ORCID Profile
0000-0002-7494-5248
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Biochemistry and Cell Biology | Structural Biology (incl. Macromolecular Modelling) | Protein Trafficking | Receptors and Membrane Biology | Proteomics and Intermolecular Interactions (excl. Medical Proteomics) | Nanotechnology | Protein Targeting And Signal Transduction | Membrane Biology | Nanobiotechnology | Cell Development, Proliferation and Death | Biomaterials | Synthesis of Materials | Physiology | Pharmaceutical Sciences | Nanoscale Characterisation | Cellular Interactions (incl. Adhesion, Matrix, Cell Wall) | Nanomaterials | Enzymes | Animal Physiology - Cell | Animal Neurobiology | Cell Physiology | Functional Materials | Macromolecular and Materials Chemistry not elsewhere classified | Materials Engineering | Animal Physiology—Cell |
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Biological Sciences | Biological sciences | Expanding Knowledge in the Medical and Health Sciences | Expanding Knowledge in the Chemical Sciences | Cancer and Related Disorders | Inherited Diseases (incl. Gene Therapy) | Expanding Knowledge in Technology | Biofuel (Biomass) Energy | Diagnostic Methods | Expanding Knowledge in the Physical Sciences | Structural Metal Products | Scientific Instruments | Health and support services not elsewhere classified | Expanding Knowledge in the Information and Computing Sciences | Skeletal system and disorders (incl. arthritis) | Health not elsewhere classified | Organs, diseases and abnormal conditions not elsewhere classified | Infectious Diseases
Related Links
Publications
Fld1p, a functional homologue of human seipin, regulates the size of lipid droplets in yeast
Publisher: Rockefeller University Press
Date: 04-02-2008
Abstract: Lipid droplets (LDs) are emerging cellular organelles that are of crucial importance in cell biology and human diseases. In this study, we present our screen of ∼4,700 Saccharomyces cerevisiae mutants for abnormalities in the number and morphology of LDs we identify 17 fld (few LDs) and 116 mld (many LDs) mutants. One of the fld mutants (fld1) is caused by the deletion of YLR404W, a previously uncharacterized open reading frame. Cells lacking FLD1 contain strikingly enlarged (supersized) LDs, and LDs from fld1Δ cells demonstrate significantly enhanced fusion activities both in vivo and in vitro. Interestingly, the expression of human seipin, whose mutant forms are associated with Berardinelli-Seip congenital lipodystrophy and motoneuron disorders, rescues LD-associated defects in fld1Δ cells. Lipid profiling reveals alterations in acyl chain compositions of major phospholipids in fld1Δ cells. These results suggest that an evolutionally conserved function of seipin in phospholipid metabolism and LD formation may be functionally important in human adipogenesis.
Identification of intracellular cavin target proteins reveals cavin-PP1alpha interactions regulate apoptosis
Publisher: Springer Science and Business Media LLC
Date: 22-07-2019
DOI: 10.1038/S41467-019-11111-1
Abstract: Caveolae are specialized domains of the plasma membrane. Formation of these invaginations is dependent on the expression of Caveolin-1 or -3 and proteins of the cavin family. In response to stress, caveolae disassemble and cavins are released from caveolae, allowing cavins to potentially interact with intracellular targets. Here, we describe the intracellular (non-plasma membrane) cavin interactome using biotin affinity proteomics and mass spectrometry. We validate 47 potential cavin-interactor proteins using a cell-free expression system and protein-protein binding assays. These data, together with pathway analyses, reveal unknown roles for cavin proteins in metabolism and stress signaling. We validated the interaction between one candidate interactor protein, protein phosphatase 1 alpha (PP1α), and Cavin-1 and -3 and show that UV treatment causes release of Cavin3 from caveolae allowing interaction with, and inhibition of, PP1α. This interaction increases H2AX phosphorylation to stimulate apoptosis, identifying a pro-apoptotic signaling pathway from surface caveolae to the nucleus.
Key principles and methods for studying the endocytosis of biological and nanoparticle therapeutics
Publisher: Springer Science and Business Media LLC
Date: 03-2021
DOI: 10.1038/S41565-021-00858-8
Abstract: Endocytosis is a critical step in the process by which many therapeutic nanomedicines reach their intracellular targets. Our understanding of cellular uptake mechanisms has developed substantially in the past five years. However, these advances in cell biology have not fully translated to the nanoscience and therapeutics literature. Misconceptions surrounding the role of different endocytic pathways and how to study these pathways are hindering progress in developing improved nanoparticle therapies. Here, we summarize the latest insights into cellular uptake mechanisms and pathways. We highlight limitations of current systems to study endocytosis, particularly problems with non-specific inhibitors. We also summarize alternative genetic approaches to robustly probe these pathways and discuss the need to understand how cells endocytose particles in vivo. We hope that this critical assessment of the current methods used in studying nanoparticle uptake will guide future studies at the interface of cell biology and nanomedicine.
Identifying Optimal Lipid Raft Characteristics Required To Promote Nanoscale Protein-Protein Interactions on the Plasma Membrane
Publisher: Informa UK Limited
Date: 2006
Impaired endoplasmic reticulum-mitochondrial signaling in ataxia-telangiectasia
Publisher: Elsevier BV
Date: 2021
Author Correction: Tyrosine dephosphorylated cortactin downregulates contractility at the epithelial zonula adherens through SRGAP1.
Publisher: Springer Science and Business Media LLC
Date: 05-12-2017
DOI: 10.1038/S41467-017-02131-W
Abstract: A correction to this article has been published and is linked from the HTML version of this article.
Staurosporines Disrupt Phosphatidylserine Trafficking and Mislocalize Ras Proteins
Publisher: Elsevier BV
Date: 12-2012
Pkd1 Regulates Lymphatic Vascular Morphogenesis during Development
Publisher: Elsevier BV
Date: 05-2014
Caveolin 1 restricts Group A Streptococcus invasion of nonphagocytic host cells
Publisher: Hindawi Limited
Date: 06-09-2017
DOI: 10.1111/CMI.12772
Abstract: Caveolae are composed of 2 major proteins, caveolin 1 (CAV1) and cavin 1 or polymerase transcript release factor I (CAVIN1). Here, we demonstrate that CAV1 levels modulate invasion of Group A Streptococcus (GAS) into nonphagocytic mammalian cells. GAS showed enhanced internalisation into CAV1-knockout mouse embryonic fibroblasts and CAV1 knockdown human epithelial HEp-2 cells, whereas overexpression of CAV1 in HEp-2 cells reduced GAS invasion. This effect was not dependent on the expression of the GAS fibronectin binding protein SfbI, which had previously been implicated in caveolae-mediated uptake. Nor was this effect dependent on CAVIN1, as knockout of CAVIN1 in mouse embryonic fibroblasts resulted in reduced GAS internalisation. Although CAV1 restricted GAS invasion into host cells, we observed only minimal association of invading GAS (strain M1T1
Reactivation of Myc transcription in the mouse heart unlocks its proliferative capacity
Publisher: Springer Science and Business Media LLC
Date: 14-04-2020
DOI: 10.1038/S41467-020-15552-X
Abstract: It is unclear why some tissues are refractory to the mitogenic effects of the oncogene Myc. Here we show that Myc activation induces rapid transcriptional responses followed by proliferation in some, but not all, organs. Despite such disparities in proliferative response, Myc is bound to DNA at open elements in responsive (liver) and non-responsive (heart) tissues, but fails to induce a robust transcriptional and proliferative response in the heart. Using heart as an exemplar of a non-responsive tissue, we show that Myc-driven transcription is re-engaged in mature cardiomyocytes by elevating levels of the positive transcription elongation factor (P-TEFb), instating a large proliferative response. Hence, P-TEFb activity is a key limiting determinant of whether the heart is permissive for Myc transcriptional activation. These data provide a greater understanding of how Myc transcriptional activity is determined and indicate modification of P-TEFb levels could be utilised to drive regeneration of adult cardiomyocytes for the treatment of heart myopathies.
A Single Method for Cryofixation and Correlative Light, Electron Microscopy and Tomography of Zebrafish Embryos
Publisher: Wiley
Date: 16-01-2009
DOI: 10.1111/J.1600-0854.2008.00859.X
Abstract: The zebrafish is a powerful vertebrate system for cell and developmental studies. In this study, we have optimized methods for fast freezing and processing of zebrafish embryos for electron microscopy (EM). We show that in the absence of primary chemical fixation, excellent ultrastructure, preservation of green fluorescent protein (GFP) fluorescence, immunogold labelling and electron tomography can be obtained using a single technique involving high-pressure freezing and embedding in Lowicryl resins at low temperature. As well as being an important new tool for zebrafish research, the maintenance of GFP fluorescence after fast freezing, freeze substitution and resin embedding will be of general use for correlative light and EM of biological s les.
βIII-Tubulin Structural Domains Regulate Mitochondrial Network Architecture in an Isotype-Specific Manner.
Publisher: MDPI AG
Date: 23-02-2022
Abstract: βIII-tubulin is a neuronal microtubule protein that is aberrantly expressed in epithelial cancers. The microtubule network is implicated in regulating the architecture and dynamics of the mitochondrial network, although the isotype-specific role for β-tubulin proteins that constitute this microtubule network remains unclear. High-resolution electron microscopy revealed that manipulation of βIII-tubulin expression levels impacts the volume and shape of mitochondria. Analysis of the structural domains of the protein identifies that the C-terminal tail of βIII-tubulin, which distinguishes this protein from other β-tubulin isotypes, significantly contributes to the isotype-specific effects of βIII-tubulin on mitochondrial architecture. Mass spectrometry analysis of protein–protein interactions with β-tubulin isotypes identifies that βIII-tubulin specifically interacts with regulators of mitochondrial dynamics that may mediate these functional effects. Advanced quantitative dynamic lattice light sheet imaging of the mitochondrial network reveals that βIII-tubulin promotes a more dynamic and extended reticular mitochondrial network, and regulates mitochondrial volume. A regulatory role for the βIII-tubulin C-terminal tail in mitochondrial network dynamics and architecture has widespread implications for the maintenance of mitochondrial homeostasis in health and disease.
Characterization of a distinct plasma membrane macrodomain in differentiated adipocytes
Publisher: Elsevier BV
Date: 11-2002
Different Characteristics and Nucleotide Binding Properties of Inosine Monophosphate Dehydrogenase (IMPDH) Isoforms
Publisher: Public Library of Science (PLoS)
Date: 07-12-2012
Hybrid organic-inorganic nanoparticles: controlled incorporation of gold nanoparticles into virus-like particles and application in surface-enhanced Raman spectroscopy
Publisher: SPIE
Date: 27-12-2006
DOI: 10.1117/12.695578
Patched1 is required in neural crest cells for the prevention of orofacial clefts
Publisher: Oxford University Press (OUP)
Date: 29-07-2013
DOI: 10.1093/HMG/DDT353
Abstract: Defects such as cleft lip with or without cleft palate (CL/P) are among the most common craniofacial birth defects in humans. In many cases, the underlying molecular and cellular mechanisms that result in these debilitating anomalies remain largely unknown. Perturbed hedgehog (HH) signalling plays a major role in craniofacial development, and mutations in a number of pathway constituents underlie craniofacial disease. In particular, mutations in the gene encoding the major HH receptor and negative regulator, patched1 (PTCH1), are associated with both sporadic and familial forms of clefting, yet relatively little is known about how PTCH1 functions during craniofacial morphogenesis. To address this, we analysed the consequences of conditional loss of Ptch1 in mouse neural crest cell-derived facial mesenchyme. Using scanning electron microscopy (SEM) and live imaging of explanted facial primordia, we captured defective nasal pit invagination and CL in mouse embryos conditionally lacking Ptch1. Our analysis demonstrates interactions between HH and FGF signalling in the development of the upper lip, and reveals cell-autonomous and non-autonomous roles mediated by Ptch1. In particular, we show that deletion of Ptch1 in the facial mesenchyme alters cell morphology, specifically in the invaginating nasal pit epithelium. These findings highlight a critical link between the neural crest cells and olfactory epithelium in directing the morphogenesis of the mammalian lip and nose primordia. Importantly, these interactions are critically dependent on Ptch1 function for the prevention of orofacial clefts.
Characterization of E-cadherin Endocytosis in Isolated MCF-7 and Chinese Hamster Ovary Cells
Publisher: Elsevier BV
Date: 06-2003
A variable undecad repeat domain in cavin1 regulates caveola formation and stability
Publisher: EMBO
Date: 18-07-2018
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.
A Role for Phosphatidic Acid in the Formation of “Supersized” Lipid Droplets
Publisher: Public Library of Science (PLoS)
Date: 28-07-2011
PTRF-Cavin, a Conserved Cytoplasmic Protein Required for Caveola Formation and Function
Publisher: Elsevier BV
Date: 2008
Mutations in mouse Ift144 model the craniofacial, limb and rib defects in skeletal ciliopathies
Publisher: Oxford University Press (OUP)
Date: 06-01-2012
DOI: 10.1093/HMG/DDR613
Abnormal Nuclear Pore Formation Triggers Apoptosis in the Intestinal Epithelium of elys-Deficient Zebrafish
Publisher: Elsevier BV
Date: 03-2009
Ryanodine receptor leak triggers fiber Ca2+ redistribution to preserve force and elevate basal metabolism in skeletal muscle
Publisher: American Association for the Advancement of Science (AAAS)
Date: 29-10-2021
Abstract: RyR1 Ca 2+ leak causes a cascade of events that shifts Ca 2+ to the cytoplasm and mitochondria, supporting force generation.
Caveola-forming proteins caveolin-1 and PTRF in prostate cancer
Publisher: Springer Science and Business Media LLC
Date: 13-08-2013
Abstract: The expression of caveola-forming proteins is dysregulated in prostate cancer. Caveolae are flask-shaped invaginations of the plasma membrane that have roles in membrane trafficking and cell signalling. Members of two families of proteins--caveolins and cavins--are known to be required for the formation and functions of caveolae. Caveolin-1, the major structural protein of caveolae, is overexpresssed in prostate cancer and has been demonstrated to be involved in prostate cancer angiogenesis, growth and metastasis. Polymerase I and transcript release factor (PTRF) is the only cavin family member necessary for caveola formation. When exogenously expressed in prostate cancer cells, PTRF reduces aggressive potential, probably via both caveola-mediated and caveola-independent mechanisms. In addition, stromal PTRF expression decreases with progression of the disease. Evaluation of caveolin-1 antibodies in the clinical setting is underway and it is hoped that future studies will reveal the mechanisms of PTRF action, allowing its targeting for therapeutic purposes.
Coronin 1B Reorganizes the Architecture of F-Actin Networks for Contractility at Steady-State and Apoptotic Adherens Junctions
Publisher: Elsevier BV
Date: 04-2016
DOI: 10.1016/J.DEVCEL.2016.03.008
Abstract: In this study we sought to identify how contractility at adherens junctions influences apoptotic cell extrusion. We first found that the generation of effective contractility at steady-state junctions entails a process of architectural reorganization whereby filaments that are initially generated as poorly organized networks of short bundles are then converted into co-aligned perijunctional bundles. Reorganization requires coronin 1B, which is recruited to junctions by E-cadherin adhesion and is necessary to establish contractile tension at the zonula adherens. When cells undergo apoptosis within an epithelial monolayer, coronin 1B is also recruited to the junctional cortex at the apoptotic/neighbor cell interface in an E-cadherin-dependent fashion to support actin architectural reorganization, contractility, and extrusion. We propose that contractile stress transmitted from the apoptotic cell through E-cadherin adhesions elicits a mechanosensitive response in neighbor cells that is necessary for the morphogenetic event of apoptotic extrusion to occur.
Uptake and intracellular fate of disulfide-bonded polymer hydrogel capsules for doxorubicin delivery to colorectal cancer cells
Publisher: American Chemical Society (ACS)
Date: 26-04-2010
DOI: 10.1021/NN100173H
Abstract: Understanding the interactions between drug carriers and cells is of importance to enhance the delivery of therapeutics. The release of therapeutics into different intracellular environments, such as the lysosomes or the cell cytoplasm, will impact their pharmacological activity. Herein, we investigate the intracellular fate of layer-by-layer (LbL)-assembled, submicrometer-sized polymer hydrogel capsules in a human colon cancer derived cell line, LIM1899. The cellular uptake of the disulfide-stabilized poly(methacrylic acid) (PMA(SH)) capsules by colon cancer cells is a time-dependent process. Confocal laser scanning microscopy and transmission electron microscopy reveal that the internalized capsules are deformed in membrane-enclosed compartments, which further mature to late endosomes or lysosomes. We further demonstrate the utility of these redox-responsive PMA(SH) capsules for the delivery of doxorubicin (DOX) to colon cancer cells. The DOX-loaded PMA(SH) capsules demonstrate a 5000-fold enhanced cytotoxicity in cell viability studies compared to free DOX.
Direct visualization of Ras proteins in spatially distinct cell surface microdomains
Publisher: Rockefeller University Press
Date: 13-01-2003
Abstract: Localization of signaling complexes to specific microdomains coordinates signal transduction at the plasma membrane. Using immunogold electron microscopy of plasma membrane sheets coupled with spatial point pattern analysis, we have visualized morphologically featureless microdomains, including lipid rafts, in situ and at high resolution. We find that an inner-plasma membrane lipid raft marker displays cholesterol-dependent clustering in microdomains with a mean diameter of 44 nm that occupy 35% of the cell surface. Cross-linking an outer-leaflet raft protein results in the redistribution of inner leaflet rafts, but they retain their modular structure. Analysis of Ras microlocalization shows that inactive H-ras is distributed between lipid rafts and a cholesterol-independent microdomain. Conversely, activated H-ras and K-ras reside predominantly in nonoverlapping, cholesterol-independent microdomains. Galectin-1 stabilizes the association of activated H-ras with these nonraft microdomains, whereas K-ras clustering is supported by farnesylation, but not geranylgeranylation. These results illustrate that the inner plasma membrane comprises a complex mosaic of discrete microdomains. Differential spatial localization within this framework can likely account for the distinct signal outputs from the highly homologous Ras proteins.
Caveolae control the anti-inflammatory phenotype of senescent endothelial cells
Publisher: Wiley
Date: 19-11-2015
DOI: 10.1111/ACEL.12270
Quantitative proteomic analysis of the adipocyte plasma membrane
Publisher: American Chemical Society (ACS)
Date: 18-10-2011
DOI: 10.1021/PR200446R
Abstract: The adipocyte is a key regulator of mammalian metabolism. To advance our understanding of this important cell, we have used quantitative proteomics to define the protein composition of the adipocyte plasma membrane (PM) in the presence and absence of insulin. Using this approach, we have identified a high confidence list of 486 PM proteins, 52 of which potentially represent novel cell surface proteins, including a member of the adiponectin receptor family and an unusually high number of hydrolases with no known function. Several novel insulin-responsive proteins including the sodium/hydrogen exchanger, NHE6 and the collagens III and VI were also identified, and we provide evidence of PM-ER association suggestive of a unique functional association between these two organelles in the adipocyte. Together these studies provide a wealth of potential therapeutic targets for the manipulation of adipocyte function and a valuable resource for metabolic research and PM biology.
Caveolin-1 Plays a Critical Role in the Differentiation of Monocytes into Macrophages
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 09-2012
DOI: 10.1161/ATVBAHA.112.254151
Abstract: Monocyte to macrophage differentiation is an essential step in atherogenesis. The structure protein of caveolae, caveolin-1, is increased in primary monocytes after its adhesion to endothelium. We explore the hypothesis that caveolin-1 plays a role in monocyte differentiation to macrophages. Both phorbol myristate acetate–induced THP-1 and colony-stimulating factor–induced primary monocyte differentiation was associated with an increase in cellular caveolin-1 expression. Overexpression of caveolin-1 by transfection increased macrophage surface markers and inflammatory genes, whereas caveolin-1 knockdown by small interfering RNA or knockout reduced these. Also, caveolin-1 knockdown inhibited the differentiation–induced nuclear translocation of early growth response 1 (EGR-1) through extracellular signal-regulated kinase phosphorylation, further decreased the binding of EGR-1 to CD115 promoter, thus decreasing EGR-1 transcriptional activity. In functional assays, caveolin-1 inhibited transmigration but promoted phagocytosis in the monocyte–macrophage lineage. Decreasing caveolin-1 inhibited the uptake of modified low-density lipoprotein and reduced cellular lipid content. Finally, we showed that caveolin-1 knockout mice displayed less monocyte differentiation than wild-type mice and that EGR-1 transcription activity was also decreased in these mice because of the inhibition of extracellular signal-regulated kinase phosphorylation. Caveolin-1 promotes monocyte to macrophage differentiation through the regulation of EGR-1 transcriptional activity, suggesting that phagocytic caveolin-1 may be critical for atherogenesis.
Laser-mediated rupture of chlamydial inclusions triggers pathogen egress and host cell necrosis
Publisher: Springer Science and Business Media LLC
Date: 10-03-2017
DOI: 10.1038/NCOMMS14729
Abstract: Remarkably little is known about how intracellular pathogens exit the host cell in order to infect new hosts. Pathogenic chlamydiae egress by first rupturing their replicative niche (the inclusion) before rapidly lysing the host cell. Here we apply a laser ablation strategy to specifically disrupt the chlamydial inclusion, thereby uncoupling inclusion rupture from the subsequent cell lysis and allowing us to dissect the molecular events involved in each step. Pharmacological inhibition of host cell calpains inhibits inclusion rupture, but not subsequent cell lysis. Further, we demonstrate that inclusion rupture triggers a rapid necrotic cell death pathway independent of BAK, BAX, RIP1 and caspases. Both processes work sequentially to efficiently liberate the pathogen from the host cytoplasm, promoting secondary infection. These results reconcile the pathogen's known capacity to promote host cell survival and induce cell death.
Integrative omics identifies conserved and pathogen-specific responses of sepsis-causing bacteria
Publisher: Springer Science and Business Media LLC
Date: 18-03-2023
DOI: 10.1038/S41467-023-37200-W
Abstract: Even in the setting of optimal resuscitation in high-income countries severe sepsis and septic shock have a mortality of 20–40%, with antibiotic resistance dramatically increasing this mortality risk. To develop a reference dataset enabling the identification of common bacterial targets for therapeutic intervention, we applied a standardized genomic, transcriptomic, proteomic and metabolomic technological framework to multiple clinical isolates of four sepsis-causing pathogens: Escherichia coli , Klebsiella pneumoniae species complex, Staphylococcus aureus and Streptococcus pyogenes . Exposure to human serum generated a sepsis molecular signature containing global increases in fatty acid and lipid biosynthesis and metabolism, consistent with cell envelope remodelling and nutrient adaptation for osmoprotection. In addition, acquisition of cholesterol was identified across the bacterial species. This detailed reference dataset has been established as an open resource to support discovery and translational research.
Heterofibrins: inhibitors of lipid droplet formation from a deep-water southern Australian marine sponge, Spongia (Heterofibria) sp.
Publisher: Royal Society of Chemistry (RSC)
Date: 2010
DOI: 10.1039/C003840G
Abstract: A bioassay-guided search for inhibitors of lipid droplet formation in a deep-water southern Australian marine sponge, Spongia (Heterofibria) sp., yielded six new compounds, fatty acids heterofibrins A1 (1) and B1 (4), along with related monolactyl and dilactyl esters, heterofibrins A2 (2), B2 (5), A3 (3) and B3 (6). Heterofibrin structures were assigned on the basis of detailed spectroscopic analysis, with comparison to chiral synthetic model compounds. All heterofibrins possess a diyne-ene moiety, while the monolactyl and dilactyl moiety featured in selected heterofibrins is unprecedented in the natural products literature. SAR by co-metabolite studies on the heterofibrins confirmed them to be non-cytotoxic, with the carboxylic acids 1 and 4 inhibiting lipid droplet formation in A431 fibroblast cell lines. Such inhibitors have potential application in the management of obesity, diabetes and atherosclerosis
Dynamic microtubules regulate the local concentration of E-cadherin at cell-cell contacts
Publisher: The Company of Biologists
Date: 13-11-2015
DOI: 10.1242/JCS.02903
Abstract: In contrast to the well-established relationship between cadherins and the actin cytoskeleton, the potential link between cadherins and microtubules (MTs) has been less extensively investigated. We now identify a pool of MTs that extend radially into cell-cell contacts and are inhibited by manoeuvres that block the dynamic activity of MT plus-ends (e.g. in the presence of low concentrations of nocodazole and following expression of a CLIP-170 mutant). Blocking dynamic MTs perturbed the ability of cells to concentrate and accumulate E-cadherin at cell-cell contacts, as assessed both by quantitative immunofluorescence microscopy and fluorescence recovery after photobleaching (FRAP) analysis, but did not affect either transport of E-cadherin to the plasma membrane or the amount of E-cadherin expressed at the cell surface. This indicated that dynamic MTs allow cells to concentrate E-cadherin at cell-cell contacts by regulating the regional distribution of E-cadherin once it reaches the cell surface. Importantly, dynamic MTs were necessary for myosin II to accumulate and be activated at cadherin adhesive contacts, a mechanism that supports the focal accumulation of E-cadherin. We propose that this population of MTs represents a novel form of cadherin-MT cooperation, where cadherin adhesions recruit dynamic MTs that, in turn, support the local concentration of cadherin molecules by regulating myosin II activity at cell-cell contacts.
Nanoscape, a data-driven 3D real-time interactive virtual cell environment
Publisher: eLife Sciences Publications, Ltd
Date: 30-06-2021
DOI: 10.7554/ELIFE.64047
Abstract: Our understanding of cellular and structural biology has reached unprecedented levels of detail, and computer visualisation techniques can be used to create three-dimensional (3D) representations of cells and their environment that are useful in both teaching and research. However, extracting and integrating the relevant scientific data, and then presenting them in an effective way, can pose substantial computational and aesthetic challenges. Here we report how computer artists, experts in computer graphics and cell biologists have collaborated to produce a tool called Nanoscape that allows users to explore and interact with 3D representations of cells and their environment that are both scientifically accurate and visually appealing. We believe that using Nanoscape as an immersive learning application will lead to an improved understanding of the complexities of cellular scales, densities and interactions compared with traditional learning modalities.
Caveolae sense oxidative stress through membrane lipid peroxidation and cytosolic release of CAVIN1 to regulate NRF2
Publisher: Elsevier BV
Date: 03-2023
Presynaptic targeting of botulinum neurotoxin type A requires a tripartite PSG‐Syt1 ‐SV2 plasma membrane nanocluster for synaptic vesicle entry
Publisher: EMBO
Date: 25-05-2023
Abstract: The unique nerve terminal targeting of botulinum neurotoxin type A (BoNT/A) is due to its capacity to bind two receptors on the neuronal plasma membrane: polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Whether and how PSGs and SV2 may coordinate other proteins for BoNT/A recruitment and internalization remains unknown. Here, we demonstrate that the targeted endocytosis of BoNT/A into synaptic vesicles (SVs) requires a tripartite surface nanocluster. Live‐cell super‐resolution imaging and electron microscopy of catalytically inactivated BoNT/A wildtype and receptor‐binding‐deficient mutants in cultured hippoc al neurons demonstrated that BoNT/A must bind coincidentally to a PSG and SV2 to target synaptic vesicles. We reveal that BoNT/A simultaneously interacts with a preassembled PSG‐synaptotagmin‐1 (Syt1) complex and SV2 on the neuronal plasma membrane, facilitating Syt1‐SV2 nanoclustering that controls endocytic sorting of the toxin into synaptic vesicles. Syt1 CRISPRi knockdown suppressed BoNT/A‐ and BoNT/E‐induced neurointoxication as quantified by SNAP‐25 cleavage, suggesting that this tripartite nanocluster may be a unifying entry point for selected botulinum neurotoxins that hijack this for synaptic vesicle targeting.
Visualisation of macropinosome maturation by the recruitment of sorting nexins
Publisher: The Company of Biologists
Date: 10-2006
DOI: 10.1242/JCS.03167
Abstract: We report that phosphoinositol-binding sorting nexin 5 (SNX5) associates with newly formed macropinosomes induced by EGF stimulation. We used the recruitment of GFP-SNX5 to macropinosomes to track their maturation. Initially, GFP-SNX5 is sequestered to discrete subdomains of the macropinosome these subdomains are subsequently incorporated into highly dynamic, often branched, tubular structures. Time-lapse videomicroscopy revealed the highly dynamic extension of SNX5-labelled tubules and their departure from the macropinosome body to follow predefined paths towards the perinuclear region of the cell, before fusing with early endosomal acceptor membranes. The extension and departure of these tubular structures occurs rapidly over 5-10 minutes and is dependent upon intact microtubules. As the tubular structures depart from the macropinosome there is a reduction in the surface area and an increase in tension of the limiting membrane of the macropinosome. In addition to the recruitment of SNX5 to the macropinosome, Rab5, SNX1 and EEA1 are also recruited by newly formed macropinosomes, followed by the accumulation of Rab7. SNX5 forms heterodimers with SNX1 and this interaction is required for endosome association of SNX5. We propose that the departure of SNX5-positive tubules represents a rapid mechanism of recycling components from macropinosomes thereby promoting their maturation into Rab7-positive structures. Collectively these findings provide a detailed real-time characterisation of the maturation process of the macropinocytic endosome.
α-Actinin-3 deficiency results in reduced glycogen phosphorylase activity and altered calcium handling in skeletal muscle
Publisher: Oxford University Press (OUP)
Date: 20-01-2010
DOI: 10.1093/HMG/DDQ010
Abstract: Approximately one billion people worldwide are homozygous for a stop codon polymorphism in the ACTN3 gene (R577X) which results in complete deficiency of the fast fibre muscle protein alpha-actinin-3. ACTN3 genotype is associated with human athletic performance and alpha-actinin-3 deficient mice [Actn3 knockout (KO) mice] have a shift in the properties of fast muscle fibres towards slower fibre properties, with increased activity of multiple enzymes in the aerobic metabolic pathway and slower contractile properties. alpha-Actinins have been shown to interact with a number of muscle proteins including the key metabolic regulator glycogen phosphorylase (GPh). In this study, we demonstrated a link between alpha-actinin-3 and glycogen metabolism which may underlie the metabolic changes seen in the KO mouse. Actn3 KO mice have higher muscle glycogen content and a 50% reduction in the activity of GPh. The reduction in enzyme activity is accompanied by altered post-translational modification of GPh, suggesting that alpha-actinin-3 regulates GPh activity by altering its level of phosphorylation. We propose that the changes in glycogen metabolism underlie the downstream metabolic consequences of alpha-actinin-3 deficiency. Finally, as GPh has been shown to regulate calcium handling, we examined calcium handling in KO mouse primary mouse myoblasts and find changes that may explain the slower contractile properties previously observed in these mice. We propose that the alteration in GPh activity in the absence of alpha-actinin-3 is a fundamental mechanistic link in the association between ACTN3 genotype and human performance.
Loss of YhcB results in dysregulation of coordinated peptidoglycan, LPS and phospholipid synthesis during Escherichia coli cell growth
Publisher: Public Library of Science (PLoS)
Date: 23-12-2021
DOI: 10.1371/JOURNAL.PGEN.1009586
Abstract: The cell envelope is essential for viability in all domains of life. It retains enzymes and substrates within a confined space while providing a protective barrier to the external environment. Destabilising the envelope of bacterial pathogens is a common strategy employed by antimicrobial treatment. However, even in one of the best studied organisms, Escherichia coli , there remain gaps in our understanding of how the synthesis of the successive layers of the cell envelope are coordinated during growth and cell ision. Here, we used a whole-genome phenotypic screen to identify mutants with a defective cell envelope. We report that loss of yhcB , a conserved gene of unknown function, results in loss of envelope stability, increased cell permeability and dysregulated control of cell size. Using whole genome transposon mutagenesis strategies, we report the comprehensive genetic interaction network of yhcB , revealing all genes with a synthetic negative and a synthetic positive relationship. These genes include those previously reported to have a role in cell envelope biogenesis. Surprisingly, we identified genes previously annotated as essential that became non-essential in a Δ yhcB background. Subsequent analyses suggest that YhcB functions at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.
Subdiffractional tracking of internalized molecules reveals heterogeneous motion states of synaptic vesicles
Publisher: Rockefeller University Press
Date: 24-10-2016
Abstract: Our understanding of endocytic pathway dynamics is severely restricted by the diffraction limit of light microscopy. To address this, we implemented a novel technique based on the subdiffractional tracking of internalized molecules (sdTIM). This allowed us to image anti–green fluorescent protein Atto647N-tagged nanobodies trapped in synaptic vesicles (SVs) from live hippoc al nerve terminals expressing vesicle-associated membrane protein 2 (VAMP2)–pHluorin with 36-nm localization precision. Our results showed that, once internalized, VAMP2–pHluorin/Atto647N–tagged nanobodies exhibited a markedly lower mobility than on the plasma membrane, an effect that was reversed upon restimulation in presynapses but not in neighboring axons. Using Bayesian model selection applied to hidden Markov modeling, we found that SVs oscillated between diffusive states or a combination of diffusive and transport states with opposite directionality. Importantly, SVs exhibiting diffusive motion were relatively less likely to switch to the transport motion. These results highlight the potential of the sdTIM technique to provide new insights into the dynamics of endocytic pathways in a wide variety of cellular settings.
Reduced Plasma Membrane Expression of Dysferlin Mutants Is Attributed to Accelerated Endocytosis via a Syntaxin-4-associated Pathway
Publisher: Elsevier BV
Date: 09-2010
Cell-free formation and interactome analysis of caveolae
Publisher: Rockefeller University Press
Date: 05-2018
Abstract: Caveolae have been linked to the regulation of signaling pathways in eukaryotic cells through direct interactions with caveolins. Here, we describe a cell-free system based on Leishmania tarentolae (Lt) extracts for the biogenesis of caveolae and show its use for single-molecule interaction studies. Insertion of expressed caveolin-1 (CAV1) into Lt membranes was analogous to that of caveolin in native membranes. Electron tomography showed that caveolins generate domains of precise size and curvature. Cell-free caveolae were used in quantitative assays to test the interaction of membrane-inserted caveolin with signaling proteins and to determine the stoichiometry of interactions. Binding of membrane-inserted CAV1 to several proposed binding partners, including endothelial nitric-oxide synthase, was negligible, but a small number of proteins, including TRAF2, interacted with CAV1 in a phosphorylation-(CAV1Y14)–stimulated manner. In cells subjected to oxidative stress, phosphorylated CAV1 recruited TRAF2 to the early endosome forming a novel signaling platform. These findings lead to a novel model for cellular stress signaling by CAV1.
Mathematical modeling of K-Ras nanocluster formation on the plasma membrane
Publisher: Elsevier BV
Date: 07-2010
Cholesterol‐Sensitive Cdc42 Activation Regulates Actin Polymerization for Endocytosis via the GEEC Pathway
Publisher: Wiley
Date: 25-04-2007
DOI: 10.1111/J.1600-0854.2007.00565.X
Abstract: Glycosyl-phosphatidylinositol (GPI)-anchored proteins (GPI-APs) are present at the surface of living cells in cholesterol dependent nanoscale clusters. These clusters appear to act as sorting signals for the selective endocytosis of GPI-APs via a Cdc42-regulated, dynamin and clathrin-independent pinocytic pathway called the GPI-AP-enriched early endosomal compartments (GEECs) pathway. Here we show that endocytosis via the GEECs pathway is inhibited by mild depletion of cholesterol, perturbation of actin polymerization or overexpression of the Cdc42/Rac-interactive-binding (CRIB) motif of neural Wiskott-Aldrich syndrome protein (N-WASP). Consistent with the involvement of Cdc42-based actin nanomachinery, nascent endocytic vesicles containing cargo for the GEEC pathway co-localize with fluorescent protein-tagged isoforms of Cdc42, CRIB domain, N-WASP and actin high-resolution electron microscopy on plasma membrane sheets reveals Cdc42-labelled regions rich in green fluorescent protein-GPI. Using total internal reflection fluorescence microscopy at the single-molecule scale, we find that mild cholesterol depletion alters the dynamics of actin polymerization at the cell surface by inhibiting Cdc42 activation and consequently its stabilization at the cell surface. These results suggest that endocytosis into GEECs occurs through a cholesterol-sensitive, Cdc42-based recruitment of the actin polymerization machinery.
Zebrafish as a model for caveolin-associated muscle disease; caveolin-3 is required for myofibril organization and muscle cell patterning
Publisher: Oxford University Press (OUP)
Date: 11-05-2005
DOI: 10.1093/HMG/DDI179
Abstract: Caveolae are an abundant feature of many animal cells. However, the exact function of caveolae remains unclear. We have used the zebrafish, Danio rerio, as a system to understand caveolae function focusing on the muscle-specific caveolar protein, caveolin-3 (Cav3). We have identified caveolin-1 (alpha and beta), caveolin-2 and Cav3 in the zebrafish. Zebrafish Cav3 has 72% identity to human CAV3, and the amino acids altered in human muscle diseases are conserved in the zebrafish protein. During embryonic development, cav3 expression is apparent by early segmentation stages in the first differentiating muscle precursors, the adaxial cells and slightly later in the notochord. cav3 expression appears in the somites during mid-segmentation stages and then later in the pectoral fins and facial muscles. Cav3 and caveolae are located along the entire sarcolemma of late stage embryonic muscle fibers, whereas beta-dystroglycan is restricted to the muscle fiber ends. Down-regulation of Cav3 expression causes gross muscle abnormalities and uncoordinated movement. Ultrastructural analysis of isolated muscle fibers reveals defects in myoblast fusion and disorganized myofibril and membrane systems. Expression of the zebrafish equivalent to a human muscular dystrophy mutant, CAV3P104L, causes severe disruption of muscle differentiation. In addition, knockdown of Cav3 resulted in a dramatic up-regulation of eng1a expression resulting in an increase in the number of muscle pioneer-like cells adjacent to the notochord. These studies provide new insights into the role of Cav3 in muscle development and demonstrate its requirement for correct intracellular organization and myoblast fusion.
FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors
Publisher: Cold Spring Harbor Laboratory
Date: 12-10-2022
DOI: 10.1101/2022.10.12.511867
Abstract: Cells selectively remove damaged or excessive mitochondria through mitophagy, a specialized form of autophagy, to maintain mitochondrial quality and quantity. Mitophagy is induced in response to erse conditions, including hypoxia, cellular differentiation, and mitochondrial damage. However, the mechanisms by which cells remove specific dysfunctional mitochondria under steady-state conditions to fine-tune mitochondrial content are not well understood. Here, we report that SCF FBXL4 , an SKP1/CUL1/F-box protein ubiquitin ligase complex, localizes to the mitochondrial outer membrane in unstressed cells and mediates the constitutive ubiquitylation and degradation of the mitophagy receptors NIX and BNIP3 to suppress basal levels of mitophagy. We demonstrate that, unlike wild-type FBXL4, pathogenic variants of FBXL4 that cause encephalopathic mtDNA depletion syndrome (MTDPS13), do not efficiently interact with the core SCF ubiquitin ligase machinery or mediate the degradation of NIX and BNIP3. Thus, we reveal a molecular mechanism that actively suppresses mitophagy via preventing NIX and BNIP3 accumulation and propose that excessive basal mitophagy in the FBXL4-associated mtDNA depletion syndrome is caused by dysregulation of NIX and BNIP3 turnover.
Caveolin Regulates Endocytosis of the Muscle Repair Protein, Dysferlin
Publisher: Elsevier BV
Date: 03-2008
Proinflammatory impact of putative viral-specific CD8+ T cells infiltrating new-onset seropositive rheumatoid arthritis synovium
Publisher: Research Square Platform LLC
Date: 07-06-2023
DOI: 10.21203/RS.3.RS-3001427/V1
Abstract: New-onset HLA-DR-associated anti-citrullinated protein autoantibody (ACPA) + rheumatoid arthritis (RA) synovial tissue (ST) contains highly-expanded TCR-αβ clonotypes, some viral-antigen-reactive. However, it is unknown how viral-specific T-cells sustain ACPA + RA. We studied paired peripheral blood (PB) and ST TCR repertoires in drug-naïve ACPA + HLA-DRB1*04:01 + diffuse-myeloid RA ST pathology. To model effects of viral infection, we induced ovalbumin antigen-induced arthritis (AIA) in mice with latent murine-cytomegalovirus or recovered from acute lymphocytic-choriomeningitis virus. We show that most clonally-expanded CD8 + T cells had polyfunctional TNF + IFNγ + cytotoxic T-lymphocyte (CTL) signatures. Transcriptomic profiles of ST CD4 + T-cell clonotypes were Th2-like and IL-6-signaled central memory-like. CMV-specific tetramers confirmed in-silico prediction of viral-epitope recognition by CTL. Perivascular GZMB + CD8 + T cells co-localized with CD4 + T-cells, dendritic cells, fibroblasts and IL-6. After viral infection, AIA severity increased with enhanced viral and ovalbumin-specific TNF + IFN-γ + T-cell cytokines, suggesting that in the diffuse-myeloid rheumatoid-synovial perivascular niche, polyfunctional bystander-CTL reinforce myeloid- and CD4 + T-cell activation.
Caveolin-1 is necessary for hepatic oxidative lipid metabolism: Evidence for crosstalk between caveolin-1 and bile acid signaling
Publisher: Elsevier BV
Date: 07-2013
DOI: 10.1016/J.CELREP.2013.06.017
Abstract: Caveolae and caveolin-1 (CAV1) have been linked to several cellular functions. However, a model explaining their roles in mammalian tissues in vivo is lacking. Unbiased expression profiling in several tissues and cell types identified lipid metabolism as the main target affected by CAV1 deficiency. CAV1-/- mice exhibited impaired hepatic peroxisome proliferator-activated receptor α (PPARα)-dependent oxidative fatty acid metabolism and ketogenesis. Similar results were recapitulated in CAV1-deficient AML12 hepatocytes, suggesting at least a partial cell-autonomous role of hepatocyte CAV1 in metabolic adaptation to fasting. Finally, our experiments suggest that the hepatic phenotypes observed in CAV1-/- mice involve impaired PPARα ligand signaling and attenuated bile acid and FXRα signaling. These results demonstrate the significance of CAV1 in (1) hepatic lipid homeostasis and (2) nuclear hormone receptor (PPARα, FXRα, and SHP) and bile acid signaling.
High‐Resolution 3D Quantitative Analysis of Caveolar Ultrastructure and Caveola–Cytoskeleton Interactions
Publisher: Wiley
Date: 07-03-2008
DOI: 10.1111/J.1600-0854.2008.00733.X
Abstract: Caveolae are characteristic invaginations of the mammalian plasma membrane (PM) implicated in lipid regulation, signal transduction and endocytosis. We have employed electron microscope tomography (ET) to quantify caveolae structure-function relationships in three-dimension (3D) at high resolution both in conventionally fixed and in fast-frozen/freeze-substituted (intact) cells as well as immunolabelled PM lawns. Our findings provide a detailed quantitative comparison of the average caveola dimensions for different cell types including tissue endothelial cells and cultured 3T3-L1 adipocytes. These studies revealed the presence of a spiked caveolar coat and a wide caveolar neck open to the extracellular milieu that is sensitive to conventional fixation the neck region appeared to form a specialized microdomain with associated cytoplasmic material. In endothelial cells in situ in pancreatic islets of Langerhans, the diaphragm spanning the caveolar opening was clearly resolved by ET, and the involuted 3D topology of the cell surface mapped to measure the contribution of caveolar membranes to local increases in the surface area of the PM. The complexity of connections among caveolae and to the actin cytoskeleton and microtubules suggests that in idual caveolae may be interconnected through a complex filamentous network to form a single functional unit.
Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence
Publisher: Springer Science and Business Media LLC
Date: 28-11-2016
DOI: 10.1038/SREP37630
Abstract: Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer’s and Parkinson’s disease. Parkinson’s disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson’s disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps. In these conditions, we could detect the formation of oligomeric and pre-fibrillar species at very short time scale and low micromolar concentrations. The pathogenic mutants surprisingly segregated into two classes: one group forming large aggregates and fibrils while the other tending to form mostly oligomers. Strikingly, co-expression experiments reveal that members from the different groups do not generally interact with each other, both at the fibril and monomer levels. Together, this data paints a completely different picture of α-synuclein aggregation, with two possible pathways leading to the development of fibrils.
Molecules, mechanisms, and cellular roles of clathrin-independent endocytosis
Publisher: Elsevier BV
Date: 08-2010
DOI: 10.1016/J.CEB.2010.04.001
Abstract: Eukaryotic cells deftly coordinate an array of endocytic pathways beyond the classical clathrin-mediated endocytic route. Although the existence of clathrin-independent endocytic pathways has been accepted for some time, only recently have tools been developed that specifically delineate their fine details, including molecular composition and ultrastructural morphology. Identification of the salient features of distinct pathways has concomitantly attributed them with specific roles during important cellular processes. Insight from model organisms confirms these roles and suggests maintenance of crucially adapted functions across species. Among other roles, clathrin-independent endocytosis has now been linked to plasma membrane repair, cellular spreading, cellular polarization, and modulation of intercellular signaling. The field is now primed to identify how these pathways function within physiologically relevant environments.
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
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.
Frontline Science: LPS-inducible SLC30A1 drives human macrophage-mediated zinc toxicity against intracellular Escherichia coli
Publisher: Oxford University Press (OUP)
Date: 22-05-2021
Abstract: TLR-inducible zinc toxicity is an antimicrobial mechanism utilized by macrophages, however knowledge of molecular mechanisms mediating this response is limited. Here, we show that E. coli exposed to zinc stress within primary human macrophages reside in membrane-bound vesicular compartments. Since SLC30A zinc exporters can deliver zinc into the lumen of vesicles, we examined LPS-regulated mRNA expression of Slc30a/SLC30A family members in primary mouse and human macrophages. A number of these transporters were dynamically regulated in both cell populations. In human monocyte-derived macrophages, LPS strongly up-regulated SLC30A1 mRNA and protein expression. In contrast, SLC30A1 was not LPS-inducible in macrophage-like PMA-differentiated THP-1 cells. We therefore ectopically expressed SLC30A1 in these cells, finding that this was sufficient to promote zinc-containing vesicle formation. The response was similar to that observed following LPS stimulation. Ectopically expressed SLC30A1 localized to both the plasma membrane and intracellular zinc-containing vesicles within LPS-stimulated THP-1 cells. Inducible overexpression of SLC30A1 in THP-1 cells infected with the Escherichia coli K-12 strain MG1655 augmented the zinc stress response of intracellular bacteria and promoted clearance. Furthermore, in THP-1 cells infected with an MG1655 zinc stress reporter strain, all bacteria contained within SLC30A1-positive compartments were subjected to zinc stress. Thus, SLC30A1 marks zinc-containing compartments associated with TLR-inducible zinc toxicity in human macrophages, and its ectopic over-expression is sufficient to initiate this antimicrobial pathway in these cells. Finally, SLC30A1 silencing did not compromise E. coli clearance by primary human macrophages, suggesting that other zinc exporters may also contribute to the zinc toxicity response.
Myosin II isoforms identify distinct functional modules that support integrity of the epithelial zonula adherens
Publisher: Springer Science and Business Media LLC
Date: 13-06-2010
DOI: 10.1038/NCB2072
ORP5 and ORP8 bind phosphatidylinositol-4, 5-biphosphate (PtdIns(4,5)P 2) and regulate its level at the plasma membrane
Publisher: Springer Science and Business Media LLC
Date: 02-10-2017
DOI: 10.1038/S41467-017-00861-5
Abstract: ORP5 and ORP8, members of the oxysterol-binding protein (OSBP)-related proteins (ORP) family, are endoplasmic reticulum membrane proteins implicated in lipid trafficking. ORP5 and ORP8 are reported to localize to endoplasmic reticulum–plasma membrane junctions via binding to phosphatidylinositol-4-phosphate (PtdIns(4) P ), and act as a PtdIns(4) P hosphatidylserine counter exchanger between the endoplasmic reticulum and plasma membrane. Here we provide evidence that the pleckstrin homology domain of ORP5/8 via PtdIns(4,5) P 2 , and not PtdIns(4) P binding mediates the recruitment of ORP5/8 to endoplasmic reticulum–plasma membrane contact sites. The OSBP-related domain of ORP8 can extract and transport multiple phosphoinositides in vitro, and knocking down both ORP5 and ORP8 in cells increases the plasma membrane level of PtdIns(4,5) P 2 with little effect on PtdIns(4) P . Overall, our data show, for the first time, that phosphoinositides other than PtdIns(4) P can also serve as co-exchangers for the transport of cargo lipids by ORPs.
Mechanotransduction activates RhoA in the neighbors of apoptotic epithelial cells to engage apical extrusion
Publisher: Elsevier BV
Date: 03-2021
DOI: 10.1016/J.CUB.2021.01.003
Abstract: Epithelia must eliminate apoptotic cells to preserve tissue barriers and prevent inflammation.
Redistribution of caveolae during mitosis
Publisher: The Company of Biologists
Date: 15-06-2011
DOI: 10.1242/JCS.076570
Abstract: Caveolae form a specialized platform within the plasma membrane that is crucial for an array of important biological functions, ranging from signaling to endocytosis. Using total internal reflection fluorescence (TIRF) and 3D fast spinning-disk confocal imaging to follow caveola dynamics for extended periods, and electron microscopy to obtain high resolution snapshots, we found that the vast majority of caveolae are dynamic with lifetimes ranging from a few seconds to several minutes. Use of these methods revealed a change in the dynamics and localization of caveolae during mitosis. During interphase, the equilibrium between the arrival and departure of caveolae from the cell surface maintains the steady-state distribution of caveolin-1 (Cav1) at the plasma membrane. During mitosis, increased dynamics coupled to an imbalance between the arrival and departure of caveolae from the cell surface induces a redistribution of Cav1 from the plasma membrane to intracellular compartments. These changes are reversed during cytokinesis. The observed redistribution of Cav1 was reproduced by treatment of interphase cells with nocodazole, suggesting that microtubule rearrangements during mitosis can mediate caveolin relocalization. This study provides new insights into the dynamics of caveolae and highlights precise regulation of caveola budding and recycling during mitosis.
Reassessing the Role of Phosphocaveolin‐1 in Cell Adhesion and Migration
Publisher: Wiley
Date: 14-09-2007
DOI: 10.1111/J.1600-0854.2007.00653.X
Abstract: Although phosphorylation on tyrosine 14 was identified early in the discovery of caveolin‐1, the functional significance of this modification still remains elusive. Recent evidence points to a role of caveolin‐1 tyrosine 14 phosphorylation in cell adhesion and migration. These results are based on a variety of tools, including a widely used mouse monoclonal anti‐phosphocaveolin‐1 antibody, which labels, in cultured cells, a protein localized at or near focal adhesions. We here report results from three independent laboratories, showing that this antibody recognizes phosphocaveolin‐1 amongst other proteins in immunoblot analyses and that the signal obtained with this antibody in immunostaining experiments is in part due to labeling of paxillin. Published data need to be interpreted keeping in mind that images of phosphocaveolin‐1 cellular localization obtained using this antibody are not valid. We re‐evaluate the current knowledge about the role of caveolin‐1 in cell adhesion and migration in view of this new information.
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
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.
Critical role of CAV1/caveolin-1 in cell stress responses in human breast cancer cells via modulation of lysosomal function and autophagy
Publisher: Informa UK Limited
Date: 04-05-2015
Author response: Nanoscape, a data-driven 3D real-time interactive virtual cell environment
Publisher: eLife Sciences Publications, Ltd
Date: 22-03-2021
The GTPase-Activating Protein GRAF1 Regulates the CLIC/GEEC Endocytic Pathway
Publisher: Elsevier BV
Date: 11-2008
Therapeutic Levels of the Hydroxmethylglutaryl-Coenzyme A Reductase Inhibitor Lovastatin Activate Ras Signaling via Phospholipase D2
Publisher: Informa UK Limited
Date: 03-2011
DOI: 10.1128/MCB.00989-10
Minimum information reporting in bio-nano experimental literature
Publisher: Springer Science and Business Media LLC
Date: 09-2018
Nicotinamide riboside attenuates age-associated metabolic and functional changes in hematopoietic stem cells.
Publisher: Springer Science and Business Media LLC
Date: 11-05-2021
DOI: 10.1038/S41467-021-22863-0
Abstract: With age, hematopoietic stem cells (HSC) undergo changes in function, including reduced regenerative potential and loss of quiescence, which is accompanied by a significant expansion of the stem cell pool that can lead to haematological disorders. Elevated metabolic activity has been implicated in driving the HSC ageing phenotype. Here we show that nicotinamide riboside (NR), a form of vitamin B3, restores youthful metabolic capacity by modifying mitochondrial function in multiple ways including reduced expression of nuclear encoded metabolic pathway genes, d ing of mitochondrial stress and a decrease in mitochondrial mass and network-size. Metabolic restoration is dependent on continuous NR supplementation and accompanied by a shift of the aged transcriptome towards the young HSC state, more youthful bone marrow cellular composition and an improved regenerative capacity in a transplant setting. Consequently, NR administration could support healthy ageing by re-establishing a more youthful hematopoietic system.
Caveolin-1 is required for lateral line neuromast and notochord development
Publisher: The Company of Biologists
Date: 07-2007
DOI: 10.1242/JCS.003830
Abstract: Caveolae have been linked to erse cellular functions and to many disease states. In this study we have used zebrafish to examine the role of caveolin-1 and caveolae during early embryonic development. During development, expression is apparent in a number of tissues including Kupffer's vesicle, tailbud, intersomite boundaries, heart, branchial arches, pronephric ducts and periderm. Particularly strong expression is observed in the sensory organs of the lateral line, the neuromasts and in the notochord where it overlaps with expression of caveolin-3. Morpholino-mediated downregulation of Cav1α caused a dramatic inhibition of neuromast formation. Detailed ultrastructural analysis, including electron tomography of the notochord, revealed that the central regions of the notochord has the highest density of caveolae of any embryonic tissue comparable to the highest density observed in any vertebrate tissue. In addition, Cav1α downregulation caused disruption of the notochord, an effect that was enhanced further by Cav3 knockdown. These results indicate an essential role for caveolin and caveolae in this vital structural and signalling component of the embryo.
In vivo proteomic mapping through GFP-directed proximity-dependent biotin labelling in zebrafish
Publisher: Cold Spring Harbor Laboratory
Date: 06-11-2020
DOI: 10.1101/2020.11.05.370585
Abstract: Protein interaction networks are crucial for complex cellular processes. However, the elucidation of protein interactions occurring within highly specialised cells and tissues is challenging. Here we describe the development, and application, of a new method for proximity-dependent biotin labelling in whole zebrafish. Using a conditionally stabilised GFP-binding nanobody to target a biotin ligase to GFP-labelled proteins of interest, we show tissue-specific proteomic profiling using existing GFP-tagged transgenic zebrafish lines. We demonstrate the applicability of this approach, termed BLITZ (Biotin Labelling In Tagged Zebrafish), in erse cell types such as neurons and vascular endothelial cells. We applied this methodology to identify interactors of caveolar coat protein, cavins, in skeletal muscle. Using this system, we defined specific interaction networks within in vivo muscle cells for the closely related but functionally distinct Cavin4 and Cavin1 proteins.
Author response: In vivo proteomic mapping through GFP-directed proximity-dependent biotin labelling in zebrafish
Publisher: eLife Sciences Publications, Ltd
Date: 10-02-2021
Glucose principally regulates insulin secretion in mouse islets by controlling the numbers of granule fusion events per cell
Publisher: Springer Science and Business Media LLC
Date: 09-2013
Individual Palmitoyl Residues Serve Distinct Roles in H-Ras Trafficking, Microlocalization, and Signaling
Publisher: Informa UK Limited
Date: 08-2005
FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors
Publisher: EMBO
Date: 10-05-2023
Abstract: To maintain both mitochondrial quality and quantity, cells selectively remove damaged or excessive mitochondria through mitophagy, which is a specialised form of autophagy. Mitophagy is induced in response to erse conditions, including hypoxia, cellular differentiation and mitochondrial damage. However, the mechanisms that govern the removal of specific dysfunctional mitochondria under steady‐state conditions to fine‐tune mitochondrial content are not well understood. Here, we report that SCF FBXL4 , an SKP1/CUL1/F‐box protein ubiquitin ligase complex, localises to the mitochondrial outer membrane in unstressed cells and mediates the constitutive ubiquitylation and degradation of the mitophagy receptors NIX and BNIP3 to suppress basal levels of mitophagy. We demonstrate that the pathogenic variants of FBXL4 that cause encephalopathic mtDNA depletion syndrome (MTDPS13) do not efficiently interact with the core SCF ubiquitin ligase machinery or mediate the degradation of NIX and BNIP3. Thus, we reveal a molecular mechanism whereby FBXL4 actively suppresses mitophagy by preventing NIX and BNIP3 accumulation. We propose that the dysregulation of NIX and BNIP3 turnover causes excessive basal mitophagy in FBXL4‐associated mtDNA depletion syndrome.
Constitutive Formation of Caveolae in a Bacterium
Publisher: Elsevier BV
Date: 08-2012
DOI: 10.1016/J.CELL.2012.06.042
Abstract: Caveolin plays an essential role in the formation of characteristic surface pits, caveolae, which cover the surface of many animal cells. The fundamental principles of caveola formation are only slowly emerging. Here we show that caveolin expression in a prokaryotic host lacking any intracellular membrane system drives the formation of cytoplasmic vesicles containing polymeric caveolin. Vesicle formation is induced by expression of wild-type caveolins, but not caveolin mutants defective in caveola formation in mammalian systems. In addition, cryoelectron tomography shows that the induced membrane domains are equivalent in size and caveolin density to native caveolae and reveals a possible polyhedral arrangement of caveolin oligomers. The caveolin-induced vesicles or heterologous caveolae (h-caveolae) form by budding in from the cytoplasmic membrane, generating a membrane domain with distinct lipid composition. Periplasmic solutes are encapsulated in the budding h-caveola, and purified h-caveolae can be tailored to be targeted to specific cells of interest.
Non-caveolar caveolin-1 expression in prostate cancer cells promotes lymphangiogenesis
Publisher: Impact Journals, LLC
Date: 03-08-2015
Abstract: Lymphangiogenesis allows prostate cancer (PCa) lymphatic metastasis, which is associated with poor prognosis and short survival rates. Caveolin-1 (Cav-1) is a membrane protein localized in caveolae, but also exists in non-caveolar, cellular or extracellular forms. Cav-1 is overexpressed in PCa, promotes prostate tumour progression and metastasis. We investigated the effect of caveolar and non-caveolar Cav-1 on PCa lymphangiogenic potential. Cav-1 was down-regulated in PC3 and DU145, and ectopically expressed in LNCaP cells. The effect of PCa cell conditioned media on lymphatic endothelial cell (LEC) viability, chemotaxis, chemokinesis and differentiation was assessed. The effect of Cav-1 on PCa cell expression of lymphangiogenesis-modulators VEGF-A and VEGF-C was assessed using qPCR and ELISA of the conditioned medium. Non-caveolar Cav-1, whether exogenous or endogenous (in LNCaP and PC3 cells, respectively) enhanced LEC proliferation, migration and differentiation. In contrast, caveolar Cav-1 (in DU145 cells) did not significantly affect PCa cell lymphangiogenic potential. The effect of non-caveolar Cav-1 on LECs was mediated by increased expression of VEGF-A as demonstrated by neutralization by anti-VEGF-A antibody. This study unveils for the first time a crucial role for non-caveolar Cav-1 in modulating PCa cell expression of VEGF-A and subsequent LEC proliferation, migration and tube formation.
Vascular defects in a mouse model of hypotrichosis-lymphedema-telangiectasia syndrome indicate a role for SOX18 in blood vessel maturation
Publisher: Oxford University Press (OUP)
Date: 09-05-2009
DOI: 10.1093/HMG/DDP219
Abstract: Mutations in the transcription factor gene SOX18 cause vascular, lymphatic and hair follicle defects in humans with dominant and recessive forms of hypotrichosis-lymphedema-telangiectasia (HLT) syndrome. Here, we clarify the role of SOX18 in the vascular dysfunction in HLT by ultrastructural, immunofluorescence, molecular and functional analysis of vascular anomalies in embryos of the naturally occurring Sox18-mutant mouse strain ragged-opossum (Ra(Op)). Early genesis and patterning of vasculature was unimpaired in Ra(Op) embryos, but surface capillaries became enlarged from 12.5 dpc and embryos developed massive surface hemorrhage by 14.5 dpc. Large focal breaches in the endothelial barrier were observed, in addition to endothelial hyperplasia associated with impaired pericyte recruitment to the microvasculature. Expression of the genes encoding the endothelial factors MMP7, IL7R and N-cadherin was reduced in Ra(Op) embryos, suggesting that these are downstream targets of SOX18. Together, our results indicate that vascular anomalies in HLT arise from defects in regulation of genes required for the acquisition of structural integrity during microvascular maturation.
Caveolin-1 Is Essential for Liver Regeneration
Publisher: American Association for the Advancement of Science (AAAS)
Date: 15-09-2006
Abstract: Liver regeneration is an orchestrated cellular response that coordinates cell activation, lipid metabolism, and cell ision. We found that caveolin-1 gene–disrupted mice ( cav1 –/– mice) exhibited impaired liver regeneration and low survival after a partial hepatectomy. Hepatocytes showed dramatically reduced lipid droplet accumulation and did not advance through the cell ision cycle. Treatment of cav1 –/– mice with glucose (which is a predominant energy substrate when compared to lipids) drastically increased survival and reestablished progression of the cell cycle. Thus, caveolin-1 plays a crucial role in the mechanisms that coordinate lipid metabolism with the proliferative response occurring in the liver after cellular injury.
Caveolin‐1‐driven membrane remodelling regulates hnRNPK‐mediated exosomal microRNA sorting in cancer
Publisher: Wiley
Date: 04-2021
DOI: 10.1002/CTM2.381
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.
Examination of the Subsarcolemmal Tubular System of Mammalian Skeletal Muscle Fibers
Publisher: Elsevier BV
Date: 06-2013
G.O.4 α-Actinin-3 regulates muscle glycogen phosphorylase: A potential mechanism for the metabolic consequences of the common human null allele of ACTN3
Publisher: Elsevier BV
Date: 09-2009
Fsp27 promotes lipid droplet growth by lipid exchange and transfer at lipid droplet contact sites
Publisher: Rockefeller University Press
Date: 05-12-2011
Abstract: Lipid droplets (LDs) are dynamic cellular organelles that control many biological processes. However, molecular components determining LD growth are poorly understood. Genetic analysis has indicated that Fsp27, an LD-associated protein, is important in controlling LD size and lipid storage in adipocytes. In this paper, we demonstrate that Fsp27 is focally enriched at the LD–LD contacting site (LDCS). Photobleaching revealed the occurrence of lipid exchange between contacted LDs in wild-type adipocytes and Fsp27-overexpressing cells but not Fsp27-deficient adipocytes. Furthermore, live-cell imaging revealed a unique Fsp27-mediated LD growth process involving a directional net lipid transfer from the smaller to larger LDs at LDCSs, which is in accordance with the biophysical analysis of the internal pressure difference between the contacting LD pair. Thus, we have uncovered a novel molecular mechanism of LD growth mediated by Fsp27.
Seeing and believing: recent advances in imaging cell-cell interactions
Publisher: F1000 Research Ltd
Date: 17-07-2015
DOI: 10.12688/F1000RESEARCH.6435.1
Abstract: Advances in cell and developmental biology have often been closely linked to advances in our ability to visualize structure and function at many length and time scales. In this review, we discuss how new imaging technologies and new reagents have provided novel insights into the biology of cadherin-based cell-cell junctions. We focus on three developments: the application of super-resolution optical technologies to characterize the nanoscale organization of cadherins at cell-cell contacts, new approaches to interrogate the mechanical forces that act upon junctions, and advances in electron microscopy which have the potential to transform our understanding of cell-cell junctions.
Caveolin-1 orchestrates the balance between glucose and lipid-dependent energy metabolism: Implications for liver regeneration
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 04-04-2012
DOI: 10.1002/HEP.24810
Abstract: Caveolin-1 (CAV1) is a structural protein of caveolae involved in lipid homeostasis and endocytosis. Using newly generated pure Balb/C CAV1 null ((Balb/C)CAV1-/-) mice, CAV1-/- mice from Jackson Laboratories ((JAX)CAV1-/-), and CAV1-/- mice developed in the Kurzchalia Laboratory ((K)CAV1-/-), we show that under physiological conditions CAV1 expression in mouse tissues is necessary to guarantee an efficient progression of liver regeneration and mouse survival after partial hepatectomy. Absence of CAV1 in mouse tissues is compensated by the development of a carbohydrate-dependent anabolic adaptation. These results were supported by extracellular flux analysis of cellular glycolytic metabolism in CAV1-knockdown AML12 hepatocytes, suggesting cell autonomous effects of CAV1 loss in hepatic glycolysis. Unlike in (K)CAV1-/- livers, in (JAX)CAV1-/- livers CAV1 deficiency is compensated by activation of anabolic metabolism (pentose phosphate pathway and lipogenesis) allowing liver regeneration. Administration of 2-deoxy-glucose in (JAX)CAV1-/- mice indicated that liver regeneration in (JAX)CAV1-/- mice is strictly dependent on hepatic carbohydrate metabolism. Moreover, with the exception of regenerating (JAX)CAV1-/- livers, expression of CAV1 in mice is required for efficient hepatic lipid storage during fasting, liver regeneration, and diet-induced steatosis in the three CAV1-/- mouse strains. Furthermore, under these conditions CAV1 accumulates in the lipid droplet fraction in wildtype mouse hepatocytes. Our data demonstrate that lack of CAV1 alters hepatocyte energy metabolism homeostasis under physiological and pathological conditions.
Munc18-1 is a molecular chaperone for α-synuclein, controlling its self-replicating aggregation
Publisher: Rockefeller University Press
Date: 05-09-2016
Abstract: Munc18-1 is a key component of the exocytic machinery that controls neurotransmitter release. Munc18-1 heterozygous mutations cause developmental defects and epileptic phenotypes, including infantile epileptic encephalopathy (EIEE), suggestive of a gain of pathological function. Here, we used single-molecule analysis, gene-edited cells, and neurons to demonstrate that Munc18-1 EIEE-causing mutants form large polymers that coaggregate wild-type Munc18-1 in vitro and in cells. Surprisingly, Munc18-1 EIEE mutants also form Lewy body–like structures that contain α-synuclein (α-Syn). We reveal that Munc18-1 binds α-Syn, and its EIEE mutants coaggregate α-Syn. Likewise, removal of endogenous Munc18-1 increases the aggregative propensity of α-SynWT and that of the Parkinson’s disease–causing α-SynA30P mutant, an effect rescued by Munc18-1WT expression, indicative of chaperone activity. Coexpression of the α-SynA30P mutant with Munc18-1 reduced the number of α-SynA30P aggregates. Munc18-1 mutations and haploinsufficiency may therefore trigger a pathogenic gain of function through both the corruption of native Munc18-1 and a perturbed chaperone activity for α-Syn leading to aggregation-induced neurodegeneration.
Endocytic crosstalk: cavins, caveolins, and caveolae regulate clathrin-independent endocytosis.
Publisher: Public Library of Science (PLoS)
Date: 08-04-2014
Faceted polymersomes: a sphere-to-polyhedron shape transformation
Publisher: Royal Society of Chemistry (RSC)
Date: 2019
DOI: 10.1039/C8SC04206C
Abstract: We uncover how our polymersomes facet through a sphere-to-polyhedron shape transformation pathway that is driven by perylene aggregation confined within a topologically spherical polymersome shell.
Formation of retromer transport carriers is disrupted by the Parkinson disease‐linked Vps35 D620N variant
Publisher: Wiley
Date: 22-01-2021
DOI: 10.1111/TRA.12779
GTP-dependent segregation of H-ras from lipid rafts is required for biological activity
Publisher: Springer Science and Business Media LLC
Date: 09-03-2001
DOI: 10.1038/35070050
PTRF–cavin-1 expression decreases the migration of PC3 prostate cancer cells: Role of matrix metalloprotease 9
Publisher: Elsevier BV
Date: 02-2011
DOI: 10.1016/J.EJCB.2010.06.004
Abstract: Caveolae are specialized plasma membrane subdomains with a distinct lipid and protein composition, which play an essential role in cell physiology by performing trafficking and signalling functions. The structure and functions of caveolae have been shown to require caveolin-1, a major protein component of caveolae. Caveolin-1 expression and secretion are increased in metastatic prostate cancer, and caveolin-1 seems to contribute to prostate cancer growth and metastasis. Recently, a cytoplasmic protein named PTRF (Polymerase I and Transcript Release Factor) or cavin-1 was found to be required, in concert with caveolin-1, for the formation and functions of caveolae. Genetic ablation of PTRF results in loss of caveolae while caveolin-1 is still expressed, albeit at reduced level, but associates with flat plasma membrane. In metastatic PC3 prostate cancer cells that express abundant caveolin-1 but no PTRF, heterologous PTRF expression restores caveola formation and caveolin-1 distribution (Hill et al., 2008 Cell 132, 113-124). We now show that PTRF/cavin-1-expressing PC3 cells exhibit decreased migration, and that this effect is mediated by reduced MMP9 production. PTRF/cavin-1, and to a lesser extent, cavin-2, -3, and -4 all decreased MMP9. We further show that the PTRF/cavin-1-mediated reduction of MMP9 production is independent of caveola formation. Taken together, our results suggest that PTRF/cavin-1 expression alters prostate cancer aggressiveness.
Molecular Characterization of Caveolin-induced Membrane Curvature
Publisher: Elsevier BV
Date: 10-2015
Expression of PTRF in PC-3 cells modulates cholesterol dynamics and the actin cytoskeleton impacting secretion pathways
Publisher: Elsevier BV
Date: 02-2012
Author response: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response
Publisher: eLife Sciences Publications, Ltd
Date: 14-05-2021
Retromer has a selective function in cargo sorting via endosome transport carriers
Publisher: Rockefeller University Press
Date: 17-12-2018
Abstract: Retromer is a peripheral membrane protein complex that coordinates multiple vesicular trafficking events within the endolysosomal system. Here, we demonstrate that retromer is required for the maintenance of normal lysosomal morphology and function. The knockout of retromer subunit Vps35 causes an ultrastructural alteration in lysosomal structure and aberrant lysosome function, leading to impaired autophagy. At the whole-cell level, knockout of retromer Vps35 subunit reduces lysosomal proteolytic capacity as a consequence of the improper processing of lysosomal hydrolases, which is dependent on the trafficking of the cation-independent mannose 6-phosphate receptor (CI-M6PR). Incorporation of CI-M6PR into endosome transport carriers via a retromer-dependent process is restricted to those tethered by GCC88 but not golgin-97 or golgin-245. Finally, we show that this retromer-dependent retrograde cargo trafficking pathway requires SNX3, but not other retromer-associated cargo binding proteins, such as SNX27 or SNX-BAR proteins. Therefore, retromer does contribute to the retrograde trafficking of CI-M6PR required for maturation of lysosomal hydrolases and lysosomal function.
Human Miltons associate with mitochondria and induce microtubule-dependent remodeling of mitochondrial networks
Publisher: Elsevier BV
Date: 05-2010
DOI: 10.1016/J.BBAMCR.2010.03.006
Abstract: Proper mitochondrial distribution is crucial for cell function. In Drosophila, mitochondrial transport is facilitated by Miro and Milton, which regulate mitochondrial attachment to microtubules via kinesin heavy chain. Mammals contain two sequence orthologs of Milton however, they have been ascribed various functions in intracellular transport. In this report, we show that the human Miltons target to mitochondria irrespective of whether they are linked to GFP at their C- or N-termini. Their ectopic expression induces the formation of extended mitochondrial tubules as well as large bulbous-like mitochondria with narrow tubular membrane necks that connect them to the mitochondrial mass. The mitochondrial extensions appear highly dynamic and their formation relies on the presence of microtubules. Using the photoswitchable fluorescent protein Dendra2 targeted to the mitochondrial matrix, we found that the mitochondrial extensions and bulbous mitochondria are fused with neighboring regions of the network. Truncation analysis of huMilton1 revealed that the N-terminal region, inclusive of the coiled-coil segment could localize to microtubules, suggesting that Milton attachment to kinesin occurs independent of Miro or mitochondrial attachment. In addition, we show that the huMiltons have the capacity to self-interact and can also facilitate mitochondrial recruitment of a cytosolic Miro mutant. We conclude that the human Miltons are important mediators of the mitochondrial trafficking machinery.
A microtubule-organizing center directing intracellular transport in the early mouse embryo
Publisher: American Association for the Advancement of Science (AAAS)
Date: 09-2017
Abstract: Cell functions ranging from cell ision to morphogenesis rely on microtubules, with microtubule-organizing centers serving as anchoring sites for their outgrowth. Although the centrosome organizes the microtubule cytoskeleton in most animal cells, this organelle is absent in early development. Using live-cell imaging, Zenker et al. found that the cells of the early mouse embryo are connected by stable microtubule bridges to direct the growth of microtubules within them. Microtubules emanating from the bridges help to guide transport of key proteins, including E-cadherin, to the cell membrane to control cell polarization during early development. Science , this issue p. 925
Centrobin regulates the assembly of functional mitotic spindles
Publisher: Springer Science and Business Media LLC
Date: 03-2010
DOI: 10.1038/ONC.2010.37
Abstract: The proper function of the spindle is crucial to the high fidelity of chromosome segregation and is indispensable for tumor suppression in humans. Centrobin is a recently identified centrosomal protein that has a role in stabilizing the microtubule structure. Here we functionally characterize the defects in centrosome integrity and spindle assembly in Centrobin-depleted cells. Centrobin-depleted cells show a range of spindle abnormalities including unfocused poles that are not associated with centrosomes, S-shaped spindles and mini spindles. These cells undergo mitotic arrest and subsequently often die by apoptosis, as determined by live cell imaging. Co-depletion of Mad2 relieves the mitotic arrest, indicating that cells arrest due to a failure to silence the spindle checkpoint in metaphase. Consistent with this, Centrobin-depleted metaphase cells stained positive for BubR1 and BubR1 S676. Staining with a panel of centrosome markers showed a loss of centrosome anchoring to the mitotic spindle. Furthermore, these cells show less cold-stable microtubules and a shorter distance between kinetochore pairs. These results show a requirement of Centrobin in maintaining centrosome integrity, which in turn promotes anchoring of mitotic spindle to the centrosomes. Furthermore, this anchoring is required for the stability of microtubule-kinetochore attachments and biogenesis of tension-ridden and properly functioning mitotic spindle.
Colocation of Tpm3.1 and myosin IIa heads defines a discrete subdomain in stress fibres.
Publisher: The Company of Biologists
Date: 2019
DOI: 10.1242/JCS.228916
Abstract: Co-polymers of tropomyosin and actin make up a major fraction of the actin cytoskeleton. Tropomyosin isoforms determine the function of an actin filament by selectively enhancing or inhibiting the association of other actin binding proteins, altering the stability of an actin filament and regulating myosin activity in an isoform-specific manner. Previous work has implicated specific roles for at least five different tropomyosin isoforms in stress fibres, as depletion of any of these five isoforms results in a loss of stress fibres. Despite this, most models of stress fibres continue to exclude tropomyosins. In this study, we investigate tropomyosin organisation in stress fibres by using super-resolution light microscopy and electron microscopy with genetically tagged, endogenous tropomyosin. We show that tropomyosin isoforms are organised in subdomains within the overall domain of stress fibres. The isoforms Tpm3.1 and 3.2 (hereafter Tpm3.1/3.2, encoded by
Caveolae regulate the nanoscale organization of the plasma membrane to remotely control Ras signaling
Publisher: Rockefeller University Press
Date: 24-02-2014
Abstract: The molecular mechanisms whereby caveolae exert control over cellular signaling have to date remained elusive. We have therefore explored the role caveolae play in modulating Ras signaling. Lipidomic and gene array analyses revealed that caveolin-1 (CAV1) deficiency results in altered cellular lipid composition, and plasma membrane (PM) phosphatidylserine distribution. These changes correlated with increased K-Ras expression and extensive isoform-specific perturbation of Ras spatial organization: in CAV1-deficient cells K-RasG12V nanoclustering and MAPK activation were enhanced, whereas GTP-dependent lateral segregation of H-Ras was abolished resulting in compromised signal output from H-RasG12V nanoclusters. These changes in Ras nanoclustering were phenocopied by the down-regulation of Cavin1, another crucial caveolar structural component, and by acute loss of caveolae in response to increased osmotic pressure. Thus, we postulate that caveolae remotely regulate Ras nanoclustering and signal transduction by controlling PM organization. Similarly, caveolae transduce mechanical stress into PM lipid alterations that, in turn, modulate Ras PM organization.
Cytoskeletal Tropomyosin Tm5NM1 Is Required for Normal Excitation–Contraction Coupling in Skeletal Muscle
Publisher: American Society for Cell Biology (ASCB)
Date: 2009
Abstract: The functional ersity of the actin microfilaments relies in part on the actin binding protein tropomyosin (Tm). The muscle-specific Tms regulate actin-myosin interactions and hence contraction. However, there is less known about the roles of the numerous cytoskeletal isoforms. We have shown previously that a cytoskeletal Tm, Tm5NM1, defines a Z-line adjacent cytoskeleton in skeletal muscle. Recently, we identified a second cytoskeletal Tm in this region, Tm4. Here we show that Tm4 and Tm5NM1 define separate actin filaments the former associated with the terminal sarcoplasmic reticulum (SR) and other tubulovesicular structures. In skeletal muscles of Tm5NM1 knockout (KO) mice, Tm4 localization was unchanged, demonstrating the specificity of the membrane association. Tm5NM1 KO muscles exhibit potentiation of T-system depolarization and decreased force rundown with repeated T-tubule depolarizations consistent with altered T-tubule function. These results indicate that a Tm5NM1-defined actin cytoskeleton is required for the normal excitation–contraction coupling in skeletal muscle.
N4WBP5, a potential target for ubiquitination by the Nedd4 family of proteins, is a novel Golgi-associated protein
Publisher: Elsevier BV
Date: 03-2002
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
A novel switch region regulates H-ras membrane orientation and signal output
Publisher: Wiley
Date: 14-02-2008
Design and Application of In Vivo FRET Biosensors to Identify Protein Prenylation and Nanoclustering Inhibitors
Publisher: Elsevier BV
Date: 07-2012
DOI: 10.1016/J.CHEMBIOL.2012.05.019
Abstract: Protein prenylation is required for membrane anchorage of small GTPases. Correct membrane targeting is essential for their biological activity. Signal output of the prenylated proto-oncogene Ras in addition critically depends on its organization into nanoscale proteolipid assemblies of the plasma membrane, so called nanoclusters. While protein prenylation is an established drug target, only a handful of nanoclustering inhibitors are known, partially due to the lack of appropriate assays to screen for such compounds. Here, we describe three cell-based high-throughput screening amenable Förster resonance energy transfer NANOclustering and Prenylation Sensors (NANOPS) that are specific for Ras, Rho, and Rab proteins. Rab-NANOPS provides the first evidence for nanoclustering of Rab proteins. Using NANOPS in a cell-based chemical screen, we now identify macrotetrolides, known ionophoric antibiotics, as submicromolar disruptors of Ras nanoclustering and MAPK signaling.
Caveolin-1 and cavin1 act synergistically to generate a unique lipid environment in caveolae
Publisher: Rockefeller University Press
Date: 26-01-2021
Abstract: Caveolae are specialized domains of the vertebrate cell surface with a well-defined morphology and crucial roles in cell migration and mechanoprotection. Unique compositions of proteins and lipids determine membrane architectures. The precise caveolar lipid profile and the roles of the major caveolar structural proteins, caveolins and cavins, in selectively sorting lipids have not been defined. Here, we used quantitative nanoscale lipid mapping together with molecular dynamic simulations to define the caveolar lipid profile. We show that caveolin-1 (CAV1) and cavin1 in idually sort distinct plasma membrane lipids. Intact caveolar structures composed of both CAV1 and cavin1 further generate a unique lipid nano-environment. The caveolar lipid sorting capability includes selectivities for lipid headgroups and acyl chains. Because lipid headgroup metabolism and acyl chain remodeling are tightly regulated, this selective lipid sorting may allow caveolae to act as transit hubs to direct communications among lipid metabolism, vesicular trafficking, and signaling.
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
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.
Dynamic and Regulated Association of Caveolin with Lipid Bodies: Modulation of Lipid Body Motility and Function by a Dominant Negative Mutant
Publisher: American Society for Cell Biology (ASCB)
Date: 2004
Abstract: Caveolins are a crucial component of caveolae but have also been localized to the Golgi complex, and, under some experimental conditions, to lipid bodies (LBs). The physiological relevance and dynamics of LB association remain unclear. We now show that endogenous caveolin-1 and caveolin-2 redistribute to LBs in lipid loaded A431 and FRT cells. Association with LBs is regulated and reversible removal of fatty acids causes caveolin to rapidly leave the lipid body. We also show by subcellular fractionation, light and electron microscopy that during the first hours of liver regeneration, caveolins show a dramatic redistribution from the cell surface to the newly formed LBs. At later stages of the regeneration process (when LBs are still abundant), the levels of caveolins in LBs decrease dramatically. As a model system to study association of caveolins with LBs we have used brefeldin A (BFA). BFA causes rapid redistribution of endogenous caveolins to LBs and this association was reversed upon BFA washout. Finally, we have used a dominant negative LB-associated caveolin mutant (cav DGV ) to study LB formation and to examine its effect on LB function. We now show that the cav DGV mutant inhibits microtubule-dependent LB motility and blocks the reversal of lipid accumulation in LBs.
Nucleophosmin and Nucleolin Regulate K-Ras Plasma Membrane Interactions and MAPK Signal Transduction
Publisher: Elsevier BV
Date: 10-2009
Aberrant dysferlin trafficking in cells lacking caveolin or expressing dystrophy mutants of caveolin-3
Publisher: Oxford University Press (OUP)
Date: 30-11-2005
DOI: 10.1093/HMG/DDI434
Abstract: Mutations in the dysferlin (DYSF) and caveolin-3 (CAV3) genes are associated with muscle disease. Dysferlin is mislocalized, by an unknown mechanism, in muscle from patients with mutations in caveolin-3 (Cav-3). To examine the link between Cav-3 mutations and dysferlin mistargeting, we studied their localization at high resolution in muscle fibers, in a model muscle cell line, and upon heterologous expression of dysferlin in muscle cell lines and in wild-type or caveolin-null fibroblasts. Dysferlin shows only partial overlap with Cav-3 on the surface of isolated muscle fibers but co-localizes with Cav-3 in developing transverse (T)-tubules in muscle cell lines. Heterologously expressed dystrophy-associated mutant Cav3R26Q accumulates in the Golgi complex of muscle cell lines or fibroblasts. Cav3R26Q and other Golgi-associated mutants of both Cav-3 (Cav3P104L) and Cav-1 (Cav1P132L) caused a dramatic redistribution of dysferlin to the Golgi complex. Heterologously expressed epitope-tagged dysferlin associates with the plasma membrane in primary fibroblasts and muscle cells. Transport to the cell surface is impaired in the absence of Cav-1 or Cav-3 showing that caveolins are essential for dysferlin association with the PM. These results suggest a functional role for caveolins in a novel post-Golgi trafficking pathway followed by dysferlin.
Cortical F-actin stabilization generates apical–lateral patterns of junctional contractility that integrate cells into epithelia
Publisher: Springer Science and Business Media LLC
Date: 12-01-2014
DOI: 10.1038/NCB2900
Abstract: E-cadherin cell-cell junctions couple the contractile cortices of epithelial cells together, generating tension within junctions that influences tissue organization. Although junctional tension is commonly studied at the apical zonula adherens, we now report that E-cadherin adhesions induce the contractile actomyosin cortex throughout the apical-lateral axis of junctions. However, cells establish distinct regions of contractile activity even within in idual contacts, producing high tension at the zonula adherens but substantially lower tension elsewhere. We demonstrate that N-WASP (also known as WASL) enhances apical junctional tension by stabilizing local F-actin networks, which otherwise undergo stress-induced turnover. Further, we find that cells are extruded from monolayers when this pattern of intra-junctional contractility is disturbed, either when N-WASP redistributes into lateral junctions in H-Ras(V12)-expressing cells or on mosaic redistribution of active N-WASP itself. We propose that local control of actin filament stability regulates the landscape of intra-junctional contractility to determine whether or not cells integrate into epithelial populations.
High-density lipoprotein inhibits human M1 macrophage polarization through redistribution of caveolin-1
Publisher: Wiley
Date: 30-10-2016
DOI: 10.1111/BPH.13319
Evolutionary analysis and molecular dissection of caveola biogenesis
Publisher: The Company of Biologists
Date: 15-06-2008
DOI: 10.1242/JCS.024588
Abstract: Caveolae are an abundant feature of mammalian cells. Integral membrane proteins called caveolins drive the formation of caveolae but the precise mechanisms underlying caveola formation, and the origin of caveolae and caveolins during evolution, are unknown. Systematic evolutionary analysis shows conservation of genes encoding caveolins in metazoans. We provide evidence for extensive and ancient, local and genomic gene duplication, and classify distinct caveolin gene families. Vertebrate caveolin-1 and caveolin-3 isoforms, as well as an invertebrate (Apis mellifera, honeybee) caveolin, all form morphologically identical caveolae in caveolin-1-null mouse cells, demonstrating that caveola formation is a conserved feature of evolutionarily distant caveolins. However, coexpression of flotillin-1 and flotillin-2 did not cause caveola biogenesis in this system. In contrast to the other tested caveolins, C. elegans caveolin is efficiently transported to the plasma membrane but does not generate caveolae, providing evidence of ersity of function in the caveolin gene family. Using C. elegans caveolin as a template to generate hybrid caveolin constructs we now define domains of caveolin required for caveolae biogenesis. These studies lead to a model for caveola formation and novel insights into the evolution of caveolin function.
Flotillin-1/reggie-2 traffics to surface raft domains via a novel Golgi-independent pathway. Identification of a novel membrane targeting domain and a role for palmitoylation
Publisher: Elsevier BV
Date: 12-2002
De novo macrocyclic peptides for inhibiting, stabilizing, and probing the function of the retromer endosomal trafficking complex.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 03-12-2021
Abstract: Novel macrocyclic peptides are found that bind and modulate the function of the retromer membrane trafficking complex.
Cargo-specific recruitment in clathrin- and dynamin-independent endocytosis
Publisher: Springer Science and Business Media LLC
Date: 10-2021
DOI: 10.1038/S41556-021-00767-X
Abstract: Spatially controlled, cargo-specific endocytosis is essential for development, tissue homeostasis and cancer invasion. Unlike cargo-specific clathrin-mediated endocytosis, the clathrin- and dynamin-independent endocytic pathway (CLIC-GEEC, CG pathway) is considered a bulk internalization route for the fluid phase, glycosylated membrane proteins and lipids. While the core molecular players of CG-endocytosis have been recently defined, evidence of cargo-specific adaptors or selective uptake of proteins for the pathway are lacking. Here we identify the actin-binding protein Swiprosin-1 (Swip1, EFHD2) as a cargo-specific adaptor for CG-endocytosis. Swip1 couples active Rab21-associated integrins with key components of the CG-endocytic machinery-Arf1, IRSp53 and actin-and is critical for integrin endocytosis. Through this function, Swip1 supports integrin-dependent cancer-cell migration and invasion, and is a negative prognostic marker in breast cancer. Our results demonstrate a previously unknown cargo selectivity for the CG pathway and a role for specific adaptors in recruitment into this endocytic route.
A robust method for particulate detection of a genetic tag for 3D electron microscopy
Publisher: eLife Sciences Publications, Ltd
Date: 27-04-2021
DOI: 10.7554/ELIFE.64630
Abstract: Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy (EM). Here, we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal-to-noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins, and cytoskeletal proteins. The method can be combined with different EM techniques including fast freezing and freeze substitution, focussed ion beam scanning EM, and electron tomography. Quantitation of expressed APEX-fusion proteins is achievable using membrane vesicles generated by a cell-free expression system. These membrane vesicles possess a defined quantum of signal, which can act as an internal standard for determination of the absolute density of expressed APEX-fusion proteins. Detection of fusion proteins expressed at low levels in cells from CRISPR-edited mice demonstrates the high sensitivity of the APEX-Gold method.
In vivo cell biological screening identifies an endocytic capture mechanism for T-tubule formation
Publisher: Springer Science and Business Media LLC
Date: 24-07-2020
DOI: 10.1038/S41467-020-17486-W
Abstract: The skeletal muscle T-tubule is a specialized membrane domain essential for coordinated muscle contraction. However, in the absence of genetically tractable systems the mechanisms involved in T-tubule formation are unknown. Here, we use the optically transparent and genetically tractable zebrafish system to probe T-tubule development in vivo. By combining live imaging of transgenic markers with three-dimensional electron microscopy, we derive a four-dimensional quantitative model for T-tubule formation. To elucidate the mechanisms involved in T-tubule formation in vivo, we develop a quantitative screen for proteins that associate with and modulate early T-tubule formation, including an overexpression screen of the entire zebrafish Rab protein family. We propose an endocytic capture model involving firstly, formation of dynamic endocytic tubules at transient nucleation sites on the sarcolemma, secondly, stabilization by myofibrils/sarcoplasmic reticulum and finally, delivery of membrane from the recycling endosome and Golgi complex.
In vivo proteomic mapping through GFP-directed proximity-dependent biotin labelling in zebrafish
Publisher: eLife Sciences Publications, Ltd
Date: 16-02-2021
DOI: 10.7554/ELIFE.64631
Abstract: Protein interaction networks are crucial for complex cellular processes. However, the elucidation of protein interactions occurring within highly specialised cells and tissues is challenging. Here, we describe the development, and application, of a new method for proximity-dependent biotin labelling in whole zebrafish. Using a conditionally stabilised GFP-binding nanobody to target a biotin ligase to GFP-labelled proteins of interest, we show tissue-specific proteomic profiling using existing GFP-tagged transgenic zebrafish lines. We demonstrate the applicability of this approach, termed BLITZ (Biotin Labelling In Tagged Zebrafish), in erse cell types such as neurons and vascular endothelial cells. We applied this methodology to identify interactors of caveolar coat protein, cavins, in skeletal muscle. Using this system, we defined specific interaction networks within in vivo muscle cells for the closely related but functionally distinct Cavin4 and Cavin1 proteins.
Cholesterol and Fatty Acids Regulate Dynamic Caveolin Trafficking through the Golgi Complex and between the Cell Surface and Lipid Bodies
Publisher: American Society for Cell Biology (ASCB)
Date: 04-2005
Abstract: Caveolins are a crucial component of plasma membrane (PM) caveolae but have also been localized to intracellular compartments, including the Golgi complex and lipid bodies. Mutant caveolins associated with human disease show aberrant trafficking to the PM and Golgi accumulation. We now show that the Golgi pool of mainly newly synthesized protein is detergent-soluble and predominantly in a monomeric state, in contrast to the surface pool. Caveolin at the PM is not recognized by specific caveolin antibodies unless PM cholesterol is depleted. Exit from the Golgi complex of wild-type caveolin-1 or -3, but not vesicular stomatitis virus-G protein, is modulated by changing cellular cholesterol levels. In contrast, a muscular dystrophy-associated mutant of caveolin-3, Cav3P104L, showed increased accumulation in the Golgi complex upon cholesterol treatment. In addition, we demonstrate that in response to fatty acid treatment caveolin can follow a previously undescribed pathway from the PM to lipid bodies and can move from lipid bodies to the PM in response to removal of fatty acids. The results suggest that cholesterol is a rate-limiting component for caveolin trafficking. Changes in caveolin flux through the exocytic pathway can therefore be an indicator of cellular cholesterol and fatty acid levels.
Mammalian lipid droplets are innate immune hubs integrating cell metabolism and host defense
Publisher: American Association for the Advancement of Science (AAAS)
Date: 15-10-2020
Abstract: Lipid droplets (LDs) are the major lipid storage organelles of eukaryotic cells and a source of nutrients for intracellular pathogens. We demonstrate that mammalian LDs are endowed with a protein-mediated antimicrobial capacity, which is up-regulated by danger signals. In response to lipopolysaccharide (LPS), multiple host defense proteins, including interferon-inducible guanosine triphosphatases and the antimicrobial cathelicidin, assemble into complex clusters on LDs. LPS additionally promotes the physical and functional uncoupling of LDs from mitochondria, reducing fatty acid metabolism while increasing LD-bacterial contacts. Thus, LDs actively participate in mammalian innate immunity at two levels: They are both cell-autonomous organelles that organize and use immune proteins to kill intracellular pathogens as well as central players in the local and systemic metabolic adaptation to infection.
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.
Tyrosine dephosphorylated cortactin downregulates contractility at the epithelial zonula adherens through SRGAP1.
Publisher: Springer Science and Business Media LLC
Date: 05-10-2017
DOI: 10.1038/S41467-017-00797-W
Abstract: Contractile adherens junctions support cell−cell adhesion, epithelial integrity, and morphogenesis. Much effort has been devoted to understanding how contractility is established however, less is known about whether contractility can be actively downregulated at junctions nor what function this might serve. We now identify such an inhibitory pathway that is mediated by the cytoskeletal scaffold, cortactin. Mutations of cortactin that prevent its tyrosine phosphorylation downregulate RhoA signaling and compromise the ability of epithelial cells to generate a contractile zonula adherens. This is mediated by the RhoA antagonist, SRGAP1. We further demonstrate that this mechanism is co-opted by hepatocyte growth factor to promote junctional relaxation and motility in epithelial collectives. Together, our findings identify a novel function of cortactin as a regulator of RhoA signaling that can be utilized by morphogenetic regulators for the active downregulation of junctional contractility.
A phosphoinositide-binding cluster in cavin1 acts as a molecular sensor for cavin1 degradation.
Publisher: American Society for Cell Biology (ASCB)
Date: 15-10-2015
Abstract: Caveolae are abundant surface organelles implicated in a range of cellular processes. Two classes of proteins work together to generate caveolae: integral membrane proteins termed caveolins and cytoplasmic coat proteins called cavins. Caveolae respond to membrane stress by releasing cavins into the cytosol. A crucial aspect of this model is tight regulation of cytosolic pools of cavin under resting conditions. We now show that a recently identified region of cavin1 that can bind phosphoinositide (PI) lipids is also a major site of ubiquitylation. Ubiquitylation of lysines within this site leads to rapid proteasomal degradation. In cells that lack caveolins and caveolae, cavin1 is cytosolic and rapidly degraded as compared with cells in which cavin1 is associated with caveolae. Membrane stretching causes caveolar disassembly, release of cavin complexes into the cytosol, and increased proteasomal degradation of wild-type cavin1 but not mutant cavin1 lacking the major ubiquitylation site. Release of cavin1 from caveolae thus leads to exposure of key lysine residues in the PI-binding region, acting as a trigger for cavin1 ubiquitylation and down-regulation. This mutually exclusive PI-binding/ubiquitylation mechanism may help maintain low levels of cytosolic cavin1 in resting cells, a prerequisite for cavins acting as signaling modules following release from caveolae.
Caveolae respond to acute oxidative stress through membrane lipid peroxidation, cytosolic release of CAVIN1, and downstream regulation of NRF2
Publisher: Research Square Platform LLC
Date: 22-10-2021
DOI: 10.21203/RS.3.RS-963423/V1
Abstract: Caveolae have been linked to many biological functions, but their precise roles are unclear. Using quantitative whole cell proteomics of genome-edited cells, we show that the oxidative stress response is the major pathway dysregulated in cells lacking the key caveola structural protein, CAVIN1. CAVIN1 deletion compromised sensitivity to oxidative stress in cultured cells and in animals. Wound-induced accumulation of reactive oxygen species and apoptosis were suppressed in Cavin1-null zebrafish, negatively affecting regeneration. Oxidative stress triggered lipid peroxidation (LPO) and induced caveolar disassembly. The resulting release of CAVIN1 from caveolae allowed direct interaction between CAVIN1 and NRF2, a key regulator of the antioxidant response, facilitating NRF2 degradation. CAVIN1-null cells with impaired negative regulation of NRF2 showed resistance to LPO-induced ferroptosis. Thus, caveolae, via LPO and CAVIN1 release, maintain cellular susceptibility to oxidative stress-induced cell death demonstrating a crucial role for this enigmatic organelle in cellular homeostasis and wound response.
Three Separable Domains Regulate GTP-Dependent Association of H-ras with the Plasma Membrane
Publisher: Informa UK Limited
Date: 08-2004
The caveolin–cavin system plays a conserved and critical role in mechanoprotection of skeletal muscle
Publisher: Rockefeller University Press
Date: 31-08-2015
Abstract: Dysfunction of caveolae is involved in human muscle disease, although the underlying molecular mechanisms remain unclear. In this paper, we have functionally characterized mouse and zebrafish models of caveolae-associated muscle disease. Using electron tomography, we quantitatively defined the unique three-dimensional membrane architecture of the mature muscle surface. Caveolae occupied around 50% of the sarcolemmal area predominantly assembled into multilobed rosettes. These rosettes were preferentially disassembled in response to increased membrane tension. Caveola-deficient cavin-1−/− muscle fibers showed a striking loss of sarcolemmal organization, aberrant T-tubule structures, and increased sensitivity to membrane tension, which was rescued by muscle-specific Cavin-1 reexpression. In vivo imaging of live zebrafish embryos revealed that loss of muscle-specific Cavin-1 or expression of a dystrophy-associated Caveolin-3 mutant both led to sarcolemmal damage but only in response to vigorous muscle activity. Our findings define a conserved and critical role in mechanoprotection for the unique membrane architecture generated by the caveolin–cavin system.
AarF Domain Containing Kinase 3 (ADCK3) Mutant Cells Display Signs of Oxidative Stress, Defects in Mitochondrial Homeostasis and Lysosomal Accumulation.
Publisher: Public Library of Science (PLoS)
Date: 11-02-2016
MURC/Cavin-4 and cavin family members form tissue-specific caveolar complexes
Publisher: Rockefeller University Press
Date: 22-06-2009
Abstract: Polymerase I and transcript release factor (PTRF)/Cavin is a cytoplasmic protein whose expression is obligatory for caveola formation. Using biochemistry and fluorescence resonance energy transfer–based approaches, we now show that a family of related proteins, PTRF/Cavin-1, serum deprivation response (SDR)/Cavin-2, SDR-related gene product that binds to C kinase (SRBC)/Cavin-3, and muscle-restricted coiled-coil protein (MURC)/Cavin-4, forms a multiprotein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and associate with caveolin at plasma membrane caveolae. Cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The tissue-restricted expression of cavins suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. Cavin-4 is expressed predominantly in muscle, and its distribution is perturbed in human muscle disease associated with Caveolin-3 dysfunction, identifying Cavin-4 as a novel muscle disease candidate caveolar protein.
Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response
Publisher: eLife Sciences Publications, Ltd
Date: 18-06-2021
DOI: 10.7554/ELIFE.61407
Abstract: Caveolae-associated protein 3 (cavin3) is inactivated in most cancers. We characterized how cavin3 affects the cellular proteome using genome-edited cells together with label-free quantitative proteomics. These studies revealed a prominent role for cavin3 in DNA repair, with BRCA1 and BRCA1 A-complex components being downregulated on cavin3 deletion. Cellular and cell-free expression assays revealed a direct interaction between BRCA1 and cavin3 that occurs when cavin3 is released from caveolae that are disassembled in response to UV and mechanical stress. Overexpression and RNAi-depletion revealed that cavin3 sensitized various cancer cells to UV-induced apoptosis. Supporting a role in DNA repair, cavin3-deficient cells were sensitive to PARP inhibition, where concomitant depletion of 53BP1 restored BRCA1-dependent sensitivity to PARP inhibition. We conclude that cavin3 functions together with BRCA1 in multiple cancer-related pathways. The loss of cavin3 function may provide tumor cell survival by attenuating apoptotic sensitivity and hindering DNA repair under chronic stress conditions.
EHD2 regulates caveolar dynamics via ATP-driven targeting and oligomerization
Publisher: American Society for Cell Biology (ASCB)
Date: 04-2012
Abstract: Eps15 homology domain–containing 2 (EHD2) belongs to the EHD-containing protein family of dynamin-related ATPases involved in membrane remodeling in the endosomal system. EHD2 dimers oligomerize into rings on highly curved membranes, resulting in stimulation of the intrinsic ATPase activity. In this paper, we report that EHD2 is specifically and stably associated with caveolae at the plasma membrane and not involved in clathrin-mediated endocytosis or endosomal recycling, as previously suggested. EHD2 interacts with pacsin2 and cavin1, and ordered membrane assembly of EHD2 is dependent on cavin1 and caveolar integrity. While the EHD of EHD2 is dispensable for targeting, we identified a loop in the nucleotide-binding domain that, together with ATP binding, is required for caveolar localization. EHD2 was not essential for the formation or shaping of caveolae, but high levels of EHD2 caused distortion and loss of endogenous caveolae. Assembly of EHD2 stabilized and constrained caveolae to the plasma membrane to control turnover, and depletion of EHD2, resulting in endocytic and more dynamic and short-lived caveolae. Thus, following the identification of caveolin and cavins, EHD2 constitutes a third structural component of caveolae involved in controlling the stability and turnover of this organelle.
Modular Detection of GFP-Labeled Proteins for Rapid Screening by Electron Microscopy in Cells and Organisms
Publisher: Elsevier BV
Date: 11-2015
DOI: 10.1016/J.DEVCEL.2015.10.016
Abstract: Reliable and quantifiable high-resolution protein localization is critical for understanding protein function. However, the time required to clone and characterize any protein of interest is a significant bottleneck, especially for electron microscopy (EM). We present a modular system for enzyme-based protein tagging that allows for improved speed and s ling for analysis of subcellular protein distributions using existing clone libraries to EM-resolution. We demonstrate that we can target a modified soybean ascorbate peroxidase (APEX) to any GFP-tagged protein of interest by engineering a GFP-binding peptide (GBP) directly to the APEX-tag. We demonstrate that APEX-GBP (1) significantly reduces the time required to characterize subcellular protein distributions of whole libraries to less than 3 days, (2) provides remarkable high-resolution localization of proteins to organelle subdomains, and (3) allows EM localization of GFP-tagged proteins, including proteins expressed at endogenous levels, in vivo by crossing existing GFP-tagged transgenic zebrafish lines with APEX-GBP transgenic lines.
ORP5 localizes to ER-lipid droplet contacts and regulates the level of PI(4)P on lipid droplets
Publisher: Rockefeller University Press
Date: 25-10-2020
Abstract: Lipid droplets (LDs) are evolutionarily conserved organelles that play important roles in cellular metabolism. Each LD is enclosed by a monolayer of phospholipids, distinct from bilayer membranes. During LD biogenesis and growth, this monolayer of lipids expands by acquiring phospholipids from the endoplasmic reticulum (ER) through nonvesicular mechanisms. Here, in a mini-screen, we find that ORP5, an integral membrane protein of the ER, can localize to ER–LD contact sites upon oleate loading. ORP5 interacts with LDs through its ligand-binding domain, and ORP5 deficiency enhances neutral lipid synthesis and increases the size of LDs. Importantly, there is significantly more phosphatidylinositol-4-phosphate (PI(4)P) and less phosphatidylserine (PS) on LDs in ORP5-deficient cells than in normal cells. The increased presence of PI(4)P on LDs in ORP5-deficient cells requires phosphatidylinositol 4-kinase 2-α. Our results thus demonstrate the existence of PI(4)P on LDs and suggest that LD-associated PI(4)P may be primarily used by ORP5 to deliver PS to LDs.
Caveola mechanotransduction reinforces the cortical cytoskeleton to promote epithelial resilience
Publisher: Cold Spring Harbor Laboratory
Date: 29-03-2023
DOI: 10.1101/2023.03.29.534729
Abstract: As physical barriers, epithelia must preserve their integrity when challenged by mechanical stresses. Cell-cell junctions linked to the cortical cytoskeleton play key roles in this process, often with mechanotransduction mechanisms that reinforce tissues. Caveolae are mechanosensitive organelles that buffer tension via disassembly. Loss of caveolae, through caveolin-1 or cavin1 depletion, causes activation of PtdIns(4, 5)P 2 signalling, recruitment of FMNL2 formin, and enhanced cortical actin assembly. How this equates to physiological responses in epithelial cells containing endogenous caveolae is unknown. Here we examined the effect of mechanically-inducing acute disassembly of caveolae in epithelia. We show that perturbation of caveolae, through direct mechanical stress, reinforces the actin cortex at adherens junctions. Increasing interactions with membrane lipids by introducing multiple phosphatidylserine-binding undecad cavin1 (UC1) repeat domains into cavin1 rendered caveolae more stable to mechanical stimuli. This molecular stabilization blocked cortical reinforcement in response to mechanical stress. Cortical reinforcement elicited by the mechanically-induced disassembly of caveolae increased epithelial resilience against tensile stresses. These findings identify the actin cortex as a target of caveola mechanotransduction that contributes to epithelial integrity.
Apicobasal RNA asymmetries regulate cell fate in the early mouse embryo
Publisher: Springer Science and Business Media LLC
Date: 30-05-2023
DOI: 10.1038/S41467-023-38436-2
Abstract: The spatial sorting of RNA transcripts is fundamental for the refinement of gene expression to distinct subcellular regions. Although, in non-mammalian early embryogenesis, differential RNA localisation presages cell fate determination, in mammals it remains unclear. Here, we uncover apical-to-basal RNA asymmetries in outer blastomeres of 16-cell stage mouse preimplantation embryos. Basally directed RNA transport is facilitated in a microtubule- and lysosome-mediated manner. Yet, despite an increased accumulation of RNA transcripts in basal regions, higher translation activity occurs at the more dispersed apical RNA foci, demonstrated by spatial heterogeneities in RNA subtypes, RNA-organelle interactions and translation events. During the transition to the 32-cell stage, the biased inheritance of RNA transcripts, coupled with differential translation capacity, regulates cell fate allocation of trophectoderm and cells destined to form the pluripotent inner cell mass. Our study identifies a paradigm for the spatiotemporal regulation of post-transcriptional gene expression governing mammalian preimplantation embryogenesis and cell fate.
Rab18 promotes lipid droplet (LD) growth by tethering the ER to LDs through SNARE and NRZ interactions
Publisher: Rockefeller University Press
Date: 24-01-2018
Abstract: Lipid incorporation from endoplasmic reticulum (ER) to lipid droplet (LD) is important in controlling LD growth and intracellular lipid homeostasis. However, the molecular link mediating ER and LD cross talk remains elusive. Here, we identified Rab18 as an important Rab guanosine triphosphatase in controlling LD growth and maturation. Rab18 deficiency resulted in a drastically reduced number of mature LDs and decreased lipid storage, and was accompanied by increased ER stress. Rab3GAP1/2, the GEF of Rab18, promoted LD growth by activating and targeting Rab18 to LDs. LD-associated Rab18 bound specifically to the ER-associated NAG-RINT1-ZW10 (NRZ) tethering complex and their associated SNAREs (Syntaxin18, Use1, BNIP1), resulting in the recruitment of ER to LD and the formation of direct ER–LD contact. Cells with defects in the NRZ/SNARE complex function showed reduced LD growth and lipid storage. Overall, our data reveal that the Rab18-NRZ-SNARE complex is critical protein machinery for tethering ER–LD and establishing ER–LD contact to promote LD growth.
Human immune cell targeting of protein nanoparticles-caveospheres
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C6NR00506C
Abstract: Nanotechnology has the power to transform vaccine and drug delivery through protection of payloads from both metabolism and off-target effects, while facilitating specific delivery of cargo to immune cells. However, evaluation of immune cell nanoparticle targeting is conventionally restricted to monocultured cell line models. We generated human caveolin-1 nanoparticles, termed caveospheres, which were efficiently functionalized with monoclonal antibodies. Using this platform, we investigated CD4+ T cell and CD20+ B cell targeting within physiological mixtures of primary human blood immune cells using flow cytometry, imaging flow cytometry and confocal microscopy. Antibody-functionalization enhanced caveosphere binding to targeted immune cells (6.6 to 43.9-fold) within mixed populations and in the presence of protein-containing fluids. Moreover, targeting caveospheres to CCR5 enabled caveosphere internalization by non-phagocytic CD4+ T cells--an important therapeutic target for HIV treatment. This efficient and flexible system of immune cell-targeted caveosphere nanoparticles holds promise for the development of advanced immunotherapeutics and vaccines.
Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation
Publisher: Springer Science and Business Media LLC
Date: 10-02-2021
DOI: 10.1038/S41467-021-21035-4
Abstract: Caveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Cavins are cytosolic peripheral membrane proteins with negatively charged intrinsically disordered regions that flank positively charged α-helical regions. Here, we show that the three disordered domains of Cavin1 are essential for caveola formation and dynamic trafficking of caveolae. Electrostatic interactions between disordered regions and α-helical regions promote liquid-liquid phase separation behaviour of Cavin1 in vitro, assembly of Cavin1 oligomers in solution, generation of membrane curvature, association with caveolin-1, and Cavin1 recruitment to caveolae in cells. Removal of the first disordered region causes irreversible gel formation in vitro and results in aberrant caveola trafficking through the endosomal system. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin1 and caveolin-1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat.
Colony-stimulating factor-1 (CSF-1) delivers a proatherogenic signal to human macrophages
Publisher: Oxford University Press (OUP)
Date: 12-11-2008
DOI: 10.1189/JLB.0808497
Abstract: M-CSF/CSF-1 supports the proliferation and differentiation of monocytes and macrophages. In mice, CSF-1 also promotes proinflammatory responses in vivo by regulating mature macrophage functions, but little is known about the acute effects of this growth factor on mature human macrophages. Here, we show that in contrast to its effects on mouse bone marrow-derived macrophages, CSF-1 did not induce expression of urokinase plasminogen activator mRNA, repress expression of apolipoprotein E mRNA, or prime LPS-induced TNF and IL-6 secretion in human monocyte-derived macrophages (HMDM) from several independent donors. Instead, we show by expression profiling that CSF-1 modulates the HMDM transcriptome to favor a proatherogenic environment. CSF-1 induced expression of the proatherogenic chemokines CXCL10/IFN-inducible protein 10, CCL2, and CCL7 but repressed expression of the antiatherogenic chemokine receptor CXCR4. CSF-1 also up-regulated genes encoding enzymes of the cholesterol biosynthetic pathway (HMGCR, MVD, IDI1, FDPS, SQLE, CYP51A1, EBP, NSDHL, DHCR7, and DHCR24), and expression of ABCG1, encoding a cholesterol efflux transporter, was repressed. Consistent with these effects, CSF-1 increased levels of free cholesterol in HMDM, and the selective CSF-1R kinase inhibitor GW2580 ablated this response. These data demonstrate that CSF-1 represents a further link between inflammation and cardiovascular disease and suggest two distinct mechanisms by which CSF-1, which is known to be present in atherosclerotic lesions, may contribute to plaque progression.
H-ras but Not K-ras Traffics to the Plasma Membrane through the Exocytic Pathway
Publisher: Informa UK Limited
Date: 04-2000
RORα Regulates the Expression of Genes Involved in Lipid Homeostasis in Skeletal Muscle Cells
Publisher: Elsevier BV
Date: 08-2004
Are caveolae a cellular entry route for non-viral therapeutic delivery systems?
Publisher: Elsevier BV
Date: 08-2015
DOI: 10.1016/J.ADDR.2015.01.003
Abstract: The development of novel therapies increasingly relies on sophisticated delivery systems that allow the drug or gene expression-modifying agent of interest entry into cells. These systems can promote cellular targeting and/or entry, and they vary in size, charge, and functional group chemistry. Their optimization requires an in depth knowledge of the cellular routes of entry in normal and pathological states. Caveolae are plasma membrane invaginations that have the potential to undergo endocytosis. We critically review the literature exploring whether drug or nucleic acid delivery systems exploit and/or promote cellular entry via caveolae. A vast majority of studies employ pharmacological tools, co-localization experiments and very few make use of molecular tools. We provide clarification on how results of such studies should be interpreted and make suggestions for future studies.
A kinetic view of GPCR allostery and biased agonism
Publisher: Springer Science and Business Media LLC
Date: 18-08-2017
Abstract: G-protein-coupled receptors (GPCRs) are one of the most tractable classes of drug targets. These dynamic proteins can adopt multiple active states that are linked to distinct functional outcomes. Such states can be differentially stabilized by ligands interacting with the endogenous agonist-binding orthosteric site and/or by ligands acting via spatially distinct allosteric sites, leading to the phenomena of 'biased agonism' or 'biased modulation'. These paradigms are having a major impact on modern drug discovery, but it is becoming increasingly apparent that 'kinetic context', at the level of both ligand-receptor and receptor-signal pathway kinetics, can have a profound impact on the observation and quantification of these phenomena. The concept of kinetic context thus represents an important new consideration that should be routinely incorporated into contemporary chemical biology and drug discovery studies of GPCR bias and allostery.
Correlation of the invasive potential of glioblastoma and expression of caveola-forming proteins caveolin-1 and CAVIN1.
Publisher: Springer Science and Business Media LLC
Date: 04-04-2019
DOI: 10.1007/S11060-019-03161-8
Abstract: Glioblastoma (GBM) is the most common primary brain cancer. The average survival time for the majority of patients is approximately 15 months after diagnosis. A major feature of GBM that contributes to its poor prognosis is its high invasiveness. Caveolae are plasma membrane subdomains that participate in numerous biological functions. Caveolin-1 and Caveolae Associated Protein 1 (CAVIN1), formerly termed Polymerase I and Transcript Release Factor, are both necessary for caveola formation. We hypothesized that high expression of caveola-forming proteins in GBM promotes invasiveness via modulation of the production of matrix-degrading enzymes. The mRNA expression of caveola-forming proteins and matrix proteases in GBM s les, and survival after stratifying patients according to caveolin-1 or CAVIN1 expression, were analyzed from TCGA and REMBRANDT databases. The proteolytic profile of cell lines expressing or devoid of caveola-forming proteins was investigated using zymography and real-time qPCR. Invasion through basement membrane-like protein was investigated in vitro. Expression of both caveolin-1 and CAVIN1 was increased in GBM compared to normal s les and correlated with expression of urokinase plasminogen activator (uPA) and gelatinases. High expression of caveola-forming proteins was associated with shorter survival time. GBM cell lines capable of forming caveolae expressed more uPA and matrix metalloproteinase-2 (MMP-2) and/or -9 (MMP-9) and were more invasive than GBM cells devoid of caveola-forming proteins. Experimental manipulation of caveolin-1 or CAVIN1 expression in GBM cells recapitulated some, but not all of these features. Caveolae modulate GBM cell invasion in part via matrix protease expression.
Resolution of Novel Pancreatic Ductal Adenocarcinoma Subtypes by Global Phosphotyrosine Profiling
Publisher: Elsevier BV
Date: 08-2016
Modular transient nanoclustering of activated β2-adrenergic receptors revealed by single-molecule tracking of conformation-specific nanobodies
Publisher: Proceedings of the National Academy of Sciences
Date: 19-11-2020
Abstract: Proteins moving freely on the plasma membrane can become transiently trapped in functionally essential clusters. This capability is likely to be influenced by subtle conformational states of the protein promoting or preventing such confinement. The downside of conventional imaging of overexpressed tagged proteins is that it precludes selective tracking of inherently minor albeit functionally essential conformer populations. Intracellular expression of single-chain nanobodies allowed us to track endogenous proteins in highly specific conformational states in live cells and small organisms. We unveiled the full scope of nanoclustering behavior of β 2 -adrenergic receptors in various conformations, along with their transient nature. This technique is broadly applicable to other proteins and will help unravel essential dynamics and organization of nanoclusters.
Revisiting caveolin trafficking: the end of the caveosome
Publisher: Rockefeller University Press
Date: 11-2010
Abstract: In this issue, a study by Hayer et al. (2010. J. Cell Biol. doi: 10.1083/jcb.201003086) provides insights into the trafficking of caveolins, the major membrane proteins of caveolae. As well as providing evidence for ubiquitin-mediated endosomal sorting and degradation of caveolin in multivesicular bodies (MVBs), the new findings question the existence of a unique organelle proposed nine years ago, the caveosome.
Hydrocephalus in Nfix−/− Mice Is Underpinned by Changes in Ependymal Cell Physiology
Publisher: MDPI AG
Date: 02-08-2022
Abstract: Nuclear factor one X (NFIX) is a transcription factor required for normal ependymal development. Constitutive loss of Nfix in mice (Nfix−/−) is associated with hydrocephalus and sloughing of the dorsal ependyma within the lateral ventricles. Previous studies have implicated NFIX in the transcriptional regulation of genes encoding for factors essential to ependymal development. However, the cellular and molecular mechanisms underpinning hydrocephalus in Nfix−/− mice are unknown. To investigate the role of NFIX in hydrocephalus, we examined ependymal cells in brains from postnatal Nfix−/− and control (Nfix+/+) mice using a combination of confocal and electron microscopy. This revealed that the ependymal cells in Nfix−/− mice exhibited abnormal cilia structure and disrupted localisation of adhesion proteins. Furthermore, we modelled ependymal cell adhesion using epithelial cell culture and revealed changes in extracellular matrix and adherens junction gene expression following knockdown of NFIX. Finally, the ablation of Nfix from ependymal cells in the adult brain using a conditional approach culminated in enlarged ventricles, sloughing of ependymal cells from the lateral ventricles and abnormal localisation of adhesion proteins, which are phenotypes observed during development. Collectively, these data demonstrate a pivotal role for NFIX in the regulation of cell adhesion within ependymal cells of the lateral ventricles.
PTRF/cavin-1 neutralizes non-caveolar caveolin-1 microdomains in prostate cancer
Publisher: Springer Science and Business Media LLC
Date: 12-08-2013
DOI: 10.1038/ONC.2013.315
Abstract: Caveolin-1 has a complex role in prostate cancer and has been suggested to be a potential biomarker and therapeutic target. As mature caveolin-1 resides in caveolae, invaginated lipid raft domains at the plasma membrane, caveolae have been suggested as a tumor-promoting signaling platform in prostate cancer. However, caveola formation requires both caveolin-1 and cavin-1 (also known as PTRF polymerase I and transcript release factor). Here, we examined the expression of cavin-1 in prostate epithelia and stroma using tissue microarray including normal, non-malignant and malignant prostate tissues. We found that caveolin-1 was induced without the presence of cavin-1 in advanced prostate carcinoma, an expression pattern mirrored in the PC-3 cell line. In contrast, normal prostate epithelia expressed neither caveolin-1 nor cavin-1, while prostate stroma highly expressed both caveolin-1 and cavin-1. Utilizing PC-3 cells as a suitable model for caveolin-1-positive advanced prostate cancer, we found that cavin-1 expression in PC-3 cells inhibits anchorage-independent growth, and reduces in vivo tumor growth and metastasis in an orthotopic prostate cancer xenograft mouse model. The expression of α-smooth muscle actin in stroma along with interleukin-6 (IL-6) in cancer cells was also decreased in tumors of mice bearing PC-3-cavin-1 tumor cells. To determine whether cavin-1 acts by neutralizing caveolin-1, we expressed cavin-1 in caveolin-1-negative prostate cancer LNCaP and 22Rv1 cells. Caveolin-1 but not cavin-1 expression increased anchorage-independent growth in LNCaP and 22Rv1 cells. Cavin-1 co-expression reversed caveolin-1 effects in caveolin-1-positive LNCaP cells. Taken together, these results suggest that caveolin-1 in advanced prostate cancer is present outside of caveolae, because of the lack of cavin-1 expression. Cavin-1 expression attenuates the effects of non-caveolar caveolin-1 microdomains partly via reduced IL-6 microenvironmental function. With circulating caveolin-1 as a potential biomarker for advanced prostate cancer, identification of the molecular pathways affected by cavin-1 could provide novel therapeutic targets.
Which Ras rides the raft? - Reply
Publisher: Springer Science and Business Media LLC
Date: 08-2001
DOI: 10.1038/35087100
Co-Regulation of Cell Polarization and Migration by Caveolar Proteins PTRF/Cavin-1 and Caveolin-1
Publisher: Public Library of Science (PLoS)
Date: 13-08-2012
Caveolin interacts with the angiotensin II type I receptor during exocytic transport but not at the plasma membrane
Publisher: Elsevier BV
Date: 06-2003
Adaptor proteins MiD49 and MiD51 can act independently of Mff and Fis1 in Drp1 recruitment and are specific for mitochondrial fission
Publisher: Elsevier BV
Date: 09-2013
A role for oxysterol-binding protein-related protein 5 in endosomal cholesterol trafficking
Publisher: Rockefeller University Press
Date: 10-01-2011
Abstract: Oxysterol-binding protein (OSBP) and its related proteins (ORPs) constitute a large and evolutionarily conserved family of lipid-binding proteins that target organelle membranes to mediate sterol signaling and/or transport. Here we characterize ORP5, a tail-anchored ORP protein that localizes to the endoplasmic reticulum. Knocking down ORP5 causes cholesterol accumulation in late endosomes and lysosomes, which is reminiscent of the cholesterol trafficking defect in Niemann Pick C (NPC) fibroblasts. Cholesterol appears to accumulate in the limiting membranes of endosomal compartments in ORP5-depleted cells, whereas depletion of NPC1 or both ORP5 and NPC1 results in luminal accumulation of cholesterol. Moreover, trans-Golgi resident proteins mislocalize to endosomal compartments upon ORP5 depletion, which depends on a functional NPC1. Our results establish the first link between NPC1 and a cytoplasmic sterol carrier, and suggest that ORP5 may cooperate with NPC1 to mediate the exit of cholesterol from endosomes/lysosomes.
Rab17 Regulates Membrane Trafficking through Apical Recycling Endosomes in Polarized Epithelial Cells
Publisher: Rockefeller University Press
Date: 09-03-1998
Abstract: A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains while selectively allowing transport of proteins and lipids from one pole to the opposite by transcytosis. The small GTPase, rab17, a member of the rab family of regulators of intracellular transport, is specifically induced during cell polarization in the developing kidney. We here examined its intracellular distribution and function in both nonpolarized and polarized cells. By confocal immunofluorescence microscopy, rab17 colocalized with internalized transferrin in the perinuclear recycling endosome of BHK-21 cells. In polarized Eph4 cells, rab17 associated with the apical recycling endosome that has been implicated in recycling and transcytosis. The localization of rab17, therefore, strengthens the proposed homology between this compartment and the recycling endosome of nonpolarized cells. Basolateral to apical transport of two membrane-bound markers, the transferrin receptor and the FcLR 5-27 chimeric receptor, was specifically increased in Eph4 cells expressing rab17 mutants defective in either GTP binding or hydrolysis. Furthermore, the mutant proteins stimulated apical recycling of FcLR 5-27. These results support a role for rab17 in regulating traffic through the apical recycling endosome, suggesting a function in polarized sorting in epithelial cells.
Cavin4 interacts with Bin1 to promote T-tubule formation and stability in developing skeletal muscle
Publisher: Rockefeller University Press
Date: 11-10-2021
Abstract: The cavin proteins are essential for caveola biogenesis and function. Here, we identify a role for the muscle-specific component, Cavin4, in skeletal muscle T-tubule development by analyzing two vertebrate systems, mouse and zebrafish. In both models, Cavin4 localized to T-tubules, and loss of Cavin4 resulted in aberrant T-tubule maturation. In zebrafish, which possess duplicated cavin4 paralogs, Cavin4b was shown to directly interact with the T-tubule–associated BAR domain protein Bin1. Loss of both Cavin4a and Cavin4b caused aberrant accumulation of interconnected caveolae within the T-tubules, a fragmented T-tubule network enriched in Caveolin-3, and an impaired Ca2+ response upon mechanical stimulation. We propose a role for Cavin4 in remodeling the T-tubule membrane early in development by recycling caveolar components from the T-tubule to the sarcolemma. This generates a stable T-tubule domain lacking caveolae that is essential for T-tubule function.
The endocytic protein GRAF1 is directed to cell-matrix adhesion sites and regulates cell spreading
Publisher: American Society for Cell Biology (ASCB)
Date: 15-11-2011
Abstract: The rho GTPase-activating protein GTPase regulator associated with focal adhesion kinase-1 (GRAF1) remodels membranes into tubulovesicular clathrin-independent carriers (CLICs) mediating lipid-anchored receptor endocytosis. However, the cell biological functions of this highly prevalent endocytic pathway are unclear. In this article, we present biochemical and cell biological evidence that GRAF1 interacted with a network of endocytic and adhesion proteins and was found enriched at podosome-like adhesions and src-induced podosomes. We further demonstrate that these sites comprise microdomains of highly ordered lipid enriched in GRAF1 endocytic cargo. GRAF1 activity was upregulated in spreading cells and uptake via CLICs was concentrated at the leading edge of migrating cells. Depletion of GRAF1, which inhibits CLIC generation, resulted in profound defects in cell spreading and migration. We propose that GRAF1 remodels membrane microdomains at adhesion sites into endocytic carriers, facilitating membrane turnover during cell morphological changes.
Cavin family proteins and the assembly of caveolae
Publisher: The Company of Biologists
Date: 04-2015
DOI: 10.1242/JCS.167866
Abstract: Caveolae are an abundant feature of the plasma membrane in many cells. Until recently, they were generally considered to be membrane invaginations whose formation primarily driven by integral membrane proteins called caveolins. However, the past decade has seen the emergence of the cavin family of peripheral membrane proteins as essential coat components and regulators of caveola biogenesis. In this Commentary, we summarise recent data on the role of cavins in caveola formation, highlighting structural studies that provide new insights into cavin coat assembly. In mammals, there are four cavin family members that associate through homo- and hetero-oligomerisation to form distinct subcomplexes on caveolae, which can be released into the cell in response to stimuli. Studies from several labs have provided a better understanding of cavin stoichiometry and the molecular basis for their oligomerisation, as well as identifying interactions with membrane phospholipids that may be important for caveola function. We propose a model in which coincident, low-affinity electrostatically controlled protein–protein and protein–lipid interactions allow the formation of caveolae, generating a meta-stable structure that can respond to plasma membrane stress by release of cavins.
Macrophage ACE2 is necessary for SARS-CoV-2 replication and subsequent cytokine responses that restrict continued virion release
Publisher: American Association for the Advancement of Science (AAAS)
Date: 25-04-2023
DOI: 10.1126/SCISIGNAL.ABQ1366
Abstract: Macrophages are key cellular contributors to the pathogenesis of COVID-19, the disease caused by the virus SARS-CoV-2. The SARS-CoV-2 entry receptor ACE2 is present only on a subset of macrophages at sites of SARS-CoV-2 infection in humans. Here, we investigated whether SARS-CoV-2 can enter macrophages, replicate, and release new viral progeny whether macrophages need to sense a replicating virus to drive cytokine release and, if so, whether ACE2 is involved in these mechanisms. We found that SARS-CoV-2 could enter, but did not replicate within, ACE2-deficient human primary macrophages and did not induce proinflammatory cytokine expression. By contrast, ACE2 overexpression in human THP-1–derived macrophages permitted SARS-CoV-2 entry, processing and replication, and virion release. ACE2-overexpressing THP-1 macrophages sensed active viral replication and triggered proinflammatory, antiviral programs mediated by the kinase TBK-1 that limited prolonged viral replication and release. These findings help elucidate the role of ACE2 and its absence in macrophage responses to SARS-CoV-2 infection.
Structure-Based Reassessment of the Caveolin Signaling Model: Do Caveolae Regulate Signaling through Caveolin-Protein Interactions?
Publisher: Elsevier BV
Date: 07-2012
Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis
Publisher: Springer Science and Business Media LLC
Date: 07-10-2015
DOI: 10.1038/NATURE15695
Abstract: The human kidney contains up to 2 million epithelial nephrons responsible for blood filtration. Regenerating the kidney requires the induction of the more than 20 distinct cell types required for excretion and the regulation of pH, and electrolyte and fluid balance. We have previously described the simultaneous induction of progenitors for both collecting duct and nephrons via the directed differentiation of human pluripotent stem cells. Paradoxically, although both are of intermediate mesoderm in origin, collecting duct and nephrons have distinct temporospatial origins. Here we identify the developmental mechanism regulating the preferential induction of collecting duct versus kidney mesenchyme progenitors. Using this knowledge, we have generated kidney organoids that contain nephrons associated with a collecting duct network surrounded by renal interstitium and endothelial cells. Within these organoids, in idual nephrons segment into distal and proximal tubules, early loops of Henle, and glomeruli containing podocytes elaborating foot processes and undergoing vascularization. When transcription profiles of kidney organoids were compared to human fetal tissues, they showed highest congruence with first trimester human kidney. Furthermore, the proximal tubules endocytose dextran and differentially apoptose in response to cisplatin, a nephrotoxicant. Such kidney organoids represent powerful models of the human organ for future applications, including nephrotoxicity screening, disease modelling and as a source of cells for therapy.
Caveolin-1 Deficiency Leads to Increased Susceptibility to Cell Death and Fibrosis in White Adipose Tissue: Characterization of a Lipodystrophic Model
Publisher: Public Library of Science (PLoS)
Date: 26-09-2012
Cholesterol manipulation by West Nile virus perturbs the cellular immune response
Publisher: Elsevier BV
Date: 10-2007
DOI: 10.1016/J.CHOM.2007.09.003
Abstract: Complex membrane structures induced by West Nile virus (WNV), an enveloped RNA virus, are required for efficient viral replication. How these membranes are induced and how they facilitate the viral life cycle are unknown. We show that WNV modulates host cell cholesterol homeostasis by upregulating cholesterol biosynthesis and redistributing cholesterol to viral replication membranes. Manipulating cholesterol levels and altering concentrations of cellular geranylgeranylated proteins had a deleterious effect on virus replication. Depletion of the key cholesterol-synthesizing enzyme 3-hydroxy-methyglutaryl-CoA reductase drastically h ered virus replication. Significantly, virus-induced redistribution of cellular cholesterol downregulated the interferon-stimulated Jak-STAT antiviral signaling response to infection. This defect could be partially restored by exogenous addition of cholesterol, which increased the ability of infected cells to respond to interferon. We propose that, by manipulating cellular cholesterol, WNV utilizes the cellular response to cholesterol deficiency and dependence of antiviral signaling pathways on cholesterol-rich microdomains to facilitate viral replication and survival.
Phosphorylation of PKCδ by FER tips the balance from EGFR degradation to recycling
Publisher: Rockefeller University Press
Date: 07-01-2021
Abstract: Receptor degradation terminates signaling by activated receptor tyrosine kinases. Degradation of EGFR occurs in lysosomes and requires the switching of RAB5 for RAB7 on late endosomes to enable their fusion with the lysosome, but what controls this critical switching is poorly understood. We show that the tyrosine kinase FER alters PKCδ function by phosphorylating it on Y374, and that phospho-Y374-PKCδ prevents RAB5 release from nascent late endosomes, thereby inhibiting EGFR degradation and promoting the recycling of endosomal EGFR to the cell surface. The rapid association of phospho-Y374-PKCδ with EGFR-containing endosomes is diminished by PTPN14, which dephosphorylates phospho-Y374-PKCδ. In triple-negative breast cancer cells, the FER-dependent phosphorylation of PKCδ enhances EGFR signaling and promotes anchorage-independent cell growth. Importantly, increased Y374-PKCδ phosphorylation correlating with arrested late endosome maturation was identified in ∼25% of triple-negative breast cancer patients, suggesting that dysregulation of this pathway may contribute to their pathology.
Spatiotemporal Regulation of Early Lipolytic Signaling in Adipocytes
Publisher: Elsevier BV
Date: 11-2009
A novel form of ataxia oculomotor apraxia characterized by oxidative stress and apoptosis resistance
Publisher: Springer Science and Business Media LLC
Date: 09-03-2007
Abstract: Several different autosomal recessive genetic disorders characterized by ataxia with oculomotor apraxia (AOA) have been identified with the unifying feature of defective DNA damage recognition and/or repair. We describe here the characterization of a novel form of AOA showing increased sensitivity to agents that cause single-strand breaks (SSBs) in DNA but having no gross defect in the repair of these breaks. Evidence for the presence of residual SSBs in DNA was provided by dramatically increased levels of poly (ADP-ribose)polymerase (PARP-1) auto-poly (ADP-ribosyl)ation, the detection of increased levels of reactive oxygen/nitrogen species (ROS/RNS) and oxidative damage to DNA in the patient cells. There was also evidence for oxidative damage to proteins and lipids. Although these cells were hypersensitive to DNA damaging agents, the mode of death was not by apoptosis. These cells were also resistant to TRAIL-induced death. Consistent with these observations, failure to observe a decrease in mitochondrial membrane potential, reduced cytochrome c release and defective apoptosis-inducing factor translocation to the nucleus was observed. Apoptosis resistance and PARP-1 hyperactivation were overcome by incubating the patient's cells with antioxidants. These results provide evidence for a novel form of AOA characterized by sensitivity to DNA damaging agents, oxidative stress, PARP-1 hyperactivation but resistance to apoptosis.
Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells
Publisher: Rockefeller University Press
Date: 16-08-2010
Abstract: Although the importance of clathrin- and caveolin-independent endocytic pathways has recently emerged, key aspects of these routes remain unknown. Using quantitative ultrastructural approaches, we show that clathrin-independent carriers (CLICs) account for approximately three times the volume internalized by the clathrin-mediated endocytic pathway, forming the major pathway involved in uptake of fluid and bulk membrane in fibroblasts. Electron tomographic analysis of the 3D morphology of the earliest carriers shows that they are multidomain organelles that form a complex sorting station as they mature. Proteomic analysis provides direct links between CLICs, cellular adhesion turnover, and migration. Consistent with this, CLIC-mediated endocytosis of key cargo proteins, CD44 and Thy-1, is polarized at the leading edge of migrating fibroblasts, while transient ablation of CLICs impairs their ability to migrate. These studies provide the first quantitative ultrastructural analysis and molecular characterization of the major endocytic pathway in fibroblasts, a pathway that provides rapid membrane turnover at the leading edge of migrating cells.
Publisher Correction: Cargo-specific recruitment in clathrin- and dynamin-independent endocytosis
Publisher: Springer Science and Business Media LLC
Date: 29-08-2022
Parkinson Disease-linked Vps35 R524W Mutation Impairs the Endosomal Association of Retromer and Induces -Synuclein Aggregation
Publisher: Elsevier BV
Date: 08-2016
Cell-to-Cell Heterogeneity in Lipid Droplets Suggests a Mechanism to Reduce Lipotoxicity
Publisher: Elsevier BV
Date: 08-2013
High-Throughput Screening of Australian Marine Organism Extracts for Bioactive Molecules Affecting the Cellular Storage of Neutral Lipids
Publisher: Public Library of Science (PLoS)
Date: 08-08-2011
Sugar transporter Slc37a2 regulates bone metabolism in mice via a tubular lysosomal network in osteoclasts
Publisher: Springer Science and Business Media LLC
Date: 21-02-2023
DOI: 10.1038/S41467-023-36484-2
Abstract: Osteoclasts are giant bone-digesting cells that harbor specialized lysosome-related organelles termed secretory lysosomes (SLs). SLs store cathepsin K and serve as a membrane precursor to the ruffled border, the osteoclast’s ‘resorptive apparatus’. Yet, the molecular composition and spatiotemporal organization of SLs remains incompletely understood. Here, using organelle-resolution proteomics, we identify member a2 of the solute carrier 37 family (Slc37a2) as a SL sugar transporter. We demonstrate in mice that Slc37a2 localizes to the SL limiting membrane and that these organelles adopt a hitherto unnoticed but dynamic tubular network in living osteoclasts that is required for bone digestion. Accordingly, mice lacking Slc37a2 accrue high bone mass owing to uncoupled bone metabolism and disturbances in SL export of monosaccharide sugars, a prerequisite for SL delivery to the bone-lining osteoclast plasma membrane. Thus, Slc37a2 is a physiological component of the osteoclast’s unique secretory organelle and a potential therapeutic target for metabolic bone diseases.
Author response: A robust method for particulate detection of a genetic tag for 3D electron microscopy
Publisher: eLife Sciences Publications, Ltd
Date: 25-04-0100
Adherens Junctions Revisualized: Organizing Cadherins as Nanoassemblies
Publisher: Elsevier BV
Date: 10-2015
DOI: 10.1016/J.DEVCEL.2015.09.012
Abstract: This Perspective considers how classical cadherin cell-cell adhesion receptors are organized at the nanoscale to generate lateral clusters. Recent advances in optical microscopy reveal that clustering constitutes a general feature of cadherin organization, but one that takes erse forms. Here we consider the molecular mechanisms responsible for cadherin clustering and their functional implications. We frame our discussion in light of what is known about how nanoscale organization is conferred upon the plasma membrane, through protein-protein interactions, regulation of the cortical actin cytoskeleton, and the lipid environment of the membrane.
Caveolae respond to acute oxidative stress through membrane lipid peroxidation, cytosolic release of CAVIN1, and downstream regulation of NRF2
Publisher: Cold Spring Harbor Laboratory
Date: 09-06-2021
DOI: 10.1101/2021.06.09.447684
Abstract: Caveolae have been linked to many biological functions, but their precise roles are unclear. Using quantitative whole cell proteomics of genome-edited cells, we show that the oxidative stress response is the major pathway dysregulated in cells lacking the key caveola structural protein, CAVIN1. CAVIN1 deletion compromised sensitivity to oxidative stress in cultured cells and in animals. Wound-induced accumulation of reactive oxygen species and apoptosis were suppressed in Cavin1-null zebrafish, negatively affecting regeneration. Oxidative stress triggered lipid peroxidation and induced caveolar disassembly. The resulting release of CAVIN1 from caveolae allowed direct interaction between CAVIN1 and NRF2, a key regulator of the antioxidant response, facilitating NRF2 degradation. CAVIN1-null cells with impaired negative regulation of NRF2 showed resistance to lipid peroxidation-induced ferroptosis. Thus, caveolae, via lipid peroxidation and CAVIN1 release, maintain cellular susceptibility to oxidative stress-induced cell death demonstrating a crucial role for this enigmatic organelle in cellular homeostasis and wound response.
Regulation of albumin endocytosis by PSD95/Dlg/ZO-1 (PDZ) scaffolds. Interaction of Na+-H+ exchange regulatory factor-2 with ClC-5.
Publisher: Elsevier BV
Date: 06-2006
Cortactin Scaffolds Arp2/3 and WAVE2 at the Epithelial Zonula Adherens
Publisher: Elsevier BV
Date: 03-2014
SEIPIN Regulates Lipid Droplet Expansion and Adipocyte Development by Modulating the Activity of Glycerol-3-phosphate Acyltransferase
Publisher: Elsevier BV
Date: 11-2016
Dynamin2 functions as an accessory protein to reduce the rate of caveola internalization
Publisher: Rockefeller University Press
Date: 02-02-2023
Abstract: Caveolae are small membrane invaginations that generally are stably attached to the plasma membrane. Their release is believed to depend on the GTPase dynamin 2 (Dyn2), in analogy with its role in fission of clathrin-coated vesicles. The mechanistic understanding of caveola fission is, however, sparse. Here, we used microscopy-based tracking of in idual caveolae in living cells to determine the role of Dyn2 in caveola dynamics. We report that Dyn2 stably associated with the bulb of a subset of caveolae, but was not required for formation or fission of caveolae. Dyn2-positive caveolae displayed longer plasma membrane duration times, whereas depletion of Dyn2 resulted in shorter duration times and increased caveola fission. The stabilizing role of Dyn2 was independent of its GTPase activity and the caveola stabilizing protein EHD2. Thus, we propose that, in contrast to the current view, Dyn2 is not a core component of the caveolae machinery, but rather functions as an accessory protein that restrains caveola internalization.
Related Organisations
University Of Leicester
Location: United Kingdom of Great Britain and Northern Ireland
The University Of Edinburgh
Location: United Kingdom of Great Britain and Northern Ireland
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