oxidation, or the change in relative electrophoretic mobility (REM) of LDL. The decrease in binding was 1.1x10(7) LDL particles/mm(2) per% oxidation for GlySerAspGlu-OH, 1.8x10(7) LDL particles/mm(2) per% oxidation for GlyCystineSerAspGlu and 2.4x10(7) LDL particles/mm(2) per% oxidation for LR5. A second group of three peptides were also selected showing increased binding with LDL oxidation: GlyCystineCysCys (1.5x10(7) LDL/mm(2) per microg/ml), GlyLysLysCys-SH (10(7) LDL/mm(2) per microg/ml) and GlyLysLys-OH (5.6x10(7) LDL/mm(2) per microg/ml). The latter gave a linear increase in LDL binding with oxidation level (1.2x10(7) LDL particles/mm(2) per% oxidation). LDL concentration is around 2-3 mg/ml in plasma compared with the low detection levels with this method (1-10 microg/ml), allowing a strategy to be developed requiring the minimum s le volume and diluting with physiological buffer prior to assay. By using a comparative reading between LDL adsorption on surfaces from the first and second group of peptides (e.g. GlyCystineSerAspGlu and GlyLysLys-OH, respectively), LDL oxidation could be determined without knowledge of LDL concentration. Higher binding was seen on GlyCystineSerAspGlu than GlyLysLys-OH below 30% LDL oxidation, whereas above 30% oxidation the binding on the latter surface was greater. Simple correlation of this form could provide good tests for atherosclerosis risk.

3D active stabilization for single-molecule imaging

Publisher: Springer Science and Business Media LLC

Date: 02-12-2021

DOI: 10.1038/S41596-020-00426-9

Abstract: A key part of any super-resolution technique involves accurately correcting for mechanical motion of the s le and setup during acquisition. If left uncorrected, drift degrades the resolution of the final reconstructed image and can introduce unwanted artifacts. Here, we describe how to implement active stabilization, thereby reducing drift to ~1 nm across all three dimensions. In this protocol, we show how to implement our method on custom and standard microscopy hardware. We detail the construction of a separate illumination and detection path, dedicated exclusively to acquiring the diffraction pattern of fiducials deposited on the imaging slide. We also show how to focus lock and adjust the focus in arbitrary nanometer step size increments. Our real-time focus locking is based on kHz calculations performed using the graphics processing unit. The fast calculations allow for rapid repositioning of the s le, which reduces drift below the photon-limited localization precision. Our approach allows for a single-molecule and/or super-resolution image acquisition free from movement artifacts and eliminates the need for complex algorithms or hardware installations. The method is also useful for long acquisitions which span over hours or days, such as multicolor super resolution. Installation does not require specialist knowledge and can be implemented in standard biological laboratories. The full protocol can be implemented within ~2 weeks.

Inhibition of Cholesterol Efflux by 7-Ketocholesterol:  Comparison between Cells, Plasma Membrane Vesicles, and Liposomes as Cholesterol Donors

Publisher: American Chemical Society (ACS)

Date: 10-2001

DOI: 10.1021/BI010833H

Abstract: Cholesterol removal from lipid-loaded macrophages is an important, potentially antiatherogenic process, and we have previously shown that an oxysterol, 7-ketocholesterol (7K), can impair efflux to lipid-free apoprotein A-1 (apoA-1). This publication investigates whether incorporation of 7K into membranes could account for this impairment of cholesterol efflux. Cholesterol efflux was studied from lipoprotein-loaded THP-1 cells, from plasma membrane vesicles obtained from these cells, and from artificial, protein-free liposomes. Impairment of cholesterol efflux by 7K was observed for all cholesterol donor systems whether measured as decline in cholesterol removal rates or as the percentage mass of total cellular cholesterol exported. 7-Ketocholesterol itself was not removed by apoA-1 from any of the cholesterol donor systems. Increasing membrane cholesterol content increased the rate of cholesterol removal by apoA-1 (as seen with plasma membrane vesicles), the quantity of cholesterol removed at equilibrium (liposomes), or both (whole cells). Although the minimum inhibitory 7K concentrations varied between the cholesterol donor systems, 7K inhibited cholesterol efflux in all systems. It was concluded that 7K induces alteration in membranes which decreased the efficiency of cholesterol efflux and the quantity of removed cholesterol induced by apoA-1. As cell membrane proteins are not essential for cholesterol efflux in these systems, the impairment of such by 7K suggests that its effect on membrane lipid composition and its structure are key regulatory elements in this efflux process.

Conformational states of the kinase Lck regulate clustering in early T cell signaling

Publisher: Springer Science and Business Media LLC

Date: 02-12-2012

DOI: 10.1038/NI.2488

Abstract: Phosphorylation of the T cell antigen receptor (TCR) by the tyrosine kinase Lck is an essential step in the activation of T cells. Because Lck is constitutively active, spatial organization may regulate TCR signaling. Here we found that Lck distributions on the molecular level were controlled by the conformational states of Lck, with the open, active conformation inducing clustering and the closed, inactive conformation preventing clustering. In contrast, association with lipid domains and protein networks were not sufficient or necessary for Lck clustering. Conformation-driven Lck clustering was highly dynamic, so that TCR triggering resulted in Lck clusters that contained phosphorylated TCRs but excluded the phosphatase CD45. Our data suggest that Lck conformational states represent an intrinsic mechanism for the intermolecular organization of early T cell signaling.

Quantifying the dynamics of the oligomeric transcription factor STAT3 by pair correlation of molecular brightness

Publisher: Springer Science and Business Media LLC

Date: 24-03-2016

DOI: 10.1038/NCOMMS11047

Abstract: Oligomerization of transcription factors controls their translocation into the nucleus and DNA-binding activity. Here we present a fluorescence microscopy analysis termed pCOMB (pair correlation of molecular brightness) that tracks the mobility of different oligomeric species within live cell nuclear architecture. pCOMB lifies the signal from the brightest species present and filters the dynamics of the extracted oligomeric population based on arrival time between two locations. We use this method to demonstrate a dependence of signal transducer and activator of transcription 3 (STAT3) mobility on oligomeric state. We find that on entering the nucleus STAT3 dimers must first bind DNA to form STAT3 tetramers, which are also DNA-bound but exhibit a different mobility signature. Examining the dimer-to-tetramer transition by a cross-pair correlation analysis (cpCOMB) reveals that chromatin accessibility modulates STAT3 tetramer formation. Thus, the pCOMB approach is suitable for mapping the impact oligomerization on transcription factor dynamics.

FSCS Reveals the Complexity of Lipid Domain Dynamics in the Plasma Membrane of Live Cells

Publisher: Elsevier BV

Date: 06-2018

DOI: 10.1016/J.BPJ.2018.04.050

Correction for Pageon et al., Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

Publisher: Proceedings of the National Academy of Sciences

Date: 17-10-2016

DOI: 10.1073/PNAS.1615763113

Quantitative Analysis of Three-Dimensional Fluorescence Localization Microscopy Data

Publisher: Elsevier BV

Date: 07-2013

DOI: 10.1016/J.BPJ.2013.05.063

How Do Cells Make Decisions: Engineering Micro- and Nanoenvironments for Cell Migration

Publisher: Hindawi Limited

Date: 2010

DOI: 10.1155/2010/363106

Abstract: Cell migration contributes to cancer metastasis and involves cell adhesion to the extracellular matrix (ECM), force generation through the cell's cytoskeletal, and finally cell detachment. Both adhesive cues from the ECM and soluble cues from neighbouring cells and tissue trigger intracellular signalling pathways that are essential for cell migration. While the machinery of many signalling pathways is relatively well understood, how hierarchies of different and conflicting signals are established is a new area of cellular cancer research. We examine the recent advances in microfabrication, microfluidics, and nanotechnology that can be utilized to engineer micro- and nanoscaled cellular environments. Controlling both adhesive and soluble cues for migration may allow us to decipher how cells become motile, choose the direction for migration, and how oncogenic transformations influences these decision-making processes.

Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

Publisher: Proceedings of the National Academy of Sciences

Date: 29-08-2016

DOI: 10.1073/PNAS.1607436113

Abstract: T-cell activation requires the translation of antigen binding to the T-cell receptor (TCR) into intracellular signaling. However, how antigen recognition and signal transduction are mechanistically linked is poorly understood. Here, we used single-molecule localization microscopy to link TCR clustering to signaling. We found that the likelihood of a single receptor to initiate signaling upon ligand binding depended on receptor-to-receptor spacing, with TCRs in dense clusters having the highest signaling efficiency. This means that antigen recognition must first be translated into a spatial reorganization of receptors into dense, signaling-competent clusters before signaling can begin. Thus, the quality of an antigen in terms of signaling is given by its ability to densely cluster receptors.

Optimisation of nanostructured porous silicon surface chemistry towards biophotonic sensors

Publisher: IEEE

Date: 2006

DOI: 10.1109/ICONN.2006.340659

Quantitative Microscopy: Protein Dynamics and Membrane Organisation

Publisher: Wiley

Date: 02-07-2009

DOI: 10.1111/J.1600-0854.2009.00908.X

Abstract: The mobility of membrane proteins is a critical determinant of their interaction capabilities and protein functions. The heterogeneity of cell membranes imparts different types of motion onto proteins immobility, random Brownian motion, anomalous sub-diffusion, 'hop' or confined diffusion, or directed flow. Quantifying the motion of proteins therefore enables insights into the lateral organisation of cell membranes, particularly membrane microdomains with high viscosity such as lipid rafts. In this review, we examine the hypotheses and findings of three main techniques for analysing protein dynamics: fluorescence recovery after photobleaching, single particle tracking and fluorescence correlation spectroscopy. These techniques, and the physical models employed in data analysis, have become increasingly sophisticated and provide unprecedented details of the biophysical properties of protein dynamics and membrane domains in cell membranes. Yet despite these advances, there remain significant unknowns in the relationships between cholesterol-dependent lipid microdomains, protein-protein interactions, and the effect of the underlying cytoskeleton. New multi-dimensional microscopy approaches may afford greater temporal and spatial resolution resulting in more accurate quantification of protein and membrane dynamics in live cells.

Protease sensing using nontoxic silicon quantum dots

Publisher: SPIE-Intl Soc Optical Eng

Date: 23-08-2017

DOI: 10.1117/1.JBO.22.8.087002

VAMP7 controls T cell activation by regulating the recruitment and phosphorylation of vesicular Lat at TCR-activation sites

Publisher: Springer Science and Business Media LLC

Date: 12-05-2013

DOI: 10.1038/NI.2609

Abstract: The mechanisms by which Lat (a key adaptor in the T cell antigen receptor (TCR) signaling pathway) and the TCR come together after TCR triggering are not well understood. We investigate here the role of SNARE proteins, which are part of protein complexes involved in the docking, priming and fusion of vesicles with opposing membranes, in this process. Here we found, by silencing approaches and genetically modified mice, that the vesicular SNARE VAMP7 was required for the recruitment of Lat-containing vesicles to TCR-activation sites. Our results indicated that this did not involve fusion of Lat-containing vesicles with the plasma membrane. VAMP7, which localized together with Lat on the subsynaptic vesicles, controlled the phosphorylation of Lat, formation of the TCR-Lat-signaling complex and, ultimately, activation of T cells. Our findings suggest that the transport and docking of Lat-containing vesicles with target membranes containing TCRs regulates TCR-induced signaling.

Plasma membrane segregation during T cell activation: probing the order of domains

Publisher: Elsevier BV

Date: 08-2007

DOI: 10.1016/J.COI.2007.05.002

Abstract: T cell activation leads to a segregation of plasma membrane domains to form TCR signalling clusters and eventually immunological synapses. At these T cell activation sites signalling protein networks reside in plasma membrane regions which adopt a highly ordered physical state. Studies of reconstituted model membranes suggest that aggregation of lipid raft-favouring membrane components may trigger this lipid ordering and condensation of membrane domains in T cells. Activation-induced protein-protein interactions such as anchorage to the cytoskeleton drive this condensation of the plasma membrane. Elucidating the functional role and specific molecular mechanisms of lipid ordering at these domains in the T cell activation cascade will be an essential element in understanding the transmission of outside signals into intracellular responses.

Annexin A6 regulates interleukin-2-mediated T-cell proliferation

Publisher: Wiley

Date: 08-03-2016

DOI: 10.1038/ICB.2016.15

Abstract: Annexin A6 (AnxA6) has been implicated in cell signalling by contributing to the organisation of the plasma membrane. Here we examined whether AnxA6 regulates signalling and proliferation in T cells. We used a contact hypersensitivity model to immune challenge wild-type (WT) and AnxA6(-/-) mice and found that the in vivo proliferation of CD4(+) T cells, but not CD8(+) T cells, was impaired in AnxA6(-/-) relative to WT mice. However, T-cell migration and signalling through the T-cell receptor ex vivo was similar between T cells isolated from AnxA6(-/-) and WT mice. In contrast, interleukin-2 (IL-2) signalling was reduced in AnxA6(-/-) compared with WT T cells. Further, AnxA6-deficient T cells had reduced membrane order and cholesterol levels. Taken together, our data suggest that AnxA6 regulates IL-2 homeostasis and sensitivity in T cells by sustaining a lipid raft-like membrane environment.

LILRA5 is expressed by synovial tissue macrophages in rheumatoid arthritis, selectively induces pro-inflammatory cytokines and IL-10 and is regulated by TNF-alphα, IL-10 and IFN-γ

Publisher: Wiley

Date: 13-11-2008

DOI: 10.1002/EJI.200838415

Abstract: Leukocyte immunoglobulin-like receptor A5 (LILRA5) belongs to a family of receptors known to regulate leukocyte activation. There are two membrane-bound and two soluble forms of LILRA5. The transmembrane LILRA5 contain a short cytoplasmic domain and a charged arginine residue within the transmembrane region. Cross-linking of LILRA5 on monocytes induced production of pro-inflammatory cytokines, suggesting that LILRA5 plays a role in inflammation. However, expression of LILRA5 in diseases with extensive inflammatory component is unknown. Rheumatoid arthritis (RA) is a chronic inflammatory synovitis characterized by unregulated activation of leukocytes leading to joint destruction. Here we demonstrate extensive LILRA5 expression on synovial tissue macrophages and in synovial fluid of patients with active RA but not in patients with osteoarthritis. We also show that LILRA5 associated with the common gamma chain of the FcR and LILRA5 cross-linking induced phosphorylation of Src tyrosine kinases and Spleen tyrosine kinase (Syk). Furthermore, LILRA5 induced selective production of pro-inflammatory cytokines as well as IL-10. LILRA5 mRNA and protein expression was tightly regulated by TNF-alpha, IL-10 and IFN-gamma. Increased expression of LILRA5 in rheumatoid tissue, together with its ability to induce key cytokines involved in RA, suggests that this novel receptor may contribute to disease pathogenesis.

S100A8 and S100A9 in human arterial wall - Implications for atherogenesis

Publisher: Elsevier BV

Date: 12-2005

DOI: 10.1074/JBC.M509442200

The integration of signaling and the spatial organization of the T cell synapse

Publisher: Frontiers Media SA

Date: 2012

DOI: 10.3389/FIMMU.2012.00352

Nanodomains in biological membranes

Publisher: Portland Press Ltd.

Date: 06-02-2015

DOI: 10.1042/BSE0570093

Abstract: Lipid rafts are defined as cholesterol- and sphingomyelin-enriched membrane domains in the plasma membrane of cells that are highly dynamic and cannot be resolved with conventional light microscopy. Membrane proteins that are embedded in the phospholipid matrix can be grouped into raft and non-raft proteins based on their association with detergent-resistant membranes in biochemical assays. Selective lipid–protein interactions not only produce heterogeneity in the membrane, but also cause the spatial compartmentalization of membrane reactions. It has been proposed that lipid rafts function as platforms during cell signalling transduction processes such as T-cell activation (see Chapter 13 (pages 165–175)). It has been proposed that raft association co-localizes specific signalling proteins that may yield the formation of the observed signalling microclusters at the immunological synapses. However, because of the nanometre size and high dynamics of lipid rafts, direct observations have been technically challenging, leading to an ongoing discussion of the lipid raft model and its alternatives. Recent developments in fluorescence imaging techniques have provided new opportunities to investigate the organization of cell membranes with unprecedented spatial resolution. In this chapter, we describe the concept of the lipid raft and alternative models and how new imaging technologies have advanced these concepts.

Integrin-mediated adhesion regulates membrane order

Publisher: Rockefeller University Press

Date: 28-08-2006

DOI: 10.1083/JCB.200603034

Abstract: The properties of cholesterol-dependent domains (lipid rafts) in cell membranes have been controversial. Because integrin-mediated cell adhesion and caveolin both regulate trafficking of raft components, we investigated the effects of adhesion and caveolin on membrane order. The fluorescent probe Laurdan and two-photon microscopy revealed that focal adhesions are highly ordered in fact, they are more ordered than caveolae or domains that stain with cholera toxin subunit B (CtxB). Membrane order at focal adhesion depends partly on phosphorylation of caveolin1 at Tyr14, which localizes to focal adhesions. Detachment of cells from the substratum triggers a rapid, caveolin-independent decrease in membrane order, followed by a slower, caveolin-dependent decrease that correlates with internalization of CtxB-stained domains. Endocytosed CtxB domains also become more fluid. Thus, membrane order is highly dependent on caveolae and focal adhesions. These results show that lipid raft properties are conferred by assembly of specific protein complexes. The ordered state within focal adhesions may have important consequences for signaling at these sites.

How does T cell receptor clustering impact on signal transduction?

Publisher: The Company of Biologists

Date: 11-02-2019

DOI: 10.1242/JCS.226423

Abstract: The essential function of the T cell receptor (TCR) is to translate the engagement of peptides on the major histocompatibility complex (pMHC) into appropriate intracellular signals through the associated cluster of differentiation 3 (CD3) complex. The spatial organization of the TCR–CD3 complex in the membrane is thought to be a key regulatory element of signal transduction, raising the question of how receptor clustering impacts on TCR triggering. How signal transduction at the TCR–CD3 complex encodes the quality and quantity of pMHC molecules is not fully understood. This question can be approached by reconstituting T cell signaling in model and cell membranes and addressed by single-molecule imaging of endogenous proteins in T cells. We highlight such methods and further discuss how TCR clustering could affect pMHC rebinding rates, the local balance between kinase and phosphatase activity and/or the lipid environment to regulate the signal efficiency of the TCR–CD3 complex. We also examine whether clustering could affect the conformation of cytoplasmic CD3 tails through a biophysical mechanism. Taken together, we highlight how the spatial organization of the TCR–CD3 complex – addressed by reconstitution approaches – has emerged as a key regulatory element in signal transduction of this archetypal immune receptor.

Dynamic organization of lymphocyte plasma membrane: lessons from advanced imaging methods

Publisher: Wiley

Date: 02-08-2010

DOI: 10.1111/J.1365-2567.2010.03319.X

Silicon-based mesoporous photonic crystals: Towards single cell optical biosensors

Publisher: SPIE

Date: 20-08-2009

DOI: 10.1117/12.826133

VCD studies on cyclic peptides assembled from L‐α‐amino acids and a trans‐2‐aminocyclopentane‐ or trans‐2‐aminocyclohexane carboxylic acid

Publisher: Wiley

Date: 16-09-2010

DOI: 10.1002/PSC.1272

Abstract: The increasing interest in peptidomimetics of biological relevance prompted us to synthesize a series of cyclic peptides comprising trans-2-aminocyclohexane carboxylic acid (Achc) or trans-2-aminocyclopentane carboxylic acid (Acpc). NMR experiments in combination with MD calculations were performed to investigate the three-dimensional structure of the cyclic peptides. These data were compared to the conformational information obtained by electronic circular dichroism (ECD) and vibrational circular dichroism (VCD) spectroscopy. Experimental VCD spectra were compared to theoretical VCD spectra computed quantum chemically at B3LYP/6-31G(d) density functional theory (DFT) level. The good agreement between the structural features derived from the VCD spectra and the NMR-based structures underlines the applicability of VCD in studying the conformation of small cyclic peptides.

Sub-resolution lipid domains exist in the plasma membrane and regulate protein diffusion and distribution

Publisher: Springer Science and Business Media LLC

Date: 04-12-2012

DOI: 10.1038/NCOMMS2273

Abstract: Lipid microdomains are postulated to regulate many membrane-associated processes but have remained highly controversial. Here we provide the first direct evidence that the plasma membrane of intact, live cells is comprised of a sub-resolution mixture of approximately 76% ordered and 24% disordered lipid domains, which correspond to liquid-ordered and -disordered model membranes. These measurements were based on the unmixing of fluorescence lifetime decays (phasor analysis) obtained from environmentally sensitive membrane dyes that report the degree of lipid packing. Using the transmembrane protein Linker for Activation of T cells (LAT) as an ex le, we demonstrate that association with ordered domains retarded LAT diffusion and decreased clustering in meso-scaled protein domains as analysed by super-resolution microscopy. Our data therefore propose a membrane model in which the majority of the plasma membrane is covered by cholesterol-dependent, ordered lipid domains that contribute to the non-random distribution and diffusion of membrane constituents.

Condensation of the plasma membrane at the site of T lymphocyte activation

Publisher: Rockefeller University Press

Date: 03-10-2005

DOI: 10.1083/JCB.200505047

Abstract: After activation, T lymphocytes restructure their cell surface to form membrane domains at T cell receptor (TCR)–signaling foci and immunological synapses (ISs). To address whether these rearrangements involve alteration in the structure of the plasma membrane bilayer, we used the fluorescent probe Laurdan to visualize its lipid order. We observed a condensation of the plasma membrane at TCR activation sites. The formation of ordered domains depends on the presence of the transmembrane protein linker for the activation of T cells and Src kinase activity. Moreover, these ordered domains are stabilized by the actin cytoskeleton. Membrane condensation occurs upon TCR stimulation alone but is prolonged by CD28 costimulation with TCR. In ISs, which are formed by conjugates of TCR transgenic T lymphocytes and cognate antigen-presenting cells, similar condensed membrane phases form first in central regions and later at the periphery of synapses. The formation of condensed membrane domains at T cell activation sites biophysically reflects membrane raft accumulation, which has potential implications for signaling at ISs.

Optical Techniques for Imaging Membrane Domains in Live Cells (Live-Cell Palm of Protein Clustering)

Publisher: Elsevier

Date: 2012

DOI: 10.1016/B978-0-12-391857-4.00011-2

Cyclic RGD peptides interfere with binding of the Helicobacter pylori protein CagL to integrins αVβ3 and α5β1

Publisher: Springer Science and Business Media LLC

Date: 14-09-2011

DOI: 10.1007/S00726-011-1066-0

Abstract: The human pathogen Helicobacter pylori that may cause different gastric diseases exploits integrins for infection of gastric cells. The H. pylori protein CagL present on the outer region of the type IV secretion pilus contains an RGD sequence (-Arg-Gly-Asp-) that enables binding to cells presenting integrins α5β1 and αVβ3. This interaction can be inhibited with conformationally designed cyclic RGD peptides derived from the CagL epitope -Ala-Leu-Arg-Gly-Asp-Leu-Ala-. The inhibition of the CagL-αVβ3 interaction by different RGD peptides strongly suggests the importance of the RGD motif for CagL binding. CagL point mutants (RAD, RGA) show decreased affinity to integrin αVβ3. Furthermore, structure-activity relationship studies with cyclic RGD peptides in a spatial screening approach show the distinct influence of the three-dimensional arrangement of RGD motif on the ability to interfere with this interaction. Resulting from these studies, similar structural requirements for the CagL epitope as previously suggested for other ligands of integrin αVβ3 are proposed.

Antibody modified porous silicon microparticles for the selective capture of cells

Publisher: American Chemical Society (ACS)

Date: 10-06-2014

DOI: 10.1021/BC500144U

Abstract: Herein, the ability of porous silicon (PSi) particles for selectively binding to specific cells is investigated. PSi microparticles with a high reflectance band in the reflectivity profile are fabricated, and subsequently passivated and modified with antibodies via the Cu(I)-catalyzed alkyne-azide cycloaddition reaction and succimidyl activation. To demonstrate the ability of the antibody-modified PSi particles to selectively bind to one cell type over others, HeLa cells were transfected with surface epitopes fused to fluorescent proteins. The antibody-functionalized PSi particles showed good selectivity for the corresponding surface protein on HeLa cells, with no significant cross-reactivity. The results are important for the application of PSi particles in cell sensing and drug delivery.

Using an Electrical Potential to Reversibly Switch Surfaces between Two States for Dynamically Controlling Cell Adhesion

Publisher: Wiley

Date: 22-06-2012

DOI: 10.1002/ANIE.201202118

Abstract: Smart surfaces presenting both antifouling molecules with a charged functional group at their distal end, and molecules that are terminated by RGD peptides for cell adhesion, were fabricated and characterized (see picture). By applying potentials of +300 or -300 mV, the surfaces could be dynamically switched to make the peptide accessible or inaccessible to cells.

Effect of surface chemistry on tropomyosin binding to actin filaments on surfaces

Publisher: Wiley

Date: 23-11-2016

DOI: 10.1002/CM.21342

Abstract: Reconstitution of actin filaments on surfaces for observation of filament-associated protein dynamics by fluorescence microscopy is currently an exciting field in biophysics. Here we examine the effects of attaching actin filaments to surfaces on the binding and dissociation kinetics of a fluorescence-labeled tropomyosin, a rod-shaped protein that forms continuous strands wrapping around the actin filament. Two attachment modalities of the actin to the surface are explored: where the actin filament is attached to the surface at multiple points along its length and where the actin filament is attached at one end and aligned parallel to the surface by buffer flow. To facilitate analysis of actin-binding protein dynamics, we have developed a software tool for the viewing, tracing and analysis of filaments and co-localized species in noisy fluorescence timelapse images. Our analysis shows that the interaction of tropomyosin with actin filaments is similar for both attachment modalities. © 2016 Wiley Periodicals, Inc.

Biologists Wanted: New Fluorescence Fluctuation Tools for Cell Biology

Publisher: Elsevier BV

Date: 08-2016

DOI: 10.1016/J.BPJ.2016.07.012

An intermolecular FRET sensor detects the dynamics of T cell receptor clustering

Publisher: Springer Science and Business Media LLC

Date: 28-04-2017

DOI: 10.1038/NCOMMS15100

Abstract: Clustering of the T-cell receptor (TCR) is thought to initiate downstream signalling. However, the detection of protein clustering with high spatial and temporal resolution remains challenging. Here we establish a Förster resonance energy transfer (FRET) sensor, named CliF, which reports intermolecular associations of neighbouring proteins in live cells. A key advantage of the single-chain FRET sensor is that it can be combined with image correlation spectroscopy (ICS), single-particle tracking (SPT) and fluorescence lifetime imaging microscopy (FLIM). We test the sensor with a light-sensitive actuator that induces protein aggregation upon radiation with blue light. When applied to T cells, the sensor reveals that TCR triggering increases the number of dense TCR–CD3 clusters. Further, we find a correlation between cluster movement within the immunological synapse and cluster density. In conclusion, we develop a sensor that allows us to map the dynamics of protein clustering in live T cells.

Secretion of Apolipoprotein E From Macrophages Occurs via a Protein Kinase A– and Calcium-Dependent Pathway Along the Microtubule Network

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 14-09-2007

DOI: 10.1161/CIRCRESAHA.107.157198

Abstract: Macrophage-specific expression of apolipoprotein (apo)E protects against atherosclerosis however, the signaling and trafficking pathways regulating secretion of apoE are unknown. We investigated the roles of the actin skeleton, microtubules, protein kinase A (PKA) and calcium (Ca 2+ ) in regulating apoE secretion from macrophages. Disrupting microtubules with vinblastine or colchicine inhibited basal secretion of apoE substantially, whereas disruption of the actin skeleton had no effect. Structurally distinct inhibitors of PKA (H89, KT5720, inhibitory peptide PKI 14–22 ) all decreased basal secretion of apoE by between 50% to 80% ( P .01). Pulse-chase experiments demonstrated that inhibition of PKA reduced the rate of apoE secretion without affecting its degradation. Confocal microscopy and live cell imaging of apoE–green fluorescent protein–transfected RAW macrophages identified apoE–green fluorescent protein in vesicles colocalized with the microtubular network, and inhibition of PKA markedly inhibited vesicular movement. Chelation of intracellular calcium ([Ca 2+ ] i ) with 1,2-bis(2-aminophenoxy)ethane- N , N , N ′, N ′-tetraacetate-acetoxymethyl ester (BAPTA-AM) inhibited apoE secretion by 77.2% ( P .01). Injection of c57Bl6 apoE +/+ bone marrow–derived macrophages into the peritoneum of apoE −/− C57Bl6 mice resulted in time-dependent secretion of apoE into plasma, which was significantly inhibited by transient exposure of macrophages to BAPTA-AM and colchicine and less effectively inhibited by H89. We conclude that macrophage secretion of apoE occurs via a PKA- and calcium-dependent pathway along the microtubule network.

Formation of tetra(ethylene oxide) terminated Si - C linked monolayers and their derivatization with glycine: An example of a generic strategy for the immobilization of biomolecules on silicon

Publisher: American Chemical Society (ACS)

Date: 28-09-2005

DOI: 10.1021/LA051191S

Abstract: Surface modification with oligo(ethylene oxide) functionalized monolayers terminated with reactive headgroups constitutes a powerful strategy to provide specific coupling of biomolecules with simultaneous protection from nonspecific adsorption on surfaces for the preparation of biorecognition interfaces. To date, oligo(ethylene oxide) functionalized monolayer-forming molecules which can be activated for attachment of biomolecules but which can selectively form monolayers onto hydrogen terminated silicon have yet to be developed. Here, self-assembled monolayers (SAMs) containing tetra(ethylene oxide) moieties protected with tert-butyl dimethylsilyl groups were formed by thermal hydrosilylation of alkenes with single-crystal Si(111)-H. The protection group was used to avoid side reactions with the hydride terminated silicon surface. Monolayer formation was carried out using solutions of the alkene in the high-boiling-point solvent 1,3,5-triethylbenzene. The protecting group was removed under very mild acidic conditions to yield a free hydroxyl functionality, a convenient surface moiety for coupling of biological entities via carbamate bond formation. The chemical composition and structure of the monolayers before and after deprotection were characterized by X-ray photoelectron spectroscopy (XPS) and X-ray reflectometry. To demonstrate the utility of this surface for covalent modification, two reagents were compared and contrasted for their ability to activate the surface hydroxyl groups for coupling of free amines, carbonyl diimidazole (CDI), and disuccinimidyl carbonate (DSC). Analysis of XP spectra before and after activation by CDI or DSC, and after subsequent reaction with glycine, provided quantitative information on the extent of activation and overall coupling efficiencies. CDI activated surfaces gave poor coupling yields under various conditions, whereas DSC mediated activation followed by aminolysis at neutral pH was found to be an efficient method for the immobilization of amines on tetra(ethylene oxide) modified surfaces.

Cryo-electron microscopy and single molecule fluorescent microscopy detect CD4 receptor induced HIV size expansion prior to cell entry

Publisher: Elsevier BV

Date: 12-2015

DOI: 10.1016/J.VIROL.2015.09.006

Abstract: Viruses are often thought to have static structure, and they only remodel after the viruses have entered target cells. Here, we detected a size expansion of virus particles prior to viral entry using cryo-electron microscopy (cryo-EM) and single molecule fluorescence imaging. HIV expanded both under cell-free conditions with soluble receptor CD4 (sCD4) targeting the CD4 binding site on the HIV-1 envelope protein (Env) and when HIV binds to receptor on cellular membrane. We have shown that the HIV Env is needed to facilitate receptor induced virus size expansions, showing that the 'lynchpin' for size expansion is highly specific. We demonstrate that the size expansion required maturation of HIV and an internal capsid core with wild type stability, suggesting that different HIV compartments are linked and are involved in remodelling. Our work reveals a previously unknown event in HIV entry, and we propose that this pre-entry priming process enables HIV particles to facilitate the subsequent steps in infection.

Phagocytosis of IgG‐Coated Polystyrene Beads by Macrophages Induces and Requires High Membrane Order

Publisher: Wiley

Date: 21-09-2011

DOI: 10.1111/J.1600-0854.2011.01272.X

Abstract: The biochemical composition and biophysical properties of cell membranes are hypothesized to affect cellular processes such as phagocytosis. Here, we examined the plasma membranes of murine macrophage cell lines during the early stages of uptake of immunoglobulin G (IgG)-coated polystyrene particles. We found that the plasma membrane undergoes rapid actin-independent condensation to form highly ordered phagosomal membranes, the biophysical hallmark of lipid rafts. Surprisingly, these membranes are depleted of cholesterol and enriched in sphingomyelin and ceramide. Inhibition of sphingomyelinase activity impairs membrane condensation, F-actin accumulation at phagocytic cups and particle uptake. Switching phagosomal membranes to a cholesterol-rich environment had no effect on membrane condensation and the rate of phagocytosis. In contrast, preventing membrane condensation with the oxysterol 7-ketocholesterol, even in the presence of ceramide, blocked F-actin dissociation from nascent phagosomes and particle uptake. In conclusion, our results suggest that ordered membranes function to co-ordinate F-actin remodelling and that the biophysical properties of phagosomal membranes are essential for phagocytosis.

Measuring membrane association and protein diffusion within membranes with supercritical angle fluorescence microscopy

Publisher: The Optical Society

Date: 29-03-2016

DOI: 10.1364/BOE.7.001561

Accumulation of raft lipids in T-cell plasma membrane domains engaged in TCR signalling

Publisher: Wiley

Date: 29-01-2009

DOI: 10.1038/EMBOJ.2009.6

Introducing distinctly different chemical functionalities onto the internal and external surfaces of mesoporous materials

Publisher: Wiley

Date: 18-03-2008

DOI: 10.1002/ANIE.200704784

Geometric regulation of histone state directs melanoma reprogramming

Publisher: Cold Spring Harbor Laboratory

Date: 11-12-2019

DOI: 10.1101/872226

Abstract: Malignant melanoma displays a high degree of cellular plasticity during disease progression, making classification of the heterogeneous population and selection of an appropriate therapy challenging. Signals in the tumor microenvironment are believed to influence melanoma plasticity through changes in the epigenetic state to guide dynamic differentiation and de-differentiation events that underlie tumorigenicity and dissemination. Here we uncover a relationship between geometric features at perimeter regions of multicellular melanoma aggregates, and reprogramming to a stem cell-like melanoma initiating cell (MIC) through histone marks H3K4Me2 and H3K9Ac. Using an in vitro tumor microengineering approach, we find concurrent expression of molecular MIC markers and spatial enrichment of these histone modifications at perimeter features. Chromatin immunoprecipitation and sequencing analysis demonstrates broad regulation of genes associated with SOX-, ETS-, and USF-families. SOX10 and PRDM14, transcriptional regulators with a putative role in several cancers, overlap with H3K9Ac and show elevated expression in cells along regions of perimeter curvature. siRNA knockdown of the epigenetic modifier PRDM14 abolishes the MIC phenotype suggesting a role in regulating melanoma heterogeneity. Our results suggest mechanotransduction at the periphery of melanoma tumors may orchestrate the activity of epigenetic modifiers to regulate histone state, cellular plasticity, and tumorigenicity.

Organic modification of mesoporous silicon rugate filters: The influence of nanoarchitecture on optical behaviour

Publisher: Inderscience Publishers

Date: 2008

DOI: 10.1504/IJNT.2008.016914

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Publisher: MyJove Corporation

Date: 04-04-2013

DOI: 10.3791/50310

Clustering and Lateral Concentration of Raft Lipids by the MAL Protein

Publisher: American Society for Cell Biology (ASCB)

Date: 15-08-2009

DOI: 10.1091/MBC.E09-02-0142

Abstract: MAL, a compact hydrophobic, four-transmembrane-domain apical protein that copurifies with detergent-resistant membranes is obligatory for the machinery that sorts glycophosphatidylinositol (GPI)-anchored proteins and others to the apical membrane in epithelia. The mechanism of MAL function in lipid-raft–mediated apical sorting is unknown. We report that MAL clusters formed by two independent procedures—spontaneous clustering of MAL tagged with the tandem dimer DiHcRED (DiHcRED-MAL) in the plasma membrane of COS7 cells and antibody-mediated cross-linking of FLAG-tagged MAL—laterally concentrate markers of sphingolipid rafts and exclude a fluorescent analogue of phosphatidylethanolamine. Site-directed mutagenesis and bimolecular fluorescence complementation analysis demonstrate that MAL forms oligomers via ϕxxϕ intramembrane protein–protein binding motifs. Furthermore, results from membrane modulation by using exogenously added cholesterol or ceramides support the hypothesis that MAL-mediated association with raft lipids is driven at least in part by positive hydrophobic mismatch between the lengths of the transmembrane helices of MAL and membrane lipids. These data place MAL as a key component in the organization of membrane domains that could potentially serve as membrane sorting platforms.

Biointerfaces on indium-tin oxide prepared from organophosphonic acid self-assembled monolayers

Publisher: American Chemical Society (ACS)

Date: 08-07-2014

DOI: 10.1021/LA501774B

Abstract: Herein we show the development of biointerfaces on indium-tin oxide (ITO) surfaces prepared from organophosphonate self-assembled monolayers. The interfaces were prepared in a stepwise fabrication procedure containing a base monolayer modified with oligo(ethylene oxide) species to which biological recognition ligands were attached. The density of ligands was controlled by varying the ratio of two oligo(ethylene oxide) species such that only one is compatible with further coupling. The final biointerface on ITO was assessed using cell adhesion studies, which showed that the biointerfaces prepared on ITO performed similarly to equivalent monolayers on gold or silicon.

Comparison of different irrigation methods to synergistically improve maize’s yield, water productivity and economic benefits in an arid irrigation area

Publisher: Elsevier BV

Date: 2021

DOI: 10.1016/J.AGWAT.2020.106497

Roles of ATP binding cassette transporters A1 and G1, scavenger receptor BI and membrane lipid domains in cholesterol export from macrophages

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 06-2006

DOI: 10.1097/01.MOL.0000226116.35555.EB

Single‐Molecule Experiments to Elucidate the Minimal Requirement for DNA Recognition by Transcription Factor Epitopes

Publisher: Wiley

Date: 19-02-2009

DOI: 10.1002/SMLL.200800945

Abstract: Interactions between proteins and DNA are essential for the regulation of cellular processes in all living organisms. In this context, it is of special interest to investigate the sequence-specific molecular recognition between transcription factors and their cognate DNA sequences. As a model system, peptide and protein epitopes of the DNA-binding domain (DBD) of the transcription factor PhoB from Escherichia coli are analyzed with respect to DNA binding at the single-molecule level. Peptides representing the hiphilic recognition helix of the PhoB DBD (amino acids 190-209) are chemically synthesized and C-terminally modified with a linker for atomic force microscopy-dynamic force spectroscopy experiments (AFM-DFS). For comparison, the entire PhoB DBD is overexpressed in E. coli and purified using an intein-mediated protein purification method. To facilitate immobilization for AFM-DFS experiments, an additional cysteine residue is ligated to the protein. Quantitative AFM-DFS analysis proves the specificity of the interaction and yields force-related properties and kinetic data, such as thermal dissociation rate constants. An alanine scan for strategic residues in both peptide and protein sequences is performed to reveal the contributions of single amino acid residues to the molecular-recognition process. Additionally, DNA binding is substantiated by electrophoretic mobility-shift experiments. Structural differences of the peptides, proteins, and DNA upon complex formation are analyzed by circular dichroism spectroscopy. This combination of techniques eventually provides a concise picture of the contribution of epitopes or single amino acids in PhoB to DNA binding.

Fluorescence spectral correlation spectroscopy (FSCS) for probes with highly overlapping emission spectra

Publisher: The Optical Society

Date: 31-01-2014

DOI: 10.1364/OE.22.002973

Distinct mechanisms regulate Lck spatial organization in activated T cells

Publisher: Frontiers Media SA

Date: 08-03-2016

DOI: 10.3389/FIMMU.2016.00083

Forming antifouling organic multilayers on porous silicon rugate filters towards in vivo/ex vivo biophotonic devices

Publisher: Wiley

Date: 15-10-2007

DOI: 10.1002/ADFM.200600790

A rapid readout for many single plasmonic nanoparticles using dark-field microscopy and digital color analysis

Publisher: Elsevier BV

Date: 10-2018

DOI: 10.1016/J.BIOS.2018.06.066

Abstract: The integration of plasmonic nanoparticles into biosensors has the potential to increase the sensitivity and dynamic range of detection, through the use of single nanoparticle assays. The analysis of the localized surface plasmon resonance (LSPR) of plasmonic nanoparticles has allowed the limit of detection of biosensors to move towards single molecules. However, due to complex equipment or slow analysis times, these technologies have not been implemented for point-of-care detection. Herein, we demonstrate an advancement in LSPR analysis by presenting a technique, which utilizes an inexpensive CMOS-equipped digital camera and a dark-field microscope, that can analyse the λ

The raft-promoting property of virion-associated cholesterol, but not the presence of virion-associated Brij 98 rafts, is a determinant of human immunodeficiency virus type 1 infectivity.

Publisher: American Society for Microbiology

Date: 10-2004

DOI: 10.1128/JVI.78.19.10556-10565.2004

Abstract: Lipid rafts are enriched in cholesterol and sphingomyelin and are isolated on the basis of insolubility in detergents, such as Brij 98 and Triton X-100. Recent work by Holm et al. has shown that rafts insoluble in Brig 98 can be found in human immunodeficiency virus type 1 (HIV-1) virus-like particles, although it is not known whether raft-like structures are present in authentic HIV-1 and it is unclear whether a virion-associated raft-like structure is required for HIV replication. Independently, it was previously reported that virion-associated cholesterol is critical for HIV-1 infectivity, although the specific requirement of virion cholesterol in HIV-1 was not examined. In the present study, we have demonstrated that infectious wild-type HIV-1 contains Brij 98 rafts but only minimal amounts of Triton X-100 rafts. To directly assess the functional requirement of virion-associated rafts and various features of cholesterol on HIV-1 replication, we replaced virion cholesterol with exogenous cholesterol analogues that have demonstrated either raft-promoting or -inhibiting capacity in model membranes. We observed that variable concentrations of exogenous analogues are required to replace a defined amount of virion-associated cholesterol, showing that structurally erse cholesterol analogues have various affinities toward HIV-1. We found that replacement of 50% of virion cholesterol with these exogenous cholesterol analogues did not eliminate the presence of Brij 98 rafts in HIV-1. However, the infectivity levels of the lipid-modified HIV-1s directly correlate with the raft-promoting capacities of these cholesterol analogues. Our data provide the first direct assessment of virion-associated Brij 98 rafts in retroviral replication and illustrate the importance of the raft-promoting property of virion-associated cholesterol in HIV-1 replication.

Expression and stability of two isoforms of ABCG1 in human vascular cells

Publisher: Elsevier BV

Date: 2010

DOI: 10.1016/J.ATHEROSCLEROSIS.2009.06.028

Abstract: To evaluate the expression of two ABCG1 isoforms that differ in the presence or absence of a 12 amino acid (AA) peptide between the ABC cassette and the transmembrane region, termed ABCG1(+12) and ABCG1(-12), respectively, in human vascular cells and tissues. mRNA for both isoforms was expressed in human macrophages, vascular endothelial and smooth muscle cells as well as whole human spleen, lung, liver and brain tissue. However, ABCG1(+12) was not expressed in mouse tissues. 2D gel electrophoresis of ABCG1 protein indicated that both protein isoforms were expressed in human macrophages. Furthermore the half-lives of the two ABCG1 protein isoforms, stably expressed in CHOK1 cells, measured under basal conditions were different, suggesting the presence of a degradation or stabilising signal in or near the 12AA region of ABCG1(+12). ABCG1(+12) is an isoform of ABCG1 exclusively expressed in human cells at the RNA and protein level. As ABCG1(+12) is not expressed in mice, although mouse models are widely used to elucidate the function of ABCG1, further investigations into the importance of this human ABCG1 isoform are warranted.

Regulated unbinding of ZAP70 at the T cell receptor by kinetic avidity

Publisher: Cold Spring Harbor Laboratory

Date: 13-02-2020

DOI: 10.1101/2020.02.12.945170

Abstract: Protein-protein binding domains are critical in signalling networks. Src homology 2 (SH2) domains are binding domains that interact with sequences containing phosphorylated tyrosines. A subset of SH2 domain-containing proteins have tandem domains, which are thought to enhance binding affinity and specificity. However, a trade-off exists between long-lived binding and the ability to rapidly reverse signalling, which is a critical requirement of noise filtering mechanisms such as kinetic proofreading. Here, we use modelling to show that the unbinding rate of tandem, but not single, SH2 domains can be accelerated by phosphatases when tandem domains bind by a kinetic, but not a static, avidity mode. We use surface plasmon resonance to show that ZAP70, a tandem SH2 domain-containing kinase, binds kinetically to biphosphorylated peptides from the T cell antigen receptor (TCR) and that the unbinding rate can be accelerated by the phosphatase CD45. An important functional prediction of regulated unbinding is that the intracellular ZAP70/TCR half-life in T cells will be correlated to the extracellular TCR/antigen half-life and we show that this is the case in both cell lines and primary T cells. The work highlights that binding by kinetic avidity breaks the trade-off between signal fidelity (requiring long half-life) and signal reversibility (requiring short half-life), which is a key requirement for T cell antigen discriminated mediated by kinetic proofreading.

FAPP2, cilium formation, and compartmentalization of the apical membrane in polarized Madin–Darby canine kidney (MDCK) cells

Publisher: Proceedings of the National Academy of Sciences

Date: 05-12-2006

DOI: 10.1073/PNAS.0608291103

Abstract: We have analyzed the role of the phosphatidylinositol-4-phosphate adaptor protein-2 (FAPP2), a component of the apical transport machinery, in cilium formation in polarized Madin–Darby canine kidney (MDCK) cells. We show that ciliogenesis is defective in FAPP2 knockdown cells. Furthermore, by using fluorescence recovery after photobleaching studies of domain connectivity and the generalized polarization spectra of Laurdan, we demonstrate that FAPP2 depletion impairs the formation of condensed apical membrane domains. Laurdan staining also revealed that the ciliary membrane has a highly condensed bilayer domain at its base that could function as a fence to separate the ciliary membrane from the surrounding apical membrane. These results indicate that the compartmentalization of the apical membrane in MDCK cells into the ciliary membrane and the surrounding membrane depends on the balance of raft and nonraft domains.

Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events

Publisher: Springer Science and Business Media LLC

Date: 05-06-2011

DOI: 10.1038/NI.2049

Abstract: Engaged T cell antigen receptors (TCRs) initiate signaling through the adaptor protein Lat. In quiescent T cells, Lat is segregated into clusters on the cell surface, which raises the question of how TCR triggering initiates signaling. Using super-resolution fluorescence microscopy, we found that pre-existing Lat domains were neither phosphorylated nor laterally transported to TCR activation sites, which suggested that these clusters do not participate in TCR signaling. Instead, TCR activation resulted in the recruitment and phosphorylation of Lat from subsynaptic vesicles. Studies of Lat mutants confirmed that recruitment preceded and was essential for phosphorylation and that both processes were independent of surface clustering of Lat. Our data suggest that TCR ligation preconditions the membrane for vesicle recruitment and bulk activation of the Lat signaling network.

Versatile click chemistry Approach to functionalizing silicon quantum dots: Applications toward fluorescent cellular imaging

Publisher: American Chemical Society (ACS)

Date: 28-04-2014

DOI: 10.1021/LA500945F

Abstract: In this study, we describe a solution procedure for the preparation and surface modification of photostable colloidal silicon quantum dots (SiQDs) for imaging of cancer cells. Photoluminescent SiQDs were synthesized by reduction of halogenated silane precursors using a microemulsion process. It was shown that 1,8-nonadiyne molecules could be grafted onto the surface of hydrogen-terminated SiQDs via ultraviolet (UV)-promoted hydrosilylation, demonstrated by Fourier transform infrared spectroscopy (FTIR) measurements. In addition, various azide molecules were coupled onto nonadiyne-functionalized particles, rendering particles dispersible in selected polar and nonpolar solvents. The photoluminescence of functionalized SiQDs was stable against photobleaching and did not vary appreciably within biologically applicable pH and temperature ranges. To demonstrate compatibility with biological systems, water-soluble SiQDs were used for fluorescent imaging of HeLa cells. In addition, the SiQDs were shown to be non-cytotoxic at concentrations up to 240 μg/mL. The results presented herein provide good evidence for the versatility of functionalized SiQDs for fluorescent bioimaging application.

Do signalling endosomes play a role in T cell activation?

Publisher: Wiley

Date: 26-07-2013

DOI: 10.1111/FEBS.12427

Abstract: Signalling endosomes represent a general mechanism for modulating and compartmentalizing cell signalling, which is achieved by delineating specific spatial environments and connecting the plasma membrane with intracellular events. The molecular composition of vesicles, together with their targeting mechanisms and endocytic routes, contributes to the outcome of signalling pathways that are initiated either at the plasma membrane or within endosomes themselves. In T cell signalling, it is now accepted that the spatial distribution of signalling proteins is central to T cell activation not only at the immunological synapse, but also in endosomes travelling to and from the plasma membrane. In addition, there is a global rearrangement of the endosome machinery upon T cell activation, and emerging experimental evidence suggests that vesicles in T cells contain key T cell signalling proteins. We review the various mechanisms by which endosomes contribute to signalling pathways and consider whether signalling endosomes play a role in T cell signalling.

Synthesis of chemically modified bioactive peptides: recent advances, challenges and developments for medicinal chemistry

Publisher: Future Science Ltd

Date: 10-2009

DOI: 10.4155/FMC.09.97

Abstract: Although not complying with Lipinski’s rule, peptides are to an increasing extent being developed into new active pharmaceutical ingredients. This is mainly due to novel application routes, formulations and chemical modifications, which confer on the peptides improved uptake and increased metabolic stability. A brief survey of currently approved peptide drugs and the present scope of the application of peptides as drugs is provided. Cyclic peptides are emerging as an interesting class of peptides with conformational rigidity and homogeneity, high receptor affinity and selectivity, increased metabolic stability and – in special cases – even oral availability. Challenges and new methodology for the synthesis of cyclic peptides are outlined and an overview of approaches toward the design of peptide conformation and peptide modification by nonproteinogenic building blocks is given.

α- and β-crystallins modulate the head group order of human lens membranes during aging

Publisher: Association for Research in Vision and Ophthalmology (ARVO)

Date: 10-2010

DOI: 10.1167/IOVS.09-4947

Molecularly engineered surfaces for cell biology: From static to dynamic surfaces

Publisher: American Chemical Society (ACS)

Date: 22-11-2014

DOI: 10.1021/LA4037919

Abstract: Surfaces with a well-defined presentation of ligands for receptors on the cell membrane can serve as models of the extracellular matrix for studying cell adhesion or as model cell surfaces for exploring cell-cell contacts. Because such surfaces can provide exquisite control over, for ex le, the density of these ligands or when the ligands are presented to the cell, they provide a very precise strategy for understanding the mechanisms by which cells respond to external adhesive cues. In the present feature article, we present an overview of the basic biology of cell adhesion before discussing surfaces that have a static presentation of immobile ligands. We outline the biological information that such surfaces have given us, before progressing to recently developed switchable surfaces and surfaces that mimic the lipid bilayer, having adhesive ligands that can move around the membrane and be remodeled by the cell. Finally, the feature article closes with some of the biological information that these new types of surfaces could provide.

UNSW Australia

Location: Australia

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UNSW Sydney

Location: Australia

View Organisation

Discovery Projects - Grant ID: DP0212068

Start Date: 01-2002

End Date: 06-2005

Amount: $231,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0556554

Start Date: 2005

End Date: 06-2010

Amount: $670,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP1095468

Start Date: 2010

End Date: 03-2013

Amount: $330,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP110100390

Start Date: 2012

End Date: 12-2017

Amount: $430,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP140100967

Start Date: 09-2015

End Date: 12-2019

Amount: $386,372.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP170102727

Start Date: 2017

End Date: 03-2020

Amount: $485,500.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP100200021

Start Date: 03-2011

End Date: 12-2014

Amount: $425,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0772356

Start Date: 04-2007

End Date: 12-2010

Amount: $370,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0450387

Start Date: 05-2004

End Date: 12-2007

Amount: $420,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP160100979

Start Date: 10-2016

End Date: 12-2020

Amount: $335,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP170103599

Start Date: 2017

End Date: 12-2019

Amount: $398,500.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE150100132

Start Date: 02-2016

End Date: 12-2017

Amount: $860,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989078

Start Date: 09-2009

End Date: 12-2009

Amount: $400,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE120100224

Start Date: 01-2012

End Date: 12-2013

Amount: $250,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0775598

Start Date: 10-2008

End Date: 11-2008

Amount: $1,000,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE180100157

Start Date: 2018

End Date: 12-2018

Amount: $600,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE100100089

Start Date: 2010

End Date: 04-2012

Amount: $700,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE150100163

Start Date: 2015

End Date: 12-2016

Amount: $560,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989744

Start Date: 05-2010

End Date: 06-2010

Amount: $500,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE100100096

Start Date: 2010

End Date: 06-2011

Amount: $450,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP130100269

Start Date: 2013

End Date: 10-2016

Amount: $465,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE110100092

Start Date: 04-2011

End Date: 03-2013

Amount: $300,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0882855

Start Date: 2008

End Date: 12-2009

Amount: $900,000.00

Funder: Australian Research Council