Jennifer Stow

The t-SNARE syntaxin 4 is regulated during macrophage activation to function in membrane traffic and cytokine secretion

Publisher: Elsevier BV

Date: 2003

DOI: 10.1016/S0960-9822(03)00006-X

Abstract: Activation of macrophages with lipopolysaccharide (LPS) induces the rapid synthesis and secretion of proinflammatory cytokines, such as tumor necrosis factor (TNFalpha), for priming the immune response. TNFalpha plays a key role in inflammatory disease yet, little is known of the intracellular trafficking events leading to its secretion. In order to identify molecules involved in this secretory pathway, we asked whether any of the known trafficking proteins are regulated by LPS. We found that the levels of SNARE proteins were rapidly and significantly up- or downregulated during macrophage activation. A subset of t-SNAREs (Syntaxin 4/SNAP23/Munc18c) known to control regulated exocytosis in other cell types was substantially increased by LPS in a temporal pattern coinciding with peak TNFalpha secretion. Syntaxin 4 formed a complex with Munc18c at the cell surface of macrophages. Functional studies involving the introduction of Syntaxin 4 cDNA or peptides into macrophages implicate this t-SNARE in a rate-limiting step of TNFalpha secretion and in membrane ruffling during macrophage activation. We conclude that, in macrophages, SNAREs are regulated in order to accommodate the rapid onset of cytokine secretion and for membrane traffic associated with the phenotypic changes of immune activation. This represents a novel regulatory role for SNAREs in regulated secretion and in macrophage-mediated host defense.

Salmonella effector SopD2 interferes with Rab34 function

Publisher: Wiley

Date: 27-02-2017

DOI: 10.1002/CBIN.10739

Abstract: Many intracellular pathogens have evolved highly specialized mechanisms to isolate themselves from the host cell's innate immune response while still obtaining the necessary nutrients to survive. Salmonella utilizes type 3 secretion systems (T3SSs) to deliver bacterial proteins called effectors, across the encompassing Salmonella Containing vacuole (SCV) membrane, to subvert the host's membrane trafficking pathways and alter other cellular processes. The Salmonella Pathogenicity Island (SPI)-2 effector SopD2 has recently been demonstrated to modulate multiple members of the Rab GTPase family such as Rab7, Rab8, Rab10, and Rab32 (D'Costa et al., , Cell Reports, 12:1508-18 Spano et al., , Cell Host & Microbe, 19:216-26). Here, we demonstrate the additional capacity of SopD2 to bind Rab34 and modulate its function. Our data indicate that depletion of Rab34 delays maturation of the SCV, and consequently, inhibits intracellular Salmonella enterica serotype typhimurium (S. typhimurium) growth. Interestingly, intracellular growth of the S. typhimurium lacking SopD2 was severely impaired in Rab34-depleted cells, suggesting a compounding virulence effect. Overall this study reveals an additional member of the Rab GTPase family, Rab34, that is modulated by SopD2 and provides insight into its role in Salmonella biology.

RAB27A promotes melanoma cell invasion and metastasis via regulation of pro‐invasive exosomes

Publisher: Wiley

Date: 03-01-2019

DOI: 10.1002/IJC.32064

Abstract: Despite recent advances in targeted and immune-based therapies, advanced stage melanoma remains a clinical challenge with a poor prognosis. Understanding the genes and cellular processes that drive progression and metastasis is critical for identifying new therapeutic strategies. Here, we found that the GTPase RAB27A was overexpressed in a subset of melanomas, which correlated with poor patient survival. Loss of RAB27A expression in melanoma cell lines inhibited 3D spheroid invasion and cell motility in vitro, and spontaneous metastasis in vivo. The reduced invasion phenotype was rescued by RAB27A-replete exosomes, but not RAB27A-knockdown exosomes, indicating that RAB27A is responsible for the generation of pro-invasive exosomes. Furthermore, while RAB27A loss did not alter the number of exosomes secreted, it did change exosome size and altered the composition and abundance of exosomal proteins, some of which are known to regulate cancer cell movement. Our data suggest that RAB27A promotes the biogenesis of a distinct pro-invasive exosome population. These findings support RAB27A as a key cancer regulator, as well as a potential prognostic marker and therapeutic target in melanoma.

Abstract B4: Tumor-educated CD11c+/CD11bint/Gr-1- regulatory dendritic cells show a mutated pattern of trafficking molecules implicated in cytokine secretion.

Publisher: American Association for Cancer Research (AACR)

Date: 2013

DOI: 10.1158/1538-7445.TUMIMM2012-B4

Abstract: Dendritic cells (DCs) play a pivotal role in regulating innate and adaptive immunity and are key elements in the immunological response against tumors. Several specialized features make DCs uniquely efficient in priming anti-cancer T cell responses. DCs possess specialized pathways to acquire tumor antigens from apoptotic and necrotic cells and to present them to tumor specific T cells. Efficient T cell activation depends on the density of T cell receptors (TCR) ligands and co-stimulatory molecules on the cell surface, and on secretion of cytokines that determine T cell differentiation. It is well documented that DCs properties are suppressed by the tumor microenvironment and many evidences suggest that tolerogenic DCs may be a crucial element in the development of tumor-mediated immune anergy. The tumor microenvironment suppresses DCs functions by targeting different steps in the antigen presentation process, such as phagocytosis, antigen processing and presentation. Suppression also causes an important skewing in the pattern of secreted cytokines. In particular, how secretion of inflammatory cytokines becomes impaired in tumor-exposed DCs remains unclear. We have recently undergone a study to identify the trafficking proteins SNAREs (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) implicated in secretion of cytokines in DCs and to examine whether they are modified by the tumor microenvironment. Using imaging and functional assays we identified the SNARE VAMP7 as a specific mediator of the secretion of interleukine-12 (IL-12), the major inflammatory cytokine implicated in T cell priming. Interestingly, depletion of VAMP7 does not affect secretion of the inhibitory cytokine IL-10, indicating that distinct secretory pathways direct the release of mediators with opposite cellular functions. We next analyzed lung DCs in healthy mice and in mice bearing Lewis lung carcinoma (LLC). We observed a consistent tumor-induced differentiation of the subpopulation CD11chigh/CD11b-/Gr-1- into a CD11bint phenotype, which was also confirmed in vitro by co-culturing conventional DCs on LLC monolayer. Both in vitro and in vivo, these cells show a normal maturation capacity and expression of surface co-stimulatory molecules, but a strong impairment in inflammatory cytokines secretion, in particular IL-12, upon restimulation with TLR agonists. We isolated CD11chigh/CD11b-/int/Gr-1- cells to high purity to analyze the expression of trafficking proteins. We found that tumor-exposed DCs downregulate, both at mRNA and protein level, the SNAREs VAMP7 and VAMP3, and Rab27. In addition, VAMP7 in tumor-educated DCs displays a different molecular weight suggesting post-transcriptional modification or expression of a different isoform. These preliminary observations suggest that regulation/modification of trafficking proteins in DCs may represent a novel pathway targeted during suppression. Ongoing studies aim at extending the analysis to other trafficking proteins potentially involved in modulation of cytokine secretion. Citation Format: Giulia Chiaruttini, Irene Soncin, Jennifer L. Stow, Andrew A. Peden, Federica Benvenuti. Tumor-educated CD11c+/CD11bint/Gr-1- regulatory dendritic cells show a mutated pattern of trafficking molecules implicated in cytokine secretion. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances Dec 2-5, 2012 Miami, FL. Philadelphia (PA): AACR Cancer Res 2013 (1 Suppl):Abstract nr B4.

Acute epithelial injury in the rat small intestine in vivo is associated with expanded expression of transforming growth factor alpha and beta

Publisher: BMJ

Date: 05-1996

DOI: 10.1136/GUT.38.5.687

Abstract: Previous studies have shown the importance of transforming growth factors alpha and beta (TGF alpha and TGF beta) in modulating epithelial cell restitution after injury in vitro. To investigate the role of the growth factors TGF alpha and TGF beta after acute epithelial injury in vivo. An in vivo model of phytohaemagglutinin (PHA) induced acute epithelial injury in rat small intestine was used. Epithelial cell turnover was assessed by autoradiography and liquid scintillation counting of thymidine uptake. Expression of TGF alpha and TGF beta was assessed by immunohistochemistry. An expansion of the proliferative compartment and increased turnover of intestinal epithelial cells was seen in rats with PHA induced intestinal epithelial injury. Expression of TGF alpha and TGF beta peptides was shown in both the epithelial cell and lamina propria compartment. Different patterns of TGF alpha and TGF beta expression were seen, however, within the epithelium of rats with acute intestinal injury compared with untreated controls, while the expression of these peptides within the lamina propria was not changed. These findings suggest that acute intestinal epithelial cell injury in vivo is associated with compensatory changes in expression of TGF alpha and TGF beta in the epithelial cell compartment, while the lamina propria does not seem to be significantly affected.

On linear models and parameter identifiability in experimental biological systems

Publisher: Elsevier BV

Date: 10-2014

DOI: 10.1016/J.JTBI.2014.05.028

Abstract: A key problem in the biological sciences is to be able to reliably estimate model parameters from experimental data. This is the well-known problem of parameter identifiability. Here, methods are developed for biologists and other modelers to design optimal experiments to ensure parameter identifiability at a structural level. The main results of the paper are to provide a general methodology for extracting parameters of linear models from an experimentally measured scalar function - the transfer function - and a framework for the identifiability analysis of complex model structures using linked models. Linked models are composed by letting the output of one model become the input to another model which is then experimentally measured. The linked model framework is shown to be applicable to designing experiments to identify the measured sub-model and recover the input from the unmeasured sub-model, even in cases that the unmeasured sub-model is not identifiable. Applications for a set of common model features are demonstrated, and the results combined in an ex le application to a real-world experimental system. These applications emphasize the insight into answering "where to measure" and "which experimental scheme" questions provided by both the parameter extraction methodology and the linked model framework. The aim is to demonstrate the tools' usefulness in guiding experimental design to maximize parameter information obtained, based on the model structure.

Heterologous expression of delta F508 CFTR results in decreased sialylation of membrane glycoconjugates

Publisher: American Physiological Society

Date: 02-1994

DOI: 10.1152/AJPCELL.1994.266.2.C360

Abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) is commonly mutated in cystic fibrosis to the delta F508 CFTR. CFTR has been shown to function as a adenosine 3',5'-cyclic monophosphate-dependent Cl- channel at the cell surface, and there is evidence to suggest that CFTR may also have a role in transmembrane Cl- conductance in intracellular membrane compartments. Studies using cells from cystic fibrosis patients or heterologous expression systems have demonstrated that defective Cl- conductance at the cell surface and defective acidification of the Golgi compartment are associated with the presence of mutant forms of CFTR. It is possible that mutation of CFTR could also result in altered Golgi function, consistent with reports of changes in the glycosylation of cell surface and secreted glycoproteins in cystic fibrosis. Glycosylation of cell surface glycoproteins, particularly levels of sialylation, may also be related to the increased binding of Pseudomonas to cystic fibrosis cells. The current study was undertaken to compare the sialylation of cell surface glycoconjugates in heterologous cells overexpressing normal CFTR or delta F508 CFTR. The presence of sialylated residues on cells was assessed by the surface binding of the specific lectins, wheat germ agglutinin and elderberry bark lectin. A fluorescent cholera toxin B subunit probe was used to measure surface binding to sialylated gangliosides in transfected cells. Our studies show that cells lacking CFTR and cells expressing normal CFTR have unaltered levels of sialylation. In contrast, cells expressing the delta F508 CFTR have significantly decreased amounts of sialylated glycoproteins and gangliosides on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

LLAMA: a robust and scalable machine learning pipeline for analysis of cell surface projections in large scale 4D microscopy data

Publisher: Cold Spring Harbor Laboratory

Date: 11-12-2020

DOI: 10.1101/2020.12.10.420414

Abstract: We present LLAMA, a pipeline for systematic analysis of terabyte scale 4D microscopy datasets. Analysis of in idual biological structures in imaging at this scale requires efficient and robust methods which do not require human micromanagement or editing of outputs. To meet this challenge, we use a machine learning method for semantic segmentation, followed by a robust and configurable object separation and tracking algorithm, and the generation of detailed object level statistics. Advanced visualisation is a key element of LLAMA: we provide a specialised software tool which supports quality control and optimisation as well as visualisation of outputs. LLAMA was used in a quantitative analysis of macrophage surface membrane projections (filopodia, ruffles, tent-pole ruffles) examining the differential effects of two interventions: lipopolysaccharide (LPS) and macrophage colony stimulating factor (CSF-1). Distinct patterns of increased activity were identified. In addition, a continuity of behaviour was found between tent pole ruffling and wave-like ruffling, further defining the role of filopodia in ruffling.

Role of myristoylation in membrane attachment and function of G alpha i-3 on Golgi membranes

Publisher: American Physiological Society

Date: 05-1996

DOI: 10.1152/AJPCELL.1996.270.5.C1362

Abstract: Heterotrimeric G protein alpha-subunits localized on the cytoplasmic face of Golgi membranes are involved in regulating vesicle trafficking and protein secretion. We investigated the role of myristoylation in attachment of the G alpha i-3 subunit to Golgi membranes. G alpha i-3 was epitope-tagged by insertion of a FLAG sequence at an NH2-terminal site predicted to interfere with myristoylation, and the resulting NT-alpha i-3 construct was stably transfected and expressed in polarized epithelial LLC-PK1 cells. Metabolic labeling confirmed that the translation product of NT-alpha i-3 was not myristoylated. In contrast to endogenous G alpha 1-3, which is tightly bound to Golgi membranes, the unmyristoylated FLAG-tagged NT-alpha i-3 did not attach to membranes it was localized by immunofluorescence in the cytoplasm of LLC-PK1 cells and was detected only in the cytosol fraction of cell homogenates. Pertussis toxin-dependent ADP-ribosylation was used to test the ability of NT-alpha i-3 to interact with membrane-bound beta gamma-subunits. In both in vitro and in vivo assays, cytosolic NT-alpha i-3 alone was not ADP-ribosylated, although in the presence of membranes it could interact with G beta gamma-subunits to form heterotrimers. The expression of NT-alpha i-3 in LLC-PK1 cells altered the rate of basolateral secretion of sulfated proteoglycans, consistent with the demonstrated function of endogenous G alpha i-3. These data are consistent with a model in which G alpha i-3 utilizes NH2-terminal myristoylation to bind to Golgi membranes and to maximize its interaction with G beta gamma-subunits. Furthermore, our results show that stable attachment of G alpha i-3 to Golgi membranes is not required for it to participate as a regulatory element in vesicle trafficking in the secretory pathway.

Automated organelle‐based colocalization in whole‐cell imaging

Publisher: Wiley

Date: 10-09-2009

DOI: 10.1002/CYTO.A.20786

Abstract: The use of fluorescence microscopy to investigate protein colocalization is an invaluable tool for understanding subcellular structures and their associated proteins. However, current techniques are largely limited to two-dimensional (2D) imaging and often require manual segmentation. Here, we present OBCOL, a methodology to automatically segment and quantify protein colocalization not within an image as a whole but on all in idual punctuate organelles within a 3D multichannel image. A wide variety of colocalization statistics may then be calculated on the objects found, and features reported for each such as position, degree of overlap between channels, and number of component objects. OBCOL was validated on imaging of two fluorescent markers (Dextran, EGF) in 3D microscopy imaging. OBCOL's application was then exemplified by investigating the colocalization of three fluorescently tagged proteins (VAMP3, Rab11, and transferrin) on recycling endosomes in mammalian cells. The methodology showed for the first time the ersity of endosomes labeled with one or more of these proteins and quantitatively demonstrated the degree of overlap among these proteins in in idual recycling endosomes. The consistent segregation of these markers provides novel evidence for the subcompartmentalization of recycling endosomes. OBCOL is a flexible methodology for 3D multifluorophore image analysis. This study clearly demonstrated its value for investigating subcellular structures and their constituent proteins.

Macropinosome formation by tent pole ruffling in macrophages

Publisher: Rockefeller University Press

Date: 27-08-2018

DOI: 10.1083/JCB.201804137

Abstract: Pathogen-mediated activation of macrophages arms innate immune responses that include enhanced surface ruffling and macropinocytosis for environmental s ling and receptor internalization and signaling. Activation of macrophages with bacterial lipopolysaccharide (LPS) generates prominent dorsal ruffles, which are precursors for macropinosomes. Very rapid, high-resolution imaging of live macrophages with lattice light sheet microscopy (LLSM) reveals new features and actions of dorsal ruffles, which redefine the process of macropinosome formation and closure. We offer a new model in which ruffles are erected and supported by F-actin tent poles that cross over and twist to constrict the forming macropinosomes. This process allows for formation of large macropinosomes induced by LPS. We further describe the enrichment of active Rab13 on tent pole ruffles and show that CRISPR deletion of Rab13 results in aberrant tent pole ruffles and blocks the formation of large LPS-induced macropinosomes. Based on the exquisite temporal and spatial resolution of LLSM, we can redefine the ruffling and macropinosome processes that underpin innate immune responses.

Characterization of E-cadherin Endocytosis in Isolated MCF-7 and Chinese Hamster Ovary Cells

Publisher: Elsevier BV

Date: 06-2003

DOI: 10.1074/JBC.M300082200

Identification of a 200-kD, brefeldin-sensitive protein on Golgi membranes

Publisher: Rockefeller University Press

Date: 04-1992

DOI: 10.1083/JCB.117.1.27

Abstract: A mAb AD7, raised against canine liver Golgi membranes, recognizes a novel, 200-kD protein (p200) which is found in a wide variety of cultured cell lines. Immunofluorescence staining of cultured cells with the AD7 antibody produced intense staining of p200 in the juxtanuclear Golgi complex and more diffuse staining of p200 in the cytoplasm. The p200 protein in the Golgi complex was colocalized with other Golgi proteins, including mannosidase II and beta-COP, a coatomer protein. Localization of p200 by immunoperoxidase staining at the electron microscopic level revealed concentrations of p200 at the dilated rims of Golgi cisternae. Biochemical studies showed that p200 is a peripheral membrane protein which partitions to the aqueous phase of Triton X-114 solutions and is phosphorylated. The p200 protein is located on the cytoplasmic face of membranes, since it was accessible to trypsin digestion in microsomal preparations, and is recovered in approximately equal amounts in membrane pellets and in the cytosol of homogenized cells. Immunofluorescence staining of normal rat kidney cells exposed to the toxin brefeldin A (BFA), showed that there was very rapid redistribution of p200, which was dissociated from Golgi membranes in the presence of this drug. The effect of BFA was reversible, since upon removal of the toxin, AD7 rapidly reassociated with the Golgi complex. In the BFA-resistant cell line PtK1, BFA failed to cause redistribution of p200 from Golgi membranes. Taken together, these results indicate that the p200 Golgi membrane-associated protein has many properties in common with the coatomer protein, beta-COP.

PI3Kdelta inhibition reduces TNF secretion and neuroinflammation in a mouse cerebral stroke model

Publisher: Elsevier BV

Date: 11-2014

DOI: 10.1016/J.CYTO.2014.07.126

Targeting of a Tropomyosin Isoform to Short Microfilaments Associated with the Golgi Complex

Publisher: American Society for Cell Biology (ASCB)

Date: 2004

DOI: 10.1091/MBC.E03-03-0176

Abstract: A growing body of evidence suggests that the Golgi complex contains an actin-based filament system. We have previously reported that one or more isoforms from the tropomyosin gene Tm5NM (also known as γ-Tm), but not from either the α- or β-Tm genes, are associated with Golgi-derived vesicles (Heimann et al., ( 1999 ). J. Biol. Chem. 274, 10743-10750). We now show that Tm5NM-2 is sorted specifically to the Golgi complex, whereas Tm5NM-1, which differs by a single alternatively spliced internal exon, is incorporated into stress fibers. Tm5NM-2 is localized to the Golgi complex consistently throughout the G1 phase of the cell cycle and it associates with Golgi membranes in a brefeldin A-sensitive and cytochalasin D-resistant manner. An actin antibody, which preferentially reacts with the ends of microfilaments, newly reveals a population of short actin filaments associated with the Golgi complex and particularly with Golgi-derived vesicles. Tm5NM-2 is also found on these short microfilaments. We conclude that an alternative splice choice can restrict the sorting of a tropomyosin isoform to short actin filaments associated with Golgi-derived vesicles. Our evidence points to a role for these Golgi-associated microfilaments in vesicle budding at the level of the Golgi complex.

Guanine nucleotide binding proteins regulate epithelial Na+ channels

Publisher: Springer US

Date: 1991

DOI: 10.1007/978-1-4684-5934-0_31

Neurotoxic peptides from the venom of the giant Australian stinging tree

Publisher: American Association for the Advancement of Science (AAAS)

Date: 18-09-2020

DOI: 10.1126/SCIADV.ABB8828

Abstract: The pain-inducing components of Australian stinging tree venom are miniproteins that modulate voltage-gated sodium channels.

High-throughput quantification of early stages of phagocytosis

Publisher: Future Science Ltd

Date: 09-2013

DOI: 10.2144/000114075

Abstract: Phagocytosis—the engulfment of cells and foreign bodies—is an important cellular process in innate immunity, development, and disease. Quantification of various stages of phagocytosis, especially in a rapid screening fashion, is an invaluable tool for elucidating protein function during this process. However, current methods for assessing phagocytosis are largely limited to flow cytometry and manual image-based assays, providing limited information. Here, we present an image-based, semi-automated phagocytosis assay to rapidly quantitate three distinct stages during the early engulfment of opsonized beads. Captured images are analyzed using the image-processing software ImageJ and quantified using a macro. Modifications to this method allowed quantification of phagocytosis only in fluorescently labeled transfected cells. Additionally, the time course of bead internalization could be measured using this approach. The assay could discriminate perturbations to stages of phagocytosis induced by known pharmacological inhibitors of filamentous actin and phosphoinositol-3-kinase. Our methodology offers the ability to automatically categorize large amounts of image data into the three early stages of phagocytosis within minutes, clearly demonstrating its potential value in investigating aberrant phagocytosis when manipulating proteins of interest in drug screens and disease.

Receptor function, dominant negative activity and phenotype correlations for MC1R variant alleles (vol 16, pg 2249, 2007)

Publisher: Oxford University Press (OUP)

Date: 31-07-2007

DOI: 10.1093/HMG/DDM268

Disruption of Rorα1 and cholesterol 25-hydroxylase expression attenuates phagocytosis in male Rorα^sg/sg mice

Publisher: The Endocrine Society

Date: 2013

DOI: 10.1210/EN.2012-1889

Abstract: We and others have previously demonstrated that congenital deficiency of the nuclear hormone receptor, Rorα1, in staggerer (sg/sg) mice results in resistance to diet-induced obesity and increased insulin sensitivity. Paradoxically, the sg/sg mice are susceptible to atherosclerosis and display impaired innate immunity, underscoring the regulatory links between metabolic disease, inflammation, and susceptibility to infection. Here, we present novel evidence that Rorα1 regulates innate immune function by demonstrating impaired phagocytosis in sg/sg mice. The early stages of Fc-γ receptor-mediated phagocytosis in lipopolysaccharide-activated sg/sg bone marrow-derived macrophages (BMMs) were significantly impaired compared with wild-type cells. Moreover, in sg/sg BMMs, the phagocytic cup membranes had reduced levels of cholesterol. Expression profiling revealed dysregulated expression of genes involved in inflammation and lipid metabolism in sg/sg BMMs. Notably, we identified decreased expression of the mRNA encoding cholesterol 25-hydroxylase (Ch25h), an enzyme that converts cholesterol to 25-hydroxycholesterol (25HC), an oxysterol with emerging roles in immunity. Treatment of sg/sg BMMs with 25HC rescued phagocytosis in a dose-dependent manner, whereas small interfering RNA knockdown of Ch25h mRNA expression in wild-type cells attenuated phagocytosis. Hence, we propose that 25HC is essential for optimizing membrane internalization during phagocytosis and that aberrant Ch25h expression in Rorα1-deficient sg/sg macrophages disrupts phagocytosis. Our studies reveal new roles for Rorα1, Ch25h, and 25HC in phagocytosis. Aberrant 25HC underpins the paradoxical association between insulin sensitivity and impaired innate immunity in Rorα1-deficient mice, heralding a wider and essential role for this oxysterol at the nexus of metabolism and immunity.

Localization and post-Golgi trafficking of tumor necrosis factor-α in macrophages

Publisher: Mary Ann Liebert Inc

Date: 04-2000

DOI: 10.1089/107999000312379

Abstract: Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine secreted by activated macrophages. In this study, we examined the intracellular distribution and trafficking of TNF-alpha. Immunofluorescence and immunogold localization demonstrated that in lipopolysaccharide (LPS)-stimulated RAW264 macrophages, the greatest concentration of TNF-alpha is found in the perinuclear Golgi complex. Staining of the Golgi complex appeared 20 min after activation of cells and persisted for 2-12 h, and TNF-alpha appeared on the cell surface only transiently during this time. The rate of disappearance of Golgi staining correlated with the release of the cleaved, mature TNF-alpha into the medium. Pulse chase labeling and subcellular fractionation studies indicated that both 26-kDa and 17-kDa forms of TNF-alpha may be present at the level of the Golgi complex. Post-Golgi trafficking of TNF-alpha was modulated by agents that disrupt the cytoskeleton. Interferon-gamma (IFN-gamma), which primes macrophages for TNF-alpha-dependent cellular cytotoxicity, potentiated the effect of LPS by sustaining enhanced intracellular pools of TNF-alpha and also promoted redistribution of TNF-alpha into post-Golgi vesicular compartments. We propose that the primary pool of biologically active TNF-alpha in activated macrophages is held in the Golgi complex and that the cytokine is recruited directly from this intracellular pool for release in response to tumor cells or pathogens.

Specific isoforms of actin-binding proteins on distinct populations of Golgi-derived vesicles

Publisher: Elsevier BV

Date: 04-1999

DOI: 10.1074/JBC.274.16.10743

Inhibition of the PtdIns(5) kinase PIKfyve disrupts intracellular replication of Salmonella

Publisher: Wiley

Date: 18-03-2010

DOI: 10.1038/EMBOJ.2010.28

Elongator mutation in mice induces neurodegeneration and ataxia-like behavior

Publisher: Springer Science and Business Media LLC

Date: 10-08-2018

DOI: 10.1038/S41467-018-05765-6

Abstract: Cerebellar ataxias are severe neurodegenerative disorders with an early onset and progressive and inexorable course of the disease. Here, we report a single point mutation in the gene encoding Elongator complex subunit 6 causing Purkinje neuron degeneration and an ataxia-like phenotype in the mutant wobbly mouse. This mutation destabilizes the complex and compromises its function in translation regulation, leading to protein misfolding, proteotoxic stress, and eventual neuronal death. In addition, we show that substantial microgliosis is triggered by the NLRP3 inflammasome pathway in the cerebellum and that blocking NLRP3 function in vivo significantly delays neuronal degeneration and the onset of ataxia in mutant animals. Our data provide a mechanistic insight into the pathophysiology of a cerebellar ataxia caused by an Elongator mutation, substantiating the increasing body of evidence that alterations of this complex are broadly implicated in the onset of a number of erse neurological disorders.

E-cadherin transport from the trans-Golgi network in tubulovesicular carriers is selectively regulated by golgin-97.

Publisher: Wiley

Date: 28-09-2005

DOI: 10.1111/J.1600-0854.2005.00349.X

Abstract: E-cadherin is a cell-cell adhesion protein that is trafficked and delivered to the basolateral cell surface. Membrane-bound carriers for the post-Golgi exocytosis of E-cadherin have not been characterized. Green fluorescent protein (GFP)-tagged E-cadherin (Ecad-GFP) is transported from the trans-Golgi network (TGN) to the recycling endosome on its way to the cell surface in tubulovesicular carriers that resemble TGN tubules labeled by members of the golgin family of tethering proteins. Here, we examine the association of golgins with tubular carriers containing E-cadherin as cargo. Fluorescent GRIP domains from golgin proteins replicate the membrane binding of the full-length proteins and were coexpressed with Ecad-GFP. The GRIP domains of p230/golgin-245 and golgin-97 had overlapping but nonidentical distributions on the TGN both domains were on TGN-derived tubules but only the golgin-97 GRIP domain coincided with Ecad-GFP tubules in live cells. When the Arl1-binding endogenous golgins, p230/golgin-245 and golgin-97 were displaced from Golgi membranes by overexpression of the p230 GRIP domain, trafficking of Ecad-GFP was inhibited. siRNA knockdown of golgin-97 also inhibited trafficking of Ecad-GFP. Thus, the GRIP domains of p230/golgin-245 and golgin-97 bind discriminately to distinct membrane subdomains of the TGN. Golgin-97 is identified as a selective and essential component of the tubulovesicular carriers transporting E-cadherin out of the TGN.

GRIP Domain-mediated Targeting of Two New Coiled-coil Proteins, GCC88 and GCC185, to Subcompartments of the trans-Golgi Network

Publisher: Elsevier BV

Date: 02-2003

DOI: 10.1074/JBC.M210387200

The Recycling Endosome Protein Rab17 Regulates Melanocytic Filopodia Formation and Melanosome Trafficking

Publisher: Wiley

Date: 25-02-2011

DOI: 10.1111/J.1600-0854.2011.01172.X

Abstract: Rab GTPases including Rab27a, Rab38 and Rab32 function in melanosome maturation or trafficking in melanocytes. A screen to identify additional Rabs involved in these processes revealed the localization of GFP-Rab17 on recycling endosomes (REs) and melanosomes in melanocytic cells. Rab17 mRNA expression is regulated by microphthalmia transcription factor (MITF), a characteristic of known pigmentation genes. Rab17 siRNA knockdown in melanoma cells quantitatively increased melanosome concentration at the cell periphery. Rab17 knockdown did not inhibit melanosome maturation nor movement, but it caused accumulation of melanin inside cells. Double knockdown of Rab17 and Rab27a indicated that Rab17 acts on melanosomes downstream of Rab27a. Filopodia are known to play a role in melanosome transfer, and in Rab17 knockdown cells filopodia formation was inhibited. Furthermore, we show that stimulation of melanoma cells with α-melanocyte-stimulating hormone induces filopodia formation, supporting a role for filopodia in melanosome release. Cell stimulation also caused redistribution of REs to the periphery, and knockdown of additional RE-associated Rabs 11a and 11b produced a similar accumulation of melanosomes and melanin to that seen after loss of Rab17. Our findings reveal new functions for RE and Rab17 in pigmentation through a distal step in the process of melanosome release via filopodia.

Localization of human heparan glucosaminyl N-deacetylase/N-sulphotransferase to the trans-Golgi network

Publisher: Portland Press Ltd.

Date: 15-07-1997

DOI: 10.1042/BJ3250351

Abstract: In order to determine the intracellular location of heparan N-deacetylase/N-sulphotransferase, cDNAs encoding human heparan glucosaminyl N-deacetylase/N-sulphotransferase were cloned from human umbilical vein endothelial cells. The deduced amino acid sequence was identical to that of the human heparan N-sulphotransferase cloned previously [Dixon, Loftus, Gladwin, Scambler, Wasmuth and Dixon (1995) Genomics 26, 239–244]. RNA blot analysis indicated that two heparan N-sulphotransferase transcripts of approx. 8.5 and 4 kb were produced in all tissues. Expression was most abundant in heart, liver and pancreas. A cDNA encoding a Flag-tagged human heparan N-sulphotransferase (where Flag is an epitope with the sequence DYKDDDDK) was transfected into mouse LTA cells. Immunofluorescence detection using anti-Flag monoclonal antibodies demonstrated that the enzyme was localized to the trans-Golgi network. A truncated Flag-tagged heparan N-sulphotransferase was also retained in the Golgi, indicating that, as for many other Golgi enzymes, the N-terminal region of heparan N-sulphotransferase is sufficient for retention in the Golgi apparatus.

RORα and 25-hydroxycholesterol crosstalk regulates lipid droplet homeostasis in macrophages

Publisher: Public Library of Science (PLoS)

Date: 26-01-2016

DOI: 10.1371/JOURNAL.PONE.0147179

Differential expression of genes and receptors in monocytes from patients with cystic fibrosis

Publisher: Elsevier BV

Date: 05-2019

DOI: 10.1016/J.JCF.2018.07.012

Abstract: We previously reported defective alternative polarization (M2) of macrophages and early expression of classically polarized (M1) macrophage markers in unpolarized monocyte-derived macrophages (MDMs) in patients with cystic fibrosis (CF). The present study assessed whether the mechanism(s) underlying defective macrophage polarization resided in circulating monocytes. Monocyte subsets (classical, intermediate and non-classical), markers for monocyte activation (CD163) and recruitment (CD195), receptors/genes associated with macrophage differentiation and polarization were analyzed in CF and compared with healthy in iduals. No differences were observed in the monocyte subsets or in the expression of CD163 or CD195. Expression of the M-CSF receptor, TLR4, γC, IL-4Rα, IL-13Rα1, TIMP-1 and Cox-2 were higher in CF monocytes, albeit at low levels, whereas, LRP1, MMP9, MMP28 were downregulated compared to mooncytes from healthy in iduals. Our data suggest that differences in CF monocytes may contribute to the reported CFTR-dependent defect in macrophage differentiation, polarization and function.

Protein trafficking and polarity in kidney epithelium: from cell biology to physiology

Publisher: American Physiological Society

Date: 1996

DOI: 10.1152/PHYSREV.1996.76.1.245

Abstract: The transepithelial movement of fluids, electrolytes, and larger molecules is achieved by the activity of a host of specialized transporting proteins, including enzymes, receptors, and channels, that are located on either the apical, basal, or lateral plasma membrane domains of epithelial cells. In the kidney as well as in all other organs, this remarkable polarity of epithelial cells depends on the selective insertion of newly synthesized and recycling proteins and lipids into distinct plasma membrane domains and on the maintenance and modulation of these specialized domains once they are established during epithelial development. This review addresses the mechanisms by which epithelial cells control the movement of membrane components within the cell to ensure that they are delivered to the correct target membrane. Among the topics discussed are targeting signals within membrane proteins, the role of the cytoskeleton and the tight junctional barrier in cell polarity, and the requirement for accessory proteins in the targeting process, including GTP-binding proteins, and proteins that are involved in vesicle docking and fusion events. The final part of the review is devoted uniquely to the polarized targeting of functionally defined proteins in various kidney cell types. In concluding, ex les of how a breakdown in these trafficking pathways may be related to some disease states are presented.

Altered cell surface expression of human MC1R variant receptor alleles associated with red hair and skin cancer risk

Publisher: Oxford University Press (OUP)

Date: 22-06-2005

DOI: 10.1093/HMG/DDI219

Abstract: The human melanocortin-1 receptor gene (MC1R) encodes a G-protein coupled receptor that is primarily expressed on melanocytes, where it plays a key role in pigmentation regulation. Variant alleles are associated with red hair colour and fair skin, known as the RHC phenotype, as well as skin cancer risk. The R151C, R160W and D294H alleles, designated 'R', are strongly associated with the RHC phenotype and have been proposed to result in loss of function receptors due to impaired G-protein coupling. We recently provided evidence that the R151C and R160W variants can efficiently couple to G-proteins in response to alpha-melanocyte stimulating hormone. The possibility that altered cellular localization of the R151C and R160W variant receptors could underlie their association with RHC was therefore considered. Using immunofluorescence and ligand binding studies, we found that melanocytic cells exogenously or endogenously expressing MC1R show strong surface localization of the wild-type and D294H alleles but markedly reduced cell surface expression of the R151C and R160W receptors. In additional exogenous expression studies, the R variant D84E and the rare I155T variant, also demonstrated a significant reduction in plasma membrane receptor numbers. The V60L, V92M and R163Q weakly associated RHC alleles, designated 'r', were expressed with normal or intermediate cell surface receptor levels. These results indicate that reduced receptor coupling activity may not be the only contributing factor to the genetic association between the MC1R variants and the RHC phenotype, with MC1R polymorphisms now linked to a change in receptor localization.

Regulation of vesicular transport by GTP-binding proteins

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 09-1995

DOI: 10.1097/00041552-199509000-00009

Abstract: Intracellular protein trafficking occurs in a series of transport vesicles. Vesicle trafficking is regulated both by heterotrimeric and monomeric GTP-binding proteins (G proteins). Recent studies have explored effector systems used by heterotrimeric G proteins and by monomeric ADP-ribosylation factor G proteins for regulation of vesicle budding. New members of the Rab monomeric G protein family have been identified in polarized cells and new evidence confirms the function of Rab proteins in vesicle targeting. From these data we can begin to reconstruct the signal transduction pathways that regulate intracellular transport.

GAIP participates in budding of membrane carriers at the trans-Golgi network.

Publisher: Wiley

Date: 03-2003

DOI: 10.1034/J.1600-0854.2003.00106.X

Abstract: Galpha interacting protein (GAIP) is a regulator of G protein signaling protein that associates dynamically with vesicles and has been implicated in membrane trafficking, although its specific role is not yet known. Using an in vitro budding assay, we show that GAIP is recruited to a specific population of trans-Golgi network-derived vesicles and that these are distinct from coatomer or clathrin-coated vesicles. A truncation mutant (NT-GAIP) encoding only the N-terminal half of GAIP is recruited to trans-Golgi network membranes during the formation of vesicle carriers. Overexpression of NT-GAIP induces the formation of long, coated tubules, which are stabilized by microtubules. Results from the budding assay and from imaging in live cells show that these tubules remain attached to the Golgi stack rather than being released as carrier vesicles. NT-GAIP expression blocks membrane budding and results in the accumulation of tubular carrier intermediates. NT-GAIP-decorated tubules are competent to load vesicular stomatitis virus protein G-green fluorescent protein as post-Golgi, exocytic cargo and in cells expressing NT-GAIP there is reduced surface delivery of vesicular stomatitis virus protein G-green fluorescent protein. We conclude that GAIP functions as an essential part of the membrane budding machinery for a subset of post-Golgi exocytic carriers derived from the trans-Golgi network.

Development of SH2 probes and pull-down assays to detect pathogen-induced, site-specific tyrosine phosphorylation of the TLR adaptor SCIMP

Publisher: Wiley

Date: 14-03-2017

DOI: 10.1038/ICB.2017.10

Abstract: Protein tyrosine phosphorylation guides many molecular interactions for cellular functions. SCIMP is a transmembrane adaptor protein (TRAP) family member that mediates selective proinflammatory cytokine responses generated by pathogen-activated Toll-like receptor (TLR) pathways in macrophages. TLR activation triggers SCIMP phosphorylation and selective phosphorylation of distinct tyrosine residues on this adaptor offers the potential for regulating or biasing inflammatory responses. To analyze site-specific phosphorylation events, we developed three probes based on the SH2 domains of known SCIMP effectors, and used them for pull-downs from macrophage extracts. CRISPR-mediated SCIMP-deficient RAW264.7 macrophage-like cells were reconstituted with various phosphorylation-deficient (Y58F, Y96F, Y120F) SCIMPs, and used to demonstrate the specificity of LPS/TLR4-induced, site-specific phosphorylation of SCIMP for the temporal recruitment of the effectors Grb2, Csk and SLP65. Our findings reveal potential for differential SCIMP phosphorylation and specific effectors to influence TLR signaling and inflammatory programs. Furthermore, the use of Csk-SH2 pull-downs to identify additional known and new Csk targets in LPS-activated macrophages reveals the wider utility of our SH2 probes.

Actin filaments regulate epithelial Na+ channel activity

Publisher: American Physiological Society

Date: 11-1991

DOI: 10.1152/AJPCELL.1991.261.5.C882

Abstract: The functional role of the cytoskeleton in the control of ion channel activity is unknown. In the present study, immunocolocalization of Na+ channels with specific antibodies and fluorescein isothiocyanate-phalloidin to stain the cortical cytoskeleton indicates that actin is always present in close proximity to apical Na+ channels in A6 cells. The patch-cl technique was used to assess the effect of cortical actin networks on apical Na+ channels in these A6 epithelial cells. The actin filament disrupter, cytochalasin D (5 micrograms/ml), induced Na+ channel activity in cell-attached patches within 5 min of addition. Cytochalasin D also induced and/or increased Na+ channel activity in 90% of excised patches tested within 2 min. Addition of short actin filaments (greater than 5 microM) to excised patches also induced channel activity. This effect was enhanced by addition of ATP and/or cytochalasin D. The effect of actin on Na+ channel activity was reversed by addition of the G actin-binding protein DNase I or completely prevented by treatment of the excised patches with this enzyme. Addition of the actin-binding protein, filamin, reversibly inhibited both spontaneous and actin-induced Na+ channels. Thus actin filament networks, achieved by either depolymerizing endogenous actin filaments by treatment with cytochalasin D, the addition of exogenous short actin filaments plus ATP, or actin plus cytochalasin D, regulate apical Na+ channel activity. This conclusion was supported by the observation that the addition of short actin filaments in the form of actin-gelsolin complexes in molar ratios less than 8:1 was also effective in activating Na+ channels. We have thus demonstrated a functional role for the cortical actin network in the regulation of epithelial Na+ channels that may complement a structural role for membrane protein targetting and assembly.

Cytokine release from innate immune cells: association with diverse membrane trafficking pathways

Publisher: American Society of Hematology

Date: 07-07-2011

DOI: 10.1182/BLOOD-2010-08-265892

Abstract: Cytokines released from innate immune cells play key roles in the regulation of the immune response. These intercellular messengers are the source of soluble regulatory signals that initiate and constrain inflammatory responses to pathogens and injury. Although numerous studies describe detailed signaling pathways induced by cytokines and their specific receptors, there is little information on the mechanisms that control the release of cytokines from different cell types. Indeed, the pathways, molecules, and mechanisms of cytokine release remain a “black box” in immunology. Here, we review research findings and new approaches that have begun to generate information on cytokine trafficking and release by innate immune cells in response to inflammatory or infectious stimuli. Surprisingly complex machinery, multiple organelles, and specialized membrane domains exist in these cells to ensure the selective, temporal, and often polarized release of cytokines in innate immunity.

Heterogeneous localization of G protein alpha-subunits in rat kidney

Publisher: American Physiological Society

Date: 11-1991

DOI: 10.1152/AJPRENAL.1991.261.5.F831

Abstract: The Gs alpha and Gi alpha 1-3 subunits of GTP-binding proteins were localized in sections of rat kidney using antibodies against unique synthetic decapeptides from the different G alpha subunits. All of the G alpha subunits were found to have a polarized distribution on renal tubule epithelial cells, and staining was typically found on either basolateral or apical membranes in a given cell type. Gi alpha 1 was localized to the apical pole of both thick ascending limb cells and cells forming the papillary epithelium, Gi alpha 2 labeled the basolateral plasma membrane and the cytoplasm of collecting duct principal cells, and Gi alpha 3 was most abundant in the apical region of proximal tubule cells of the S1 segment, where it was concentrated in sub-brush-border invaginations. It was also found in the perinuclear Golgi complex in these cells. Gs alpha was heavily concentrated on the basolateral plasma membranes of thick ascending limb cells and both principal and intercalated cells of the collecting duct. Less intense subapical staining of G alpha s was also found in proximal tubule cells. The cells of the macula densa had a unique G protein distribution that was distinct from the surrounding cells of the thick ascending limb of Henle. Antibodies specific for the Gi alpha 1 and Gi alpha 3 subunits both stained intracellular vesicles clustered at the basal pole of the cell. A heterogeneous distribution of G alpha subunits was also found by Western blotting on isolated cortical membrane fractions.

Molecular analysis of common polymorphisms within the human Tyrosinase locus and genetic association with pigmentation traits.

Publisher: Wiley

Date: 12-05-2014

DOI: 10.1111/PCMR.12253

Regulation of endocytosis, nuclear translocation, and signaling of fibroblast growth factor receptor 1 by E-cadherin

Publisher: American Society for Cell Biology (ASCB)

Date: 2005

DOI: 10.1091/MBC.E04-09-0845

Abstract: Fibroblast growth factor (FGF) receptors (FGFRs) signal to modulate erse cellular functions, including epithelial cell morphogenesis. In epithelial cells, E-cadherin plays a key role in cell-cell adhesion, and its function can be regulated through endocytic trafficking. In this study, we investigated the location, trafficking, and function of FGFR1 and E-cadherin and report a novel mechanism, based on endocytic trafficking, for the coregulation of E-cadherin and signaling from FGFR1. FGF induces the internalization of surface FGFR1 and surface E-cadherin, followed by nuclear translocation of FGFR1. The internalization of both proteins is regulated by common endocytic machinery, resulting in cointernalization of FGFR1 and E-cadherin into early endosomes. By blocking endocytosis, we show that this is a requisite, initial step for the nuclear translocation of FGFR1. Overexpression of E-cadherin blocks both the coendocytosis of E-cadherin and FGFR1, the nuclear translocation of FGFR1 and FGF-induced signaling to the mitogen-activated protein kinase pathway. Furthermore, stabilization of surface adhesive E-cadherin, by overexpressing p120 ctn , also blocks internalization and nuclear translocation of FGFR1. These data reveal that conjoint endocytosis and trafficking is a novel mechanism for the coregulation of E-cadherin and FGFR1 during cell signaling and morphogenesis.

Rab8a localisation and activation by Toll-like receptors on macrophage macropinosomes

Publisher: The Royal Society

Date: 17-12-2019

DOI: 10.1098/RSTB.2018.0151

Abstract: Macropinocytosis is a prevalent and essential pathway in macrophages where it contributes to anti-microbial responses and innate immune cell functions. Cell surface ruffles give rise to phagosomes and to macropinosomes as multi-functional compartments that contribute to environmental s ling, pathogen entry, plasma membrane turnover and receptor signalling. Rapid, high resolution, lattice light sheet imaging demonstrates the dynamic nature of macrophage ruffling. Pathogen-mediated activation of surface and endosomal Toll-like receptors (TLRs) in macrophages upregulates macropinocytosis. Here, using multiple forms of imaging and microscopy, we track membrane-associated, fluorescently-tagged Rab8a expressed in live macrophages, using a variety of cell markers to demonstrate Rab8a localization and its enrichment on early macropinosomes. Production of a novel biosensor and its use for quantitative FRET analysis in live cells, pinpoints macropinosomes as the site for TLR-induced activation of Rab8a. We have previously shown that TLR signalling, cytokine outputs and macrophage programming are regulated by the GTPase Rab8a with PI3 Kγ as its effector. Finally, we highlight another effector, the phosphatase OCRL, which is located on macropinosomes and interacts with Rab8a, suggesting that Rab8a may operate on multiple levels to modulate phosphoinositides in macropinosomes. These findings extend our understanding of macropinosomes as regulatory compartments for innate immune function in macrophages. This article is part of the Theo Murphy meeting issue ‘Macropinocytosis’.

Domains of the TGN: coats, tethers and G proteins.

Publisher: Wiley

Date: 13-04-2004

DOI: 10.1111/J.1398-9219.2004.00182.X

Signalling, sorting and scaffolding adaptors for Toll-like receptors.

Publisher: The Company of Biologists

Date: 30-12-2019

DOI: 10.1242/JCS.239194

Abstract: Toll-like receptors (TLRs) are danger-sensing receptors that typically propagate self-limiting inflammatory responses, but can unleash uncontrolled inflammation in non-homeostatic or disease settings. Activation of TLRs by pathogen- and/or host-derived stimuli triggers a range of signalling and transcriptional pathways to programme inflammatory and anti-microbial responses, including the production of a suite of inflammatory cytokines and other mediators. Multiple sorting and signalling adaptors are recruited to receptor complexes on the plasma membrane or endosomes where they act as scaffolds for downstream signalling kinases and effectors at these sites. So far, seven proximal TLR adaptors have been identified: MyD88, MAL, TRIF (also known as TICAM1), TRAM (TICAM2), SARM (SARM1), BCAP (PIK3AP1) and SCIMP. Most adaptors tether directly to TLRs through homotypic Toll/interleukin-1 receptor domain (TIR)–TIR interactions, whereas SCIMP binds to TLRs through an atypical TIR–non-TIR interaction. In this Review, we highlight the key roles for these adaptors in TLR signalling, scaffolding and receptor sorting and discuss how the adaptors thereby direct the differential outcomes of TLR-mediated responses. We further summarise TLR adaptor regulation and function, and make note of human diseases that might be associated with mutations in these adaptors.

Distinct coated vesicles labeled for p200 bud from trans-Golgi network membranes

Publisher: Proceedings of the National Academy of Sciences

Date: 28-03-1995

DOI: 10.1073/PNAS.92.7.2874

Abstract: Golgi-associated cytoplasmic proteins, such as the coatomer protein complex, are required for vesicle budding and trafficking. We have previously described a cytoplasmic phosphoprotein, p200, which binds dynamically and specifically to Golgi membranes. The p200 protein is dissociated from Golgi membranes in the presence of brefeldin A and it is induced to bind to Golgi membranes by activation of guanine nucleotide binding proteins (G proteins) with guanosine 5'-[gamma-thio]triphosphate or aluminum fluoride. To establish the role of p200 in vesicle budding, we localized membrane-bound p200 in intact cells and on isolated Golgi membranes. We show that p200 is preferentially associated with vesicles in the trans-Golgi network (TGN). Activation of G proteins induced budding and accumulation of small, coated vesicles from Golgi membranes and p200 was localized on the cytoplasmic surface of some of these vesicles. Using immunogold labeling we further demonstrate that p200 and beta-COP are localized on different populations of Golgi-derived vesicles. These data establish that p200 is involved in the budding and coating of a class of Goli vesicles that are likely to be derived from the TGN. The data also show that there are distinct populations of non-clathrin-coated vesicles budded from Golgi membranes, and vesicles labeled for either beta-COP or p200 may represent transport vesicles for separate steps of protein transport.

Distribution and role of heterotrimeric G proteins in the secretory pathway of polarized epithelial cells

Publisher: The Company of Biologists

Date: 12-1993

DOI: 10.1242/JCS.1993.SUPPLEMENT_17.6

Abstract: SUMMARY The movement of newly synthesized proteins in the constitutive secretory pathway, from their site of synthesis in the endoplasmic reticulum to the cell surface or to intracellular destinations, requires an orderly sequence of transport steps between membrane-bound compartments. Until recently, the trafficking and secretion of proteins through this pathway was thought to occur as a relatively automatic, unregulated series of events. Recent studies show that protein trafficking in the constitutive secretory pathway requires GTP hydrolysis by families of GTP-binding proteins (G proteins), which at multiple steps potentially provide regulation and specificity for protein trafficking. Many monomeric G proteins are known to be localized and functional on membrane compartments in the constitutive secretory pathway. Now, members of the heterotrimeric G protein family have also been localized on intracellular membranes and compartments such as the Golgi complex. We have studied the localization and targeting of Gα subunits to distinct membrane domains in polarized epithelial cells. The distribution of different Gα subunits on very specific membrane domains in cultured epithelial cells and in epithelial cells of the kidney cortex, is highly suggestive of roles for these G proteins in intracellular trafficking pathways. One of these G protein subunits, Gαi-3, was localized on Golgi membranes. Studies on LLC-PK1 cells overexpressing Gαi-3 provided evidence for its functional role in regulating the transport of a constitutively secreted heparan sulfate proteoglycan through the Golgi complex. Inhibition or activation of heterotrimeric G proteins by pertussis toxin or by aluminium fluoride respectively, have provided further evidence for regulation of intracellular transport by pertussis toxin-sensitive G proteins. Although the functions of Golgi-associated G proteins are not yet understood at the molecular level, heterotrimeric G proteins have been implicated in the binding of cytosolic coat proteins and vesicle formation on Golgi membranes. Future studies will elucidate how multiple G proteins, of both the heterotrimeric and monomeric families, are involved in the regulation of Golgi function and protein trafficking in the secretory pathway.

Disruption of microtubules alters polarity of basement membrane proteoglycan secretion in epithelial cells

Publisher: American Physiological Society

Date: 07-1991

DOI: 10.1152/AJPCELL.1991.261.1.C691

Abstract: Basement membrane proteins such as the heparan sulfate proteoglycan (HSPG) are secreted in a polarized fashion from the basolateral membrane of epithelial cells. We have used the microtubule-disrupting drug colchicine to study the role of the microtubule network in directing constitutive secretion to the basolateral membrane of LLC-PK1 renal epithelial cells. Microtubule depolymerization induced by colchicine resulted in fragmentation and redistribution of fluorescently labeled trans-Golgi membranes. Increased immunofluorescent staining of HSPG was associated with these dispersed Golgi cisternae. The biosynthetic processing of HSPG was not significantly altered by the loss of microtubules or by the dispersal of the Golgi elements. The most striking effect of microtubule disruption was the loss of polarity of HSPG secretion. Immunoprecipitation studies showed that HSPG was secreted from both apical and basolateral surfaces of LLC-PK1 cells treated with colchicine, and a similar result was found for the delivery of laminin, another basement membrane protein. In contrast, there was no change in the distribution of an integral basolateral membrane protein, Na(+)-K(+)-ATPase, following colchicine treatment. Our results provide the first demonstration that microtubules are involved in the constitutive trafficking of basolateral secretory proteins. These data also suggest that there may be an inherent difference in the targeting or delivery of membrane and secretory proteins to the basolateral cell surface.

Editorial overview: Membrane traffic in the time of COVID-19.

Publisher: Elsevier BV

Date: 08-2020

DOI: 10.1016/J.CEB.2020.09.003

Individual Smad2 linker region phosphorylation sites determine the expression of proteoglycan and glycosaminoglycan synthesizing genes

Publisher: Elsevier BV

Date: 2019

DOI: 10.1016/J.CELLSIG.2018.11.005

Rab31 and APPL2 enhance FcγR-mediated phagocytosis through PI3K/Akt signaling in macrophages

Publisher: American Society for Cell Biology (ASCB)

Date: 03-2015

DOI: 10.1091/MBC.E14-10-1457

Abstract: Rab31 recruits APPL2 to regulate phagocytic cup closure and FcγR signaling pathways via production of PI(3,4,5)P 3 in macrophages. APPL2 is poised to activate macrophages and act as a counterpoint to APPL1 in FcγR-mediated PI3K/Akt signaling. New locations and roles are found for Rab31 and APPL2 by which they contribute to innate immune functions.

Basement membrane heparan sulfate proteoglycans are concentrated in the laminae rarae and in podocytes of the rat renal glomerulus

Publisher: Proceedings of the National Academy of Sciences

Date: 05-1985

DOI: 10.1073/PNAS.82.10.3296

Abstract: A polyclonal antibody was raised against a proteoglycan fraction purified from extracts of isolated rat renal glomeruli. The antibody was characterized by column chromatography and by NaDodSO4/PAGE analysis of immunoprecipitates and was shown to recognize specifically the core protein of a population of heparan sulfate proteoglycans (Mr 130,000) found in the glomerular basement membrane and in other renal basement membranes. The antibody was used to localize the core proteins of this population of heparan sulfate proteoglycans (HSPG) in the glomerulus by immunoelectron microscopic techniques. These HSPG were found to be concentrated in the lamina rarae interna and externa of the glomerular basement membrane (GBM) and precursor forms were detected in the endoplasmic reticulum and Golgi cisternae of glomerular visceral epithelial cells (podocytes).

Image-based analysis of phagocytosis: Measuring engulfment and internalization

Publisher: Springer New York

Date: 05-11-2017

DOI: 10.1007/978-1-4939-6581-6_13

N4WBP5A (Ndfip2), a Nedd4-interacting protein, localizes to multivesicular bodies and the Golgi, and has a potential role in protein trafficking

Publisher: The Company of Biologists

Date: 15-07-2004

DOI: 10.1242/JCS.01212

Abstract: N4WBP5A (Ndfip2) belongs to an evolutionarily conserved group of Nedd4-interacting proteins with two homologues in mammalian species. We have previously shown that N4WBP5A expression in Xenopus oocytes results in increased cell-surface expression of the epithelial sodium channel. N4WBPs are characterized by one or two amino terminal PPxY motifs and three transmembrane domains. Here we show that both PPxY motifs of N4WBP5A mediate interaction with WW domains of Nedd4 and that N4WBP5A can physically interact with the WW domains of several Nedd4-family proteins. N4WBP5A is ubiquitinated and ubiquitination does not significantly affect the turnover of N4WBP5A protein. Ubiquitination of N4WBP5A is enhanced by Nedd4 and Nedd4-2 expression. N4WBP5A localizes to the Golgi, vesicles associated with the Golgi complex and to multivesicular bodies. We show that the ectopic expression of N4WBP5A inhibits receptor-mediated endocytosis of labelled epidermal growth factor. N4WBP5A overexpression inhibits accumulation of EGF in large endocytic/lysosomal vesicles suggestive of a role for N4WBP5A in protein trafficking. We propose that N4WBP5A acts as an adaptor to recruit Nedd4 family ubiquitin-protein ligases to the protein trafficking machinery.

Involvement of the macrophage in experimental chronic immune complex glomerulonephritis

Publisher: S. Karger AG

Date: 1982

DOI: 10.1159/000182850

Abstract: Serial renal biopsies for glomerular culture, histochemical staining for beta-glucuronidase, electron microscopy (EM) and light microscopy, were used to study macrophage involvement in experimental chronic immune complex (IC) glomerulonephritis (GN) induced in rabbits by daily intravenous injections of bovine serum albumin (BSA). In the 26 animals studied, proliferative GN of variable severity was induced, with mild disease in 5 animals, moderate proliferation in 15 and crescentic GN in 6. Macrophages first appeared in glomerular culture outgrowths during the 2nd and 3rd weeks, coincident with the onset of proteinuria and rising serum creatinine concentration. Large numbers of macrophages (in excess of 20 per glomerulus) were seen by the 5th weeks and persisted to the 9th week. The number of macrophages in outgrowths was not significantly greater in animals with crescentic disease. EM demonstrated macrophages in capillary loops, and in glomeruli with crescents, macrophages could be seen in the urinary space. Histochemical staining for beta-glucuronidase also demonstrated macrophages in the glomerular tuft and in crescents when present. These results indicate that macrophages constitute a considerable proportion of the glomerular hypercellularity seen in chronic IC glomerulonephritis.

Dependence on pH of polarized sorting of secreted proteins

Publisher: Springer Science and Business Media LLC

Date: 10-1987

DOI: 10.1038/329632A0

Abstract: The plasma membranes of epithelial cells are ided into apical and basolateral domains. These two surfaces are characterized by markedly different protein compositions, reflecting the ability of the cell to target newly synthesized membrane proteins to specific regions of the cell surface. This targeting capability is also apparent in the polarized release of secretory products. Recent studies using canine renal tubule (MDCK) cells have suggested that distinct sets of secretory proteins are released from their apical and basolateral poles. We report experiments designed to examine secretory protein sorting by MDCK cells. We have shown that secretion of basement membrane components (laminin and heparan sulphate proteoglycan (HSPG] takes place from the basolateral cell surface and that this polarized release results from active sorting. The sorting process which mediates this polarized secretion requires an acidic intracellular compartment. MDCK cells treated with NH4Cl to raise the pH of their intracellular compartments, secrete laminin and HSPG by a default pathway which leads to their release in roughly equal quantities into the medium of both the apical and basolateral compartments.

Rab8a interacts directly with PI3Kγ to modulate TLR4-driven PI3K and mTOR signalling

Publisher: Springer Science and Business Media LLC

Date: 15-07-2014

DOI: 10.1038/NCOMMS5407

Abstract: Toll-like receptor 4 (TLR4) is activated by bacterial lipopolysaccharide (LPS) to mount innate immune responses. The TLR4-induced release of pro- and anti-inflammatory cytokines generates robust inflammatory responses, which must then be restrained to avoid disease. New mechanisms for the critical regulation of TLR-induced cytokine responses are still emerging. Here we find TLR4 complexes localized in LPS-induced dorsal ruffles on the surface of macrophages. We discover that the small GTPase Rab8a is enriched in these ruffles and recruits phosphatidylinositol 3-kinase (PI3Kγ) as an effector by interacting directly through its Ras-binding domain. Rab8a and PI3Kγ function to regulate Akt signalling generated by surface TLR4. Rab8a and PI3Kγ do not affect TLR4 endocytosis, but instead regulate mammalian target of rapamycin signalling as a mechanism for biasing the cytokine profile to constrain inflammation in innate immunity.

Dynamic imaging of the recycling endosomal network in macrophages

Publisher: Elsevier

Date: 2015

DOI: 10.1016/BS.MCB.2015.04.007

Abstract: Recycling endosomes (REs) form an extensive and complex network of subcompartmentalized vesicular and tubular elements that connect with the cell surface and other endosomes in macrophages. As surveillance and defense cells of the innate immune system, macrophages are highly dependent on REs for their active and voluminous cell surface turnover and endocytic, exocytic, and recycling of membrane and cargo. Here we set out three approaches for imaging and analyzing REs in macrophages, based on the expression of fluorescently labeled RE-associated proteins and the uptake of fluorescent cargo. Subcompartments of the REs are identified by co-expression and co-localization analysis of RE associated Rab GTPases. Transferrin is a well-known cargo marker as it recycles through REs and it is compared here to other cargo, revealing how different endocytic routes intersect with REs. We show how the movement of transferrin through REs can be modeled and quantified in live cells. Finally, since phagosomes are a signature organelle for macrophages, and REs fuse with the maturing phagosome, we show imaging of REs with phagosomes using a genetically encoded pH-sensitive SNARE-based probe. Together these approaches provide multiple ways to comprehensively analyze REs and the important roles they play in these immune cells and more broadly in other cell types.

Distinct localization of renin and GLUT-4 in juxtaglomerular cells of mouse kidney

Publisher: American Physiological Society

Date: 1998

DOI: 10.1152/AJPRENAL.1998.274.1.F26

Abstract: The insulin-responsive glucose transporter, GLUT-4, is found primarily in adipocytes and skeletal muscle cells, where it is sequestered in a specialized recycling compartment, from which it can be recruited to the cell surface following insulin stimulation. Lower levels of GLUT-4 are also expressed in other tissues, including the kidney, where it is present particularly in cells of the afferent arteriole and juxtaglomerular apparatus (JGA). The exact nature of GLUT-4-containing compartments and their relationship to other regulated trafficking pathways in different cells are not yet well defined. The trafficking of GLUT-4 has been studied in different cells with regulated secretory pathways, and a recent study shows that, in cardiomyocytes, GLUT-4 is sorted and packaged into multiple regulated pathways (J. W. Slot, G. Garruti, S. Martin, V. Oorschot, G. Pshuma, E. W. Kraegen, R. Laybutt, G. Thibault, and D. E. James. J. Cell Biol. 137: 1243–1254, 1997). In the kidney, cells of the JGA synthesize and secrete their major product, renin, via a well-established, regulated, secretory pathway. These cells also express GLUT-4 and thus offer the potential to directly compare the localization and trafficking of GLUT-4 and renin in a unique cell type. The present study was undertaken to investigate the intracellular distribution of GLUT-4 in mouse kidney cortex and to determine whether GLUT-4 and renin are trafficked in the same or in separate regulated pathways. Ultrathin cryosections of mouse kidney were labeled by the immunogold technique and viewed by electron microscopy, demonstrating the distribution of GLUT-4 in cells of the JGA, afferent arteriole, and distal tubule. In granular cells of the JGA, renin was localized in secretory granules of the regulated secretory pathway, whereas GLUT-4 labeling in the same cells was found in a distinct tubulovesicular compartment located adjacent to the trans-Golgi network. We show that granular cells have separate, morphologically distinct compartments for the sequestration of renin and GLUT-4, providing evidence that there may be distinct pathways for the sorting and trafficking of these two proteins.

Sequential recruitment of Rab GTPases during early stages of phagocytosis

Publisher: Informa UK Limited

Date: 02-01-2016

DOI: 10.1080/21592799.2016.1140615

Nobel Prize discovery paves the way for immunological traffic

Publisher: Springer Science and Business Media LLC

Date: 25-11-2013

DOI: 10.1038/NRI3564

Distinct Roles for APPL1 and APPL2 in Regulating Toll-like Receptor 4 Signaling in Macrophages

Publisher: Wiley

Date: 23-06-2016

DOI: 10.1111/TRA.12415

Abstract: Macrophages are activated by contact with pathogens to mount innate immune defenses against infection. Toll-like receptor 4 (TLR4) at the macrophage surface recognizes and binds bacterial lipopolysaccharide (LPS), setting off signaling and transcriptional events that lead to the secretion of pro- and anti-inflammatory cytokines these in turn control inflammatory and antimicrobial responses. Although the complex regulatory pathways downstream of TLR4 have been extensively studied, further molecules critical for modulating the resulting cytokine outputs remain to be characterized. Here we establish potential roles for APPL1 and 2 signaling adaptors as regulators of LPS/TLR4-induced signaling, transcription, and cytokine secretion. APPL1 and 2 are differentially localized to distinct signaling-competent membrane domains on the surface and in endocytic compartments of LPS-activated macrophages. By depleting cells of each adaptor respectively we show separate and opposing functions for APPL1 and 2 in Akt and MAPK signaling. Specifically, APPL2 has a dominant role in nuclear translocation of NF-KB p65 and it serves to constrain the secretion of pro- and anti-inflammatory cytokines. The APPLs, and in particular APPL2, are thus revealed as adaptors with important capacity to modulate inflammatory responses mounted by LPS/TLR4 during infection.

Budding roles for myosin II on the Golgi

Publisher: Elsevier BV

Date: 04-1998

DOI: 10.1016/S0962-8924(98)01238-0

Abstract: Myosin II--conventional myosin--has been typecast in muscle-man roles. While members of the Schwarzenegger clan from skeletal muscle have grabbed the limelight, myosin II motors in nonmuscle cells labour away in many varied and subtle roles. Recent findings show that nonmuscle myosin II, along with other myosins and cytoskeletal proteins, assembles on Golgi membranes. Nonmuscle myosin II associates transiently with membranes of the trans-Golgi network during the budding of a subpopulation of transport vesicles. The exact role of myosin II in vesicular trafficking is not yet understood, but its participation heralds a novel role for actin-based motors in vesicle budding.

Subcompartments of the macrophage recycling endosome direct the differential secretion of IL-6 and TNFα

Publisher: Rockefeller University Press

Date: 02-07-2007

DOI: 10.1083/JCB.200612131

Abstract: Activated macrophages secrete an array of proinflammatory cytokines, including tumor necrosis factor-α (TNFα) and interleukin 6 (IL-6), that are temporally secreted for sequential roles in inflammation. We have previously characterized aspects of the intracellular trafficking of membrane-bound TNFα and its delivery to the cell surface at the site of phagocytic cups for secretion (Murray, R.Z., J.G. Kay, D.G. Sangermani, and J.L. Stow. 2005. Science. 310:1492–1495). The trafficking pathway and surface delivery of IL-6, a soluble cytokine, were studied here using approaches such as live cell imaging of fluorescently tagged IL-6 and immunoelectron microscopy. Newly synthesized IL-6 accumulates in the Golgi complex and exits in tubulovesicular carriers either as the sole labeled cargo or together with TNFα, utilizing specific soluble NSF attachment protein receptor (SNARE) proteins to fuse with the recycling endosome. Within recycling endosomes, we demonstrate the compartmentalization of cargo proteins, wherein IL-6 is dynamically segregated from TNFα and from surface recycling transferrin. Thereafter, these cytokines are independently secreted, with TNFα delivered to phagocytic cups but not IL-6. Therefore, the recycling endosome has a central role in orchestrating the differential secretion of cytokines during inflammation.

A Role for the Phagosome in Cytokine Secretion

Publisher: American Association for the Advancement of Science (AAAS)

Date: 02-12-2005

DOI: 10.1126/SCIENCE.1120225

Abstract: Membrane traffic in activated macrophages is required for two critical events in innate immunity: proinflammatory cytokine secretion and phagocytosis of pathogens. We found a joint trafficking pathway linking both actions, which may economize membrane transport and augment the immune response. Tumor necrosis factor α (TNFα) is trafficked from the Golgi to the recycling endosome (RE), where vesicle-associated membrane protein 3 mediates its delivery to the cell surface at the site of phagocytic cup formation. Fusion of the RE at the cup simultaneously allows rapid release of TNFα and expands the membrane for phagocytosis.

pTRAPs: Transmembrane adaptors in innate immune signaling

Publisher: Oxford University Press (OUP)

Date: 30-03-2018

DOI: 10.1002/JLB.2RI1117-474R

Abstract: Transmembrane adaptor proteins (TRAPs) are protein scaffolds and signaling regulators with established roles in signal-induced activation of lymphocytes. A subset of the TRAP family, the palmitoylated TRAPs (pTRAPs), are increasingly emerging with additional roles in innate immune cells. Targeted to lipid rafts, tetraspannin-enriched microdomains, and protein microclusters in membranes, pTRAP scaffolds exert spatiotemporal regulation by recruiting signaling kinases, particularly Src and Syk family members, as well as Csk, and other effectors. In this way, pTRAPs modulate signaling and influence resulting cell responses, including the selective output of inflammatory cytokines and other mediators. Here, we review studies revealing that different pTRAPs work together, often with overlapping or redundant roles, for positive and negative regulation of key innate immune pathways, including Fc receptor and pattern recognition receptor signaling. Recent findings show that pTRAPs can bind directly to innate immune receptors, in addition to other transmembrane binding partners. Thus, pTRAPs are important, multifunctional scaffolds in pathways that are fundamental to erse innate immune responses.

The GRIP domain is a specific targeting sequence for a population of trans-Golgi network derived tubulo-vesicular carriers.

Publisher: Wiley

Date: 05-2001

DOI: 10.1034/J.1600-0854.2001.002005336.X

Abstract: Vesicular carriers for intracellular transport associate with unique sets of accessory molecules that dictate budding and docking on specific membrane domains. Although many of these accessory molecules are peripheral membrane proteins, in most cases the targeting sequences responsible for their membrane recruitment have yet to be identified. We have previously defined a novel Golgi targeting domain (GRIP) shared by a family of coiled-coil peripheral membrane Golgi proteins implicated in membrane trafficking. We show here that the docking site for the GRIP motif of p230 is a specific domain of Golgi membranes. By immuno-electron microscopy of HeLa cells stably expressing a green fluorescent protein (GFP)-p230GRIP fusion protein, we show binding specifically to a subset of membranes of the trans-Golgi network (TGN). Real-time imaging of live HeLa cells revealed that the GFP-p230GRIP was associated with highly dynamic tubular extensions of the TGN, which have the appearance and behaviour of transport carriers. To further define the nature of the GRIP membrane binding site, in vitro budding assays were performed using purified rat liver Golgi membranes and cytosol from GFP-p230GRIP-transfected cells. Analysis of Golgi-derived vesicles by sucrose gradient fractionation demonstrated that GFP-p230GRIP binds to a specific population of vesicles distinct from those labelled for beta-COP or gamma-adaptin. The GFP-p230GRIP fusion protein is recruited to the same vesicle population as full-length p230, demonstrating that the GRIP domain is solely proficient as a targeting signal for membrane binding of the native molecule. Therefore, p230 GRIP is a targeting signal for recruitment to a highly selective membrane attachment site on a specific population of trans-Golgi network tubulo-vesicular carriers.

Antibodies to basement membrane heparan sulfate proteoglycans bind to the laminae rarae of the glomerular basement membrane (GBM) and induce subepithelial GBM thickening

Publisher: Rockefeller University Press

Date: 05-1986

DOI: 10.1084/JEM.163.5.1064

Abstract: Antibodies specific for the core protein of basement membrane HSPG (Mr = 130,000) were administered to rats by intravenous injection, and the pathologic consequences on the kidney were determined at 3 min to 2 mo postinjection. Controls were given antibodies against gp330 (the pathogenic antigen of Heymann nephritis) or normal rabbit IgG. The injected anti-HSPG(GBM) IgG disappeared rapidly (by 1 d) from the circulation. The urinary excretion of albumin increased in a dose-dependent manner during the first 4 d, was increased 10-fold at 1-2 mo, but remained moderate (mean = 12 mg/24 h). By immunofluorescence the anti-HSPG(GBM) was seen to bind rapidly (by 3 min) to all glomerular capillaries, and by immunoperoxidase staining the anti-HSPG was seen to bind exclusively to the laminae rarae of the GBM where it remained during the entire 2-mo observation period. C3 was detected in glomeruli immediately after the injection (3 min), where it bound exclusively to the lamina rara interna the amount of C3 bound increased up to 2 h but decreased rapidly thereafter, and was not detectable after 4 d. Mononuclear and PMN leukocytes accumulated in glomerular capillaries, adhered to the capillary wall, and extended pseudopodia through the endothelial fenestrae to contact in the LRI of the GBM where the immune deposits and C3 were located. At 1 wk postinjection, staining for C3 reappeared in the glomeruli of some of the rats, and by this time most of the rats, including controls injected with normal rabbit IgG, had circulating anti-rabbit IgG (by ELISA) and linear deposits of rat IgG along the GBM (by immunofluorescence). Beginning at 9 d, there was progressive subepithelial thickening of the GBM which in some places was two to three times its normal width. This thickening was due to the laying down of a new layer of basement membrane-like material on the epithelial side of the GBM, which gradually displaced the old basement membrane layers toward the endothelium. The results show that the core proteins of this population of basement membrane HSPG (Mr = 130,000), which are ubiquitous components of basement membranes, are exposed to the circulation and can bind anti-HSPG(GBM) IgG in the laminae rarae of the GBM. Binding of these antibodies to the GBM leads to changes (C3 deposition, leukocyte adherence, moderate proteinuria, GBM thickening) considered typical of the acute phase of anti-GBM glomerulonephritis. Antibody binding interferes with the normal turnover of the GBM, presumably by affecting the biosynthesis and/or degradation of basement membrane components.

TLR Crosstalk Activates LRP1 to Recruit Rab8a and PI3Kγ for Suppression of Inflammatory Responses

Publisher: Elsevier BV

Date: 09-2018

DOI: 10.1016/J.CELREP.2018.08.028

Abstract: The multi-ligand endocytic receptor, low-density lipoprotein-receptor-related protein 1 (LRP1), has anti-inflammatory roles in disease. Here, we reveal that pathogen-activated Toll-like receptors (TLRs) activate LRP1 in human and mouse primary macrophages, resulting in phosphorylation of LRP1 at Y4507. In turn, this allows LRP1 to activate and recruit the guanosine triphosphatase (GTPase), Rab8a, with p110γ 101 as its phosphatidylinositol 3-kinase (PI3K) effector complex. PI3Kγ is a known regulator of TLR signaling and macrophage reprogramming. LRP1 coincides with Rab8a at signaling sites on macropinosomal membranes. In LRP1-deficient cells, TLR-induced Rab8 activation is abolished. CRISPR-mediated knockout of LRP1 in macrophages alters Akt/mTOR signaling and produces a pro-inflammatory bias in cytokine outputs, mimicking the Rab8a knockout and PI3Kγ-null phenotype. Thus, TLR-LRP1 crosstalk activates the Rab8a/PI3Kγ complex for reprogramming macrophages, revealing this as a key mechanism through which LRP1 helps to suppress inflammation.

Small GTPase Rab8a-recruited phosphatidylinositol 3-kinase γ regulates signaling and cytokine outputs from endosomal toll-like receptors

Publisher: Elsevier BV

Date: 03-2017

DOI: 10.1074/JBC.M116.766337

Cytokine Secretion via Cholesterol-rich Lipid Raft-associated SNAREs at the Phagocytic Cup

Publisher: Elsevier BV

Date: 04-2006

DOI: 10.1074/JBC.M600857200

Biosynthesis of proteoglycans by isolated rabbit glomeruli

Publisher: Elsevier BV

Date: 09-1983

DOI: 10.1016/0003-9861(83)90110-8

Abstract: Isolated rabbit glomeruli were incubated in vitro with 35SO4 in order to analyze the proteoglycans synthesized. Proteoglycans extracted with 4 M guanidine HCl from whole isolated glomeruli and from purified glomerular basement membrane (GBM) were analyzed by gel filtration chromatography. Two types of sulfated proteoglycans were found to be synthesized by rabbit glomeruli and these contained either heparan sulfate or chondroitin/dermatan sulfate glycosaminoglycan chains. These glycosaminoglycans were characterized by their sensitivity to selective degradation by nitrous acid or chondroitinase ABC, respectively. The major proteoglycan extracted from the whole glomeruli was a chondroitin/dermatan sulfate species (75%), while purified GBM contained mostly heparan sulfate (70%). The glycosaminoglycan chains were estimated to be about 12,000 molecular weight which is consistent with previous estimates for similar molecules extracted from the rat GBM.

Recycling endosome-dependent and -independent mechanisms for IL-10 secretion in LPS-activated macrophages

Publisher: Oxford University Press (OUP)

Date: 12-2012

DOI: 10.1189/JLB.0412191

Abstract: Two post-Golgi pathways where IL-10 is trafficked, ensures its secretion from activated macrophages under different physiological conditions. IL-10 is a key anti-inflammatory cytokine secreted by activated macrophages as a feedback control mechanism to prevent excessive inflammatory responses. Here, we define multiple intracellular trafficking pathways involved in the secretion of newly synthesized IL-10 from macrophages following TLR4 activation with LPS, and show how this relates to the previously defined trafficking pathways for IL-6 and TNF in macrophages simultaneously producing these proinflammatory cytokines. IL-10 exits the Golgi in multiple tubular carriers, including those dependent on p230GRIP. Some of the IL-10 is then delivered to recycling endosomes, where cytokine sorting may occur prior to its release. Another portion of the IL-10 is delivered to the cell surface in distinct vesicles colabeled for apoE. Thus, we show at least two post-Golgi pathways via which IL-10 is trafficked, ensuring its secretion from activated macrophages under different physiological conditions.

Dual trafficking of Slit3 to mitochondria and cell surface demonstrates novel localization for Slit protein

Publisher: American Physiological Society

Date: 08-2001

DOI: 10.1152/AJPCELL.2001.281.2.C486

Abstract: Drosophila slit is a secreted protein involved in midline patterning. Three vertebrate orthologs of the fly slit gene, Slit1, 2, and 3, have been isolated. Each displays overlapping, but distinct, patterns of expression in the developing vertebrate central nervous system, implying conservation of function. However, vertebrate Slit genes are also expressed in nonneuronal tissues where their cellular locations and functions are unknown. In this study, we characterized the cellular distribution and processing of mammalian Slit3 gene product, the least evolutionarily conserved of the vertebrate Slit genes, in kidney epithelial cells, using both cellular fractionation and immunolabeling. Slit3, but not Slit2, was predominantly localized within the mitochondria. This localization was confirmed using immunoelectron microscopy in cell lines and in mouse kidney proximal tubule cells. In confluent epithelial monolayers, Slit3 was also transported to the cell surface. However, we found no evidence of Slit3 proteolytic processing similar to that seen for Slit2. We demonstrated that Slit3 contains an NH 2 -terminal mitochondrial localization signal that can direct a reporter green fluorescent protein to the mitochondria. The equivalent region from Slit1 cannot elicit mitochondrial targeting. We conclude that Slit3 protein is targeted to and localized at two distinct sites within epithelial cells: the mitochondria, and then, in more confluent cells, the cell surface. Targeting to both locations is driven by specific NH 2 -terminal sequences. This is the first examination of Slit protein localization in nonneuronal cells, and this study implies that Slit3 has potentially unique functions not shared by other Slit proteins.

Cytokine secretion in macrophages and other cells: pathways and mediators

Publisher: Elsevier BV

Date: 07-2009

DOI: 10.1016/J.IMBIO.2008.11.005

Abstract: Cytokines and other immune mediators are secreted by cells of the immune system during immune responses and as a means of communication. While the functions of these cytokines, chemokines and mediators are well known, the intracellular pathways that lead to their secretion by different cells are only now being fully documented. Cytokines in some cells are released from secretory granules while in other cells they are released via constitutive secretory pathways that instead have more dynamic vesicular carriers. Recent studies have revealed that newly synthesized cytokines can be routed via compartments such as recycling endosomes prior to their secretion. Here we describe and show ex les of some of the pathways used for cytokine trafficking and release in macrophages, including some of the cellular machinery required for this transport. Increasingly, these trafficking pathways are revealed as having important regulatory roles in the execution of immune responses.

Nuclear translocation of cell-surface receptors: lessons from fibroblast growth factor

Publisher: Wiley

Date: 08-08-2005

DOI: 10.1111/J.1600-0854.2005.00332.X

Abstract: The nuclear localization of a number of growth factors, cytokine ligands and their receptors has been reported in various cell lines and tissues. These include members of the fibroblast growth factor (FGF), epidermal growth factor and growth hormone families. Accordingly, a number of nuclear functions have begun to emerge for these protein families. The demonstration of functional interactions of these proteins with the nuclear import machinery has further supported their functions as nuclear signal transducers. Here, we review the membrane- trafficking machinery and pathways demonstrated to regulate this cell surface to nucleus-trafficking event and highlight the many remaining unanswered questions. We focus on the FGF family, which is providing many of the clues as to the process of this unusual phenomenon.

Syntaxin 11 Binds Vti1b and Regulates Late Endosome to Lysosome Fusion in Macrophages

Publisher: Wiley

Date: 08-04-2011

DOI: 10.1111/J.1600-0854.2011.01189.X

Abstract: Syntaxin 11 (Stx11) is a SNARE protein enriched in cells of the immune system. Loss or mutation of Stx11 results in familial hemophagocytic lymphohistiocytosis type-4 (FHL-4), an autosomal recessive disorder of immune dysregulation characterized by high levels of inflammatory cytokines along with defects in T-cell and natural killer cell function. We show here Stx11 is located on endosomal membranes including late endosomes and lysosomes in macrophages. While Stx11 did not form a typical trans-SNARE complex, it did bind to the Q-SNARE Vti1b and was able to regulate the availability of Vti1b to form the Q-SNARE complexes Stx6/Stx7/Vtib and Stx7/Stx8/Vti1b. The mutant form of Stx11 sequestered Vti1b from forming the Q-SNARE complex that mediates late endosome to lysosome fusion. Depletion of Stx11 in activated macrophages leads to an accumulation of enlarged late endocytic compartments, increased trafficking to the cell surface and inhibition of late endosome to lysosome fusion. These phenotypes are rescued by the expression of an siRNA-resistant Stx11 construct in Stx11-depleted cells. Our results suggest that by regulating the availability of Vti1b, Stx11 regulates trafficking steps between late endosomes, lysosomes and the cell surface in macrophages.

ICAT is a multipotent inhibitor of beta-catenin. Focus on role for ICAT in beta-catenin-dependent nuclear signaling and cadherin functions

Publisher: American Physiological Society

Date: 04-2004

DOI: 10.1152/AJPCELL.00563.2003

Targeting of RNA Polymerase II by a nuclearLegionella pneumophilaDot/Icm effector SnpL

Publisher: Hindawi Limited

Date: 18-05-2018

DOI: 10.1111/CMI.12852

Abstract: The intracellular pathogen Legionella pneumophila influences numerous eukaryotic cellular processes through the Dot/Icm-dependent translocation of more than 300 effector proteins into the host cell. Although many translocated effectors localise to the Legionella replicative vacuole, other effectors can affect remote intracellular sites. Following infection, a subset of effector proteins localises to the nucleus where they subvert host cell transcriptional responses to infection. Here, we identified Lpw27461 (Lpp2587), Lpg2519 as a new nuclear-localised effector that we have termed SnpL. Upon ectopic expression or during L. pneumophila infection, SnpL showed strong nuclear localisation by immunofluorescence microscopy but was excluded from nucleoli. Using immunoprecipitation and mass spectrometry, we determined the host-binding partner of SnpL as the eukaryotic transcription elongation factor, Suppressor of Ty5 (SUPT5H)/Spt5. SUPT5H is an evolutionarily conserved component of the DRB sensitivity-inducing factor complex that regulates RNA Polymerase II dependent mRNA processing and transcription elongation. Protein interaction studies showed that SnpL bound to the central Kyprides, Ouzounis, Woese motif region of SUPT5H. Ectopic expression of SnpL led to massive upregulation of host gene expression and macrophage cell death. The activity of SnpL further highlights the ability of L. pneumophila to control fundamental eukaryotic processes such as transcription that, in the case of SnpL, leads to global upregulation of host gene expression.

Rab6a/a’ Are Important Golgi Regulators of Pro-Inflammatory TNF Secretion in Macrophages

Publisher: Public Library of Science (PLoS)

Date: 21-02-2013

DOI: 10.1371/JOURNAL.PONE.0057034

High-speed squeeze: Light-sheet imaging of zebrafish neutrophils

Publisher: Oxford University Press (OUP)

Date: 08-2020

DOI: 10.1002/JLB.1CE0320-082

Expression of heparan sulphate N-deacetylase/N-sulphotransferase by vascular smooth muscle cells

Publisher: Springer Science and Business Media LLC

Date: 2002

DOI: 10.1023/A:1020938430120

Abstract: Heparan sulphate is an important mediator in determining vascular smooth muscle cell (SMC) phenotype. The sulphation pattern of the heparan sulphate chains is critical to their function. We have examined the initial step in the biosynthesis of the sulphated domains mediated by the enzyme heparan sulphate N-deacetylase/N-sulphotransferase (NDST). Rabbit aortic SMC in primary culture exhibited NDST enzyme activity and expressed NDST-1 in their Golgi apparatus, with maximal expression in SMC 2 days after dispersal in primary culture confirmed by Western blot analysis. Endothelial cells, macrophages and fibroblasts expressed NDST-1 but had generally less intense staining than SMC, although SMC expression decreased with culture. The uninjured rat aorta also showed widespread expression of NDST-1. After balloon de-endothelialisation, NDST-1 could not be detected in SMC of the neointima in the early stages of neointimal formation, but was re-expressed at later time points (after 12 weeks). In human coronary arteries, SMC of the media and the diffuse intimal thickening expressed NDST-1, while SMC in the atherosclerotic plaque were negative for NDST-1. We conclude that SMC may regulate their heparan sulphate sulphation at the level of expression of the enzyme heparan sulphate NDST in a manner related to their phenotypic state.

A trans-Golgi network golgin is required for the regulated secretion of TNF in activated macrophages in vivo.

Publisher: Proceedings of the National Academy of Sciences

Date: 04-03-2008

DOI: 10.1073/PNAS.0800137105

Abstract: The transmembrane precursor of tumor necrosis factor-α (TNF) exits the trans -Golgi network (TGN) in tubular carriers for subsequent trafficking and delivery to the cell surface however, the molecular machinery responsible for Golgi export is unknown. We previously reported that members of the TGN golgin family are associated with subdomains and tubules of the TGN. Here, we show that the TGN golgin, p230/golgin-245 (p230), is essential for intracellular trafficking and cell surface delivery of TNF in transfected HeLa cells and activated macrophages. Live-cell imaging revealed that TNF transport from the TGN is mediated selectively by tubules and carriers marked by p230. Significantly, LPS activation of macrophages resulted in a dramatic increase of p230-labeled tubules and carriers emerging from the TGN, indicating that macrophages up-regulate the transport pathway for TNF export. Depletion of p230 in LPS-stimulated macrophages reduced cell surface delivery of TNF by -fold compared with control cells. To determine whether p230 depletion blocked TNF secretion in vivo , we generated retrogenic mice expressing a microRNA-vector to silence p230. Bone-marrow stem cells were transduced with recombinant retrovirus containing microRNA constructs and transplanted into irradiated recipients. LPS-activated peritoneal macrophages from p230 miRNA retrogenic mice were depleted of p230 and had dramatically reduced levels of cell surface TNF. Overall, these studies have identified p230 as a key regulator of TNF secretion and have shown that LPS activation of macrophages results in increased Golgi carriers for export. Also, we have demonstrated a previously undescribed approach to control cytokine secretion by the specific silencing of trafficking machinery.

Mechanisms of unconventional secretion of IL-1 family cytokines

Publisher: Elsevier BV

Date: 08-2015

DOI: 10.1016/J.CYTO.2015.03.022

Abstract: One of the most poorly understood processes in cell biology is the peculiar ability of specific leaderless proteins to be secreted via ER/Golgi-independent mechanisms ('unconventional protein secretion'). One such leaderless protein is the major immune-activating cytokine, interleukin-1β (IL-1β). Unusual amongst cytokines, IL-1β is expressed in the cytosol as an inactive precursor protein. It requires maturation by the caspase-1 protease, which itself requires activation upon immune cell sensing of infection or cell stress. Despite 25 years of intensive research into IL-1β secretory mechanisms, how it exits the cell is still not well understood. Here we will review the various mechanisms by which macrophages have been proposed to secrete IL-1 family cytokines, and the potential involvement of caspase-1 therein. Since aberrant IL-1β production drives inherited and acquired human diseases (e.g. autoinflammatory diseases, arthritic diseases, gout, Alzheimer's disease), elucidation of the IL-1β secretory pathway may offer new therapeutic opportunities for treatment across this wide range of human conditions.

Macropinosomes host TLR9 signaling and regulation of inflammatory responses in microglia

Publisher: Cold Spring Harbor Laboratory

Date: 11-02-2021

DOI: 10.1101/2021.02.11.430773

Abstract: To support their innate immune and scavenging functions in the brain, microglia are equipped with Toll-like receptors (TLRs), including the intracellular receptor TLR9, which is activated by microbial CpG-rich DNA. Macropinocytosis is an abundant and inducible pathway in microglia for fluid-phase uptake and ingestion of microbes and cell debris. TLR9 signaling has been ascribed to endolysosomes, particularly lysosomes, which it accesses through direct transport or via internalization from the surface. Here, TLR9 and exogenous CpG-DNA are localized during uptake into fluid-filled macropinosomes, upon upregulated macropinocytosis, where acidic and proteolytic environments support MyD88-induced signaling. Macropinosomes represent an abundant pathway for endolysosomal traffic of TLR9 but are also a much more exposed site for nucleic acid activation of the receptor with a risk of excessive inflammation. To constrain TLR9 inflammation, macropinosomes also house the TLR9 co-receptor LRP1 and regulators Rab8a and PI3Kγ which augment Akt signaling and favor anti-inflammatory cytokine production. Macropinosomes and their inflammatory regulators are therefore important components of TLR9 pathways in microglia that are poised for surveillance and protection in the CNS.

Polarized trafficking of E-cadherin is regulated by Rac1 and Cdc42 in Madin-Darby canine kidney cells

Publisher: American Physiological Society

Date: 06-2005

DOI: 10.1152/AJPCELL.00533.2004

Abstract: E-cadherin is a major cell-cell adhesion protein of epithelia that is trafficked to the basolateral cell surface in a polarized fashion. The exact post-Golgi route and regulation of E-cadherin transport have not been fully described. The Rho GTPases Cdc42 and Rac1 have been implicated in many cell functions, including the exocytic trafficking of other proteins in polarized epithelial cells. These Rho family proteins are also associated with the cadherin-catenin complexes at the cell surface. We have used functional mutants of Rac1 and Cdc42 and inactivating toxins to demonstrate specific roles for both Cdc42 and Rac1 in the post-Golgi transport of E-cadherin. Dominant-negative mutants of Cdc42 and Rac1 accumulate E-cadherin at a distinct post-Golgi step. This accumulation occurs before p120 ctn interacts with E-cadherin, because p120 ctn localization was not affected by the Cdc42 or Rac1 mutants. Moreover, the GTPase mutants had no effect on the trafficking of a targeting mutant of E-cadherin, consistent with the selective involvement of Cdc42 and Rac1 in basolateral trafficking. These results provide a new ex le of Rho GTPase regulation of basolateral trafficking and demonstrate novel roles for Cdc42 and Rac1 in the post-Golgi transport of E-cadherin.

Recycling of E-Cadherin

Publisher: Rockefeller University Press

Date: 12-07-1999

DOI: 10.1083/JCB.146.1.219

Abstract: E-Cadherin plays critical roles in many aspects of cell adhesion, epithelial development, and the establishment and maintenance of epithelial polarity. The fate of E-cadherin once it is delivered to the basolateral cell surface, and the mechanisms which govern its participation in adherens junctions, are not well understood. Using surface biotinylation and recycling assays, we observed that some of the cell surface E-cadherin is actively internalized and is then recycled back to the plasma membrane. The pool of E-cadherin undergoing endocytosis and recycling was markedly increased in cells without stable cell-cell contacts, i.e., in preconfluent cells and after cell contacts were disrupted by depletion of extracellular Ca2+, suggesting that endocytic trafficking of E-cadherin is regulated by cell-cell contact. The reformation of cell junctions after replacement of Ca2+ was then found to be inhibited when recycling of endocytosed E-cadherin was disrupted by bafilomycin treatment. The endocytosis and recycling of E-cadherin and of the transferrin receptor were similarly inhibited by potassium depletion and by bafilomycin treatment, and both proteins were accumulated in intracellular compartments by an 18°C temperature block, suggesting that endocytosis may occur via a clathrin-mediated pathway. We conclude that a pool of surface E-cadherin is constantly trafficked through an endocytic, recycling pathway and that this may provide a mechanism for regulating the availability of E-cadherin for junction formation in development, tissue remodeling, and tumorigenesis.

Ciliopathies and the Kidney: A Review.

Publisher: Elsevier BV

Date: 03-2020

DOI: 10.1053/J.AJKD.2020.08.012

The murine neutrophil NLRP3 inflammasome is activated by soluble but not particulate or crystalline agonists

Publisher: Wiley

Date: 02-2016

DOI: 10.1002/EJI.201545943

Abstract: Neutrophils express pattern recognition receptors (PRRs) and regulate immune responses via PRR-dependent cytokine production. An emerging theme is that neutrophil PRRs often exhibit cell type-specific adaptations in their signalling pathways. This prompted us to examine inflammasome signalling by the PRR NLRP3 in murine neutrophils, in comparison to well-established NLRP3 signalling pathways in macrophages. Here, we demonstrate that while murine neutrophils can indeed signal via the NLRP3 inflammasome, neutrophil NLRP3 selectively responds to soluble agonists but not to the particulate/crystalline agonists that trigger NLRP3 activation in macrophages via phagolysosomal rupture. In keeping with this, alum did not trigger IL-1β production from human PMN, and the lysosomotropic peptide Leu-Leu-OMe stimulated only weak NLRP3-dependent IL-1β production from murine neutrophils, suggesting that lysosomal rupture is not a strong stimulus for NLRP3 activation in neutrophils. We validated our in vitro findings for poor neutrophil NLRP3 responses to particles in vivo, where we demonstrated that neutrophils do not significantly contribute to alum-induced IL-1β production in mice. In all, our studies highlight that myeloid cell identity and the nature of the danger signal can strongly influence signalling by a single PRR, thus shaping the nature of the resultant immune response.

Cavin‐1/PTRF alters prostate cancer cell‐derived extracellular vesicle content and internalization to attenuate extracellular vesicle‐mediated osteoclastogenesis and osteoblast proliferation

Publisher: Wiley

Date: 2014

DOI: 10.3402/JEV.V3.23784

Distinctive populations of basement membrane and cell membrane heparan sulfate proteoglycans are produced by cultured cell lines

Publisher: Rockefeller University Press

Date: 07-1987

DOI: 10.1083/JCB.105.1.529

Abstract: We have investigated the nature and distribution of different populations of heparan sulfate proteoglycans (HSPGs) in several cell lines in culture. Clone 9 hepatocytes and NRK and CHO cells were biosynthetically labeled with 35SO4, and proteoglycans were isolated by DEAE-Sephacel chromatography. Heterogeneous populations of HSPGs and chondroitin/dermatan proteoglycans (CSPGs) were found in the media and cell layer extracts of all cultures. HSPGs were further purified from the media and cell layers and separated from CSPGs by ion exchange chromatography after chondroitinase ABC digestion. In all cell types, HSPGs were found both in the cell layers (20-70% of the total) as well as the medium. When the purified HSPG fractions were further separated by octyl-Sepharose chromatography, very little HSPG in the incubation media bound to the octyl-Sepharose, whereas 40-55% of that in the cell layers bound and could be eluted with 1% Triton X-100. This hydrophobic population most likely consists of membrane-intercalated HSPGs. Basement membrane-type HSPGs were identified by immunoprecipitation as a component (30-80%) of the unbound (nonhydrophobic) HSPG fraction. By immunofluorescence, basement membrane-type HSPGs were distributed in a reticular network in Clone 9 and NRK cell monolayers by immunoelectron microscopy, these HSPGs were localized to irregular clumps of extracellular matrix located beneath and between cells. The cells did not produce a morphologically recognizable basement membrane layer under these culture conditions. When membrane-associated HSPGs were localized by immunoelectron microscopy, they were found in a continuous layer along the cell membrane of all cell types. The results demonstrate that two antigenically distinct populations of HSPG--an extracellular matrix and a membrane-intercalated population--are found at the surface of several different cultured cells lines these populations can be distinguished from one another by differences in their distribution in the monolayers by immunocytochemistry and can be separated by hydrophobic chromatography and basement membrane-type HSPGs are secreted and deposited in the extracellular matrix by cultured cells even though they do not produce a bona fide basement membrane-like layer.

Evaluation of left ventricle systolic and diastolic functions by tissue doppler echocardiography in children with down syndrome

Publisher: Briefland

Date: 29-08-2017

DOI: 10.5812/IJP.5735

VAMP3 regulates podosome organisation in macrophages and together with Stx4/SNAP23 mediates adhesion, cell spreading and persistent migration

Publisher: Elsevier BV

Date: 08-2011

DOI: 10.1016/J.YEXCR.2011.04.016

Abstract: The ability of cells to adhere, spread and migrate is essential to many physiological processes, particularly in the immune system where cells must traffic to sites of inflammation and injury. By altering the levels of in idual components of the VAMP3/Stx4/SNAP23 complex we show here that this SNARE complex regulates efficient macrophage adhesion, spreading and migration on fibronectin. During cell spreading this complex mediates the polarised exocytosis of VAMP3-positive recycling endosome membrane into areas of membrane expansion, where VAMP3's surface partner Q-SNARE complex Stx4/SNAP23 was found to accumulate. Lowering the levels of VAMP3 in spreading cells resulted in a more rounded cell morphology and most cells were found to be devoid of the typical ring-like podosome superstructures seen normally in spreading cells. In migrating cells lowering VAMP3 levels disrupted the polarised localisation of podosome clusters. The reduced trafficking of recycling endosome membrane to sites of cell spreading and the disorganised podosome localisation in migrating macrophages greatly reduced their ability to persistently migrate on fibronectin. Thus, this important SNARE complex facilitates macrophage adhesion, spreading, and persistent macrophage migration on fibronectin through the delivery of VAMP3-positive membrane with its cargo to expand the plasma membrane and to participate in organising adhesive podosome structures.

The cell surface environment for pathogen recognition and entry

Publisher: Wiley

Date: 04-2016

DOI: 10.1038/CTI.2016.15

Xenopus borealis as an alternative source of oocytes for biophysical and pharmacological studies of neuronal ion channels

Publisher: Springer Science and Business Media LLC

Date: 06-10-2015

DOI: 10.1038/SREP14763

Abstract: For the past 30 years, oocytes from Xenopus laevis have been extensively used to express and characterise ion channels in an easily controlled environment. Here we report the first use of oocytes from the closely related species Xenopus borealis as an alternative expression system for neuronal ion channels. Using the two-electrode voltage-cl technique, we show that a wide variety of voltage- and ligand-gated ion channels have the same channel properties and pharmacological profiles when expressed in either X. laevis or X. borealis oocytes. Potential advantages of the X. borealis oocytes include a smaller endogenous chloride current and the ability to produce more intense fluorescence signals when studied with voltage-cl fluorometry. Scanning electron microscopy revealed a difference in vitelline membrane structure between the two species, which may be related to the discrepancy in fluorescence signals observed. We demonstrate that X. borealis oocytes are a viable heterologous system for expression of neuronal ion channels with some potential advantages over X. laevis oocytes for certain applications.

A Dileucine Motif Targets E-cadherin to the Basolateral Cell Surface in Madin-Darby Canine Kidney and LLC-PK1 Epithelial Cells

Publisher: Elsevier BV

Date: 06-2001

DOI: 10.1074/JBC.M101907200

Class IIa Histone Deacetylases Drive Toll-like Receptor-Inducible Glycolysis and Macrophage Inflammatory Responses via Pyruvate Kinase M2.

Publisher: Elsevier BV

Date: 02-2020

DOI: 10.1016/J.CELREP.2020.02.007

Syntaxin 6 and Vti1b Form a Novel SNARE Complex, Which Is Up-regulated in Activated Macrophages to Facilitate Exocytosis of Tumor Necrosis Factor-α

Publisher: Elsevier BV

Date: 03-2005

DOI: 10.1074/JBC.M414420200

The Rho GTPase Rac1 is required for recycling endosome‐mediated secretion of TNF in macrophages

Publisher: Wiley

Date: 17-12-2013

DOI: 10.1038/ICB.2013.90

Abstract: Rho GTPases are required for many cellular events such as adhesion, motility, and membrane trafficking. Here we show that in macrophages, the Rho GTPases Rac1 and Cdc42 are involved in lamellipodia and filopodia formation, respectively, and that both of these Rho GTPases are essential for the efficient surface delivery of tumor necrosis factor (TNF) to the plasma membrane following TLR4 stimulation. We have previously demonstrated intracellular trafficking of TNF via recycling endosomes in lipopolysaccharide (LPS)-activated macrophages. Here, we further define a specific role for Rac1 in intracellular TNF trafficking, demonstrating impairment in TNF release following TLR4 stimulation in the presence of a Rac inhibitor, in cells expressing a dominant negative (DN) form of Rac1, and following small interfering RNA (siRNA) knockdown of Rac1. Rac1 activity was required for TNF trafficking but not for TLR4 signaling following LPS stimulation. Reduced TNF secretion was due to a defect in Rac1 activity, but not of the closely related Rho GTPase Rac2, demonstrated by the additional use of macrophages derived from Rac2-deficient mice. Labeling recycling endosomes by the uptake of fluorescent transferrin enabled us to show that Rac1 was required for the final stages of TNF trafficking and delivery from recycling endosomes to the plasma membrane. Thus, actin remodeling by the Rho GTPase Rac1 is required for TNF cell surface delivery and release from macrophages.

Macrophage Nrf 2 the rescue

Publisher: Rockefeller University Press

Date: 22-05-2023

DOI: 10.1083/JCB.202305036

Abstract: The exuberant phagocytosis of apoptotic cell corpses by macrophages in Drosophila embryos creates highly oxidative environments. Stow and Sweet discuss work from Clemente and Weavers (2023. J. Cell Biol.0.1083/jcb.202203062) showing for the first time how macrophage Nrf2 is primed to help sustain immune function and mitigate bystander oxidative damage.

SCIMP is a transmembrane non-TIR TLR adaptor that promotes proinflammatory cytokine production from macrophages

Publisher: Springer Science and Business Media LLC

Date: 18-01-2017

DOI: 10.1038/NCOMMS14133

Abstract: Danger signals activate Toll-like receptors (TLRs), thereby initiating inflammatory responses. Canonical TLR signalling, via Toll/Interleukin-1 receptor domain (TIR)-containing adaptors and proinflammatory transcription factors such as NF-κB, occurs in many cell types however, additional mechanisms are required for specificity of inflammatory responses in innate immune cells. Here we show that SCIMP, an immune-restricted, transmembrane adaptor protein (TRAP), promotes selective proinflammatory cytokine responses by direct modulation of TLR4. SCIMP is a non-TIR-containing adaptor, binding directly to the TLR4-TIR domain in response to lipopolysaccharide. In macrophages, SCIMP is constitutively associated with the Lyn tyrosine kinase, is required for tyrosine phosphorylation of TLR4, and facilitates TLR-inducible production of the proinflammatory cytokines IL-6 and IL-12p40. Point mutations in SCIMP abrogating TLR4 binding also prevent SCIMP-mediated cytokine production. SCIMP is, therefore, an immune-specific TLR adaptor that shapes host defence and inflammation.

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

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 14-09-2007

DOI: 10.1161/CIRCRESAHA.107.157198

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

Rab11 in recycling endosomes regulates the sorting and basolateral transport of E-cadherin.

Publisher: American Society for Cell Biology (ASCB)

Date: 04-2005

DOI: 10.1091/MBC.E04-10-0867

Abstract: E-cadherin plays an essential role in cell polarity and cell-cell adhesion however, the pathway for delivery of E-cadherin to the basolateral membrane of epithelial cells has not been fully characterized. We first traced the post-Golgi, exocytic transport of GFP-tagged E-cadherin (Ecad-GFP) in unpolarized cells. In live cells, Ecad-GFP was found to exit the Golgi complex in pleiomorphic tubulovesicular carriers, which, instead of moving directly to the cell surface, most frequently fused with an intermediate compartment, subsequently identified as a Rab11-positive recycling endosome. In MDCK cells, basolateral targeting of E-cadherin relies on a dileucine motif. Both E-cadherin and a targeting mutant, ΔS1-E-cadherin, colocalized with Rab11 and fused with the recycling endosome before erging to basolateral or apical membranes, respectively. In polarized and unpolarized cells, coexpression of Rab11 mutants disrupted the cell surface delivery of E-cadherin and caused its mistargeting to the apical membrane, whereas apical ΔS1-E-cadherin was unaffected. We thus demonstrate a novel pathway for Rab11 dependent, dileucine-mediated, μ1B-independent sorting and basolateral trafficking, exemplified by E-cadherin. The recycling endosome is identified as an intermediate compartment for the post-Golgi trafficking and exocytosis of E-cadherin, with a potentially important role in establishing and maintaining cadherin-based adhesion.

The cyclic cystine knot miniprotein MCoTI-II is internalized into cells by macropinocytosis

Publisher: Elsevier BV

Date: 2007

DOI: 10.1016/J.BIOCEL.2007.06.016

Abstract: The cyclotides are macrocyclic knotted proteins characterized by a compact topology and exceptional stability. Accordingly it has been hypothesized that they may be useful as protein engineering frameworks for the stabilization and delivery of bioactive peptide sequences. This study examined the internalization of cyclotides into mammalian cells, a vital step for the delivery of bioactive peptide sequences to intracellular targets. Although the entry of various linear peptides into cells has been reported previously, this is the first report of internalization of a macrocyclic peptide. Cell uptake was examined for representatives of two cyclotide subfamilies the first was MCoTI-II, a member of the trypsin inhibitor subfamily, which was internalized by a macrophage and breast cancer cell line and the second, the prototypic cyclotide kalata B1 from the Möbius subfamily, which remained extracellular. Biotin labeled MCoTI-II entered macrophages by macropinocytosis, resulting in vesicular encapsulation without trafficking to lysosomes for degradation. The ready uptake, coupled with low cytotoxicity, indicates that MCoTI-II has the potential to transport grafted bioactivities to intracellular targets, making it a potentially valuable framework in drug design applications.

GAIP, a Galphai-3-binding protein, is associated with Golgi-derived vesicles and protein trafficking

Publisher: American Physiological Society

Date: 02-1999

DOI: 10.1152/AJPCELL.1999.276.2.C497

Abstract: Proteins of the regulators of G protein signaling (RGS) family bind to Gα subunits to downregulate their signaling in a variety of systems. Gα-interacting protein (GAIP) is a mammalian RGS protein that shows high affinity for the activated state of Gα i-3 , a protein known to regulate post-Golgi trafficking of secreted proteins in kidney epithelial cells. This study aimed to localize GAIP in epithelial cells and to investigate its potential role in the regulation of membrane trafficking. LLC-PK 1 cells were stably transfected with a c- myc-tagged GAIP cDNA. In the transfected and untransfected cells, GAIP was found in the cytosol and on cell membranes. Immunogold labeling showed that membrane-bound GAIP was localized on budding vesicles around Golgi stacks. When an in vitro assay was used to generate vesicles from isolated rat liver and Madin-Darby canine kidney cell Golgi membranes, GAIP was found to be concentrated in fractions of newly budded Golgi vesicles. Finally, the constitutive trafficking and secretion of sulfated proteoglycans was measured in cell lines overexpressing GAIP. We show evidence for GAIP regulation of secretory trafficking before the level of the trans-Golgi network but not in post-Golgi secretion. The location and functional effects of GAIP overlap only partially with those of Gα i-3 and suggest multiple roles for GAIP in epithelial cells.

Active Rab11 and functional recycling endosome are required for E-cadherin trafficking and lumen formation during epithelial morphogenesis

Publisher: American Physiological Society

Date: 08-2008

DOI: 10.1152/AJPCELL.00097.2008

Abstract: The correct targeting and trafficking of the adherens junction protein epithelial cadherin (E-cadherin) is a major determinant for the acquisition of epithelial cell polarity and for the maintenance of epithelial integrity. The compartments and trafficking components required to sort and transport E-cadherin to the basolateral cell surface remain to be fully defined. On the basis of previous data, we know that E-cadherin is trafficked via the recycling endosome (RE) in nonpolarized and newly polarized cells. Here we explore the role of the RE throughout epithelial morphogenesis in MDCK monolayers and cysts. Time-lapse microscopy in live cells, altering RE function biochemically, and expressing a dominant-negative form of Rab11 (DN-Rab11), each showed that the RE is always requisite for E-cadherin sorting and trafficking. The RE remained important for E-cadherin trafficking in MDCK cells from a nonpolarized state through to fully formed, polarized epithelial monolayers. During the development of epithelial cysts, DN-Rab11 disrupted E-cadherin targeting and trafficking, the subapical localization of pERM and actin, and cyst lumen formation. This final effect demonstrated an early and critical interdependence of Rab11 and the RE for E-cadherin targeting, apical membrane formation, and cell polarity in cysts.

Guanine nucleotide exchange factors activate Rab8a for Toll-like receptor signalling

Publisher: Informa UK Limited

Date: 07-03-2019

DOI: 10.1080/21541248.2019.1587278

Interleukin-1β Maturation Triggers Its Relocation to the Plasma Membrane for Gasdermin-D-Dependent and -Independent Secretion

Publisher: Elsevier BV

Date: 08-2018

DOI: 10.1016/J.CELREP.2018.07.027

Abstract: IL-1β requires processing by caspase-1 to generate the active, pro-inflammatory cytokine. Acute IL-1β secretion from inflammasome-activated macrophages requires caspase-1-dependent GSDMD cleavage, which also induces pyroptosis. Mechanisms of IL-1β secretion by pyroptotic and non-pyroptotic cells, and the precise functions of caspase-1 and GSDMD therein, are unresolved. Here, we show that, while efficient early secretion of endogenous IL-1β from primary non-pyroptotic myeloid cells in vitro requires GSDMD, later IL-1β release in vitro and in vivo proceeds independently of GSDMD. IL-1β maturation is sufficient for slow, caspase-1/GSDMD-independent secretion of ectopic IL-1β from resting, non-pyroptotic macrophages, but the speed of IL-1β release is boosted by inflammasome activation, via caspase-1 and GSDMD. IL-1β cleavage induces IL-1β enrichment at PIP2-enriched plasma membrane ruffles, and this is a prerequisite for IL-1β secretion and is mediated by a polybasic motif within the cytokine. We thus reveal a mechanism in which maturation-induced IL-1β trafficking facilitates its unconventional secretion.

Receptor function, dominant negative activity and phenotype correlations for MC1R variant alleles

Publisher: Oxford University Press (OUP)

Date: 27-06-2007

DOI: 10.1093/HMG/DDM177

Abstract: The human melanocortin-1 receptor (MC1R) is a G-protein coupled receptor involved in the regulation of pigmentation. Several MC1R variant alleles are associated with red hair, fair skin and increased skin cancer risk. We have performed a systematic functional analysis of nine common MC1R variants and correlated these results with the strength of the genetic association of each variant allele with pigmentation phenotypes. In vitro expression studies revealed that variant receptors with reduced cell surface expression, including V60L, D84E, R151C, I155T, R160W and R163Q, showed a corresponding impairment in cAMP coupling. The R142H and D294H variants demonstrated normal cell surface expression, but had reduced functional responses, indicating that altered G-protein coupling may be responsible for this loss of function. The V92M variant cAMP activation was equal to or higher than that for wild-type MC1R. In co-expression studies, the D84E, R151C, I155T and R160W variants showed a dominant negative effect on wild-type receptor cell surface expression, which was reflected in a decreased ability to elevate intracellular cAMP levels. The D294H variant also demonstrated a dominant negative effect on wild-type MC1R cAMP signalling, but had no effect on wild-type surface expression. Importantly, comparison of the in vitro receptor characteristics with skin and hair colour data of in iduals both homozygous and heterozygous for MC1R variant alleles revealed parallels between variant MC1R cell surface expression, functional ability, dominant negative activity and their effects on human pigmentation. These findings show the first direct correlations between variant MC1R biochemical properties and pigmentation phenotype.

Intracellular trafficking and secretion of inflammatory cytokines

Publisher: Elsevier BV

Date: 06-2013

DOI: 10.1016/J.CYTOGFR.2013.04.001

Abstract: The secretion of cytokines by immune cells plays a significant role in determining the course of an inflammatory response. The levels and timing of each cytokine released are critical for mounting an effective but confined response, whereas excessive or dysregulated inflammation contributes to many diseases. Cytokines are both culprits and targets for effective treatments in some diseases. The multiple points and mechanisms that have evolved for cellular control of cytokine secretion highlight the potency of these mediators and the fine tuning required to manage inflammation. Cytokine production in cells is regulated by cell signaling, and at mRNA and protein synthesis levels. Thereafter, the intracellular transport pathways and molecular trafficking machinery have intricate and essential roles in dictating the release and activity of cytokines. The trafficking machinery and secretory (exocytic) pathways are complex and highly regulated in many cells, involving specialized membranes, molecules and organelles that enable these cells to deliver cytokines to often-distinct areas of the cell surface, in a timely manner. This review provides an overview of secretory pathways - both conventional and unconventional - and key families of trafficking machinery. The prevailing knowledge about the trafficking and secretion of a number of in idual cytokines is also summarized. In conclusion, we present emerging concepts about the functional plasticity of secretory pathways and their modulation for controlling cytokines and inflammation.

The myotubularin phosphatase MTMR4 regulates sorting from early endosomes

Publisher: The Company of Biologists

Date: 15-09-2010

DOI: 10.1242/JCS.060103

Abstract: Phosphatidylinositol 3-phosphate [PtdIns(3)P] regulates endocytic trafficking and the sorting of receptors through early endosomes, including the rapid recycling of transferrin (Tfn). However, the phosphoinositide phosphatase that selectively opposes this function is unknown. The myotubularins are a family of eight catalytically active and six inactive enzymes that hydrolyse PtdIns(3)P to form PtdIns. However, the role each myotubularin family member plays in regulating endosomal PtdIns(3)P and thereby endocytic trafficking is not well established. Here, we identify the myotubularin family member MTMR4, which localizes to early endosomes and also to Rab11- and Sec15-positive recycling endosomes. In cells with MTMR4 knockdown, or following expression of the catalytically inactive MTMR4, MTMR4C407A, the number of PtdIns(3)P-decorated endosomes significantly increased. MTMR4 overexpression delayed the exit of Tfn from early endosomes and its recycling to the plasma membrane. By contrast, expression of MTMR4C407A, which acts as a dominant-negative construct, significantly accelerated Tfn recycling. However, in MTMR4 knockdown cells Tfn recycling was unchanged, suggesting that other MTMs might also contribute to recycling. MTMR4 regulated the subcellular distribution of Rab11 and, in cells with RNAi-mediated knockdown of MTMR4, Rab11 was directed away from the pericentriolar recycling compartment. The subcellular distribution of VAMP3, a v-SNARE protein that resides in recycling endosomes and endosome-derived transport vesicles, was also regulated by MTMR4. Therefore, MTMR4 localizes at the interface of early and recycling endosomes to regulate trafficking through this pathway.

Protein kinase C regulates endocytosis and recycling of E-cadherin

Publisher: American Physiological Society

Date: 08-2002

DOI: 10.1152/AJPCELL.00566.2001

Abstract: E-cadherin is a major component of adherens junctions in epithelial cells. We showed previously that a pool of cell surface E-cadherin is constitutively internalized and recycled back to the surface. In the present study, we investigated the potential role of protein kinase C (PKC) in regulating the trafficking of surface E-cadherin in Madin-Darby canine kidney cells. Using surface biotinylation and immunofluorescence, we found that treatment of cells with phorbol esters increased the rate of endocytosis of E-cadherin, resulting in accumulation of E-cadherin in apically localized early or recycling endosomes. The recycling of E-cadherin back to the surface was also decreased in the presence of phorbol esters. Phorbol ester-induced endocytosis of E-cadherin was blocked by specific inhibitors, implicating novel PKC isozymes, such as PKC-ε in this pathway. PKC activation led to changes in the actin cytoskeleton facilitating E-cadherin endocytosis. Depolymerization of actin increased endocytosis of E-cadherin, whereas the PKC-induced uptake of E-cadherin was blocked by the actin stabilizer jasplakinolide. Our findings show that PKC regulates vital steps of E-cadherin trafficking, its endocytosis, and its recycling.

The trans‐Golgi Network Golgin, GCC185, is Required for Endosome‐to‐Golgi Transport and Maintenance of Golgi Structure

Publisher: Wiley

Date: 04-05-2007

DOI: 10.1111/J.1600-0854.2007.00563.X

Abstract: Four mammalian golgins are specifically targeted to the trans-Golgi network (TGN) membranes via their C-terminal GRIP domains. The TGN golgins, p230/golgin-245 and golgin-97, are recruited via the GTPase Arl1, whereas the TGN golgin GCC185 is recruited independently of Arl1. Here we show that GCC185 is localized to a region of the TGN distinct from Arl1 and plays an essential role in maintaining the organization of the Golgi apparatus. Using both small interfering RNA (siRNA) and microRNA (miRNA), we show that depletion of GCC185 in HeLa cells frequently resulted in fragmentation of the Golgi apparatus. Golgi apparatus fragments were dispersed throughout the cytoplasm and contained both cis and trans markers. Trafficking of anterograde and retrograde cargo was analysed over an extended period following GCC185 depletion. Early effects of GCC185 depletion included a perturbation in the distribution of the mannose-6-phosphate receptor and a block in shiga toxin trafficking to the Golgi apparatus, which occurred in parallel with the fragmentation of the Golgi ribbon. Internalized shiga toxin accumulated in Rab11-positive endosomes, indicating GCC185 is essential for transport between the recycling endosome and the TGN. In contrast, the plasma membrane-TGN recycling protein TGN38 was efficiently transported into GCC185-depleted Golgi apparatus fragments throughout a 96-h period, and anterograde transport of E-cadherin was functional until a late stage of GCC185 depletion. This study demonstrated (i) a more effective long-term depletion of GCC185 using miRNA than siRNA and (ii) a dual role for the GCC185 golgin in the regulation of endosome-to-TGN membrane transport and in the organization of the Golgi apparatus.

Fluid-phase markers in the basolateral endocytic pathway accumulate in response to the actin assembly-promoting drug Jasplakinolide

Publisher: American Society for Cell Biology (ASCB)

Date: 04-1998

DOI: 10.1091/MBC.9.4.957

Abstract: To investigate the role of filamentous actin in the endocytic pathway, we used the cell-permeant drug Jasplakinolide (JAS) to polymerize actin in intact polarized Madin–Darby canine kidney (MDCK) cells. The uptake and accumulation of the fluid-phase markers fluorescein isothiocyanate (FITC)-dextran and horseradish peroxidase (HRP) were followed in JAS-treated or untreated cells with confocal fluorescence microscopy, biochemical assays, and electron microscopy. Pretreatment with JAS increased the uptake and accumulation of fluid-phase markers in MDCK cells. JAS increased endocytosis in a polarized manner, with a marked effect on fluid-phase uptake from the basolateral surface but not from the apical surface of polarized MDCK cells. The early uptake of FITC-dextran and HRP was increased more than twofold in JAS-treated cells. At later times, FITC-dextran and HRP accumulated in clustered endosomes in the basal and middle regions of JAS-treated cells. The large accumulated endosomes were similar to late endosomes but they were not colabeled for other late endosome markers, such as rab7 or mannose-6-phosphate receptor. JAS altered transport in the endocytic pathway at a later stage than the microtubule-dependent step affected by nocodazole. JAS also had a notable effect on cell morphology, inducing membrane bunching at the apical pole of MDCK cells. Although other studies have implicated actin in endocytosis at the apical cell surface, our results provide novel evidence that filamentous actin is also involved in the endocytosis of fluid-phase markers from the basolateral membrane of polarized cells.

Different NK cell–activating receptors preferentially recruit Rab27a or Munc13-4 to perforin-containing granules for cytotoxicity

Publisher: American Society of Hematology

Date: 05-11-2009

DOI: 10.1182/BLOOD-2009-06-225359

Abstract: The autosomal recessive immunodeficiencies Griscelli syndrome type 2 (GS2) and familial hemophagocytic lymphohistiocytosis type 3 (FHL3) are associated with loss-of-function mutations in RAB27A (encoding Rab27a) and UNC13D (encoding Munc13-4). Munc13-4 deficiency abrogates NK-cell release of perforin-containing lytic granules induced by signals for natural and antibody-dependent cellular cytotoxicity. We demonstrate here that these signals fail to induce degranulation in resting NK cells from Rab27a-deficient patients. In resting NK cells from healthy subjects, endogenous Rab27a and Munc13-4 do not colocalize extensively with perforin. However, phorbol 12-myristate 13-acetate and ionomycin stimulation or conjugation to susceptible target cells induced myosin-dependent colocalization of Rab27a and Munc13-4 with perforin. Unexpectedly, in idual engagement of receptors leukocyte functional antigen-1, NKG2D, or 2B4 induced colocalization of Rab27a, but not Munc13-4, with perforin. Conversely, engagement of antibody-dependent cellular cytotoxicity receptor CD16 induced colocalization of Munc13-4, but not Rab27a, with perforin. Furthermore, colocalization of Munc13-4 with perforin was Rab27a-dependent. In conclusion, Rab27a or Munc13-4 recruitment to lytic granules is preferentially regulated by different receptor signals, demonstrating that in idual target cell ligands regulate discrete molecular events for lytic granule maturation. The data suggest Rab27a facilitates degranulation at an early step yet highlight a reciprocal relationship between Munc13-4 and Rab27a for degranulation.

Heparan sulfate proteoglycans are concentrated on the sinusoidal plasmalemmal domain and in intracellular organelles of hepatocytes

Publisher: Rockefeller University Press

Date: 03-1985

DOI: 10.1083/JCB.100.3.975

Abstract: The distribution of cell surface heparan sulfate proteoglycans (HSPGs) was determined in rat liver by immunocytochemistry. A polyclonal antibody was raised against HSPGs purified from rat liver microsomes which specifically immunoprecipitated liver membrane HSPGs. It was shown to recognize both the heparin-releasable and membrane-intercalated form of membrane HSPGs and to recognize determinants on the core protein of these HSPGs. By immunocytochemistry membrane HSPGs were localized to hepatocytes. The distribution of HSPGs at the cell surface of the hepatocyte was restricted to the sinusoidal domain of the plasmalemma there was little or no staining of the lateral or bile canalicular domains. Intracellularly, HSPGs were occasionally detected in cisternae of the rough endoplasmic reticulum and were regularly found in Golgi cisternae--usually distributed across the entire Golgi stack. HSPGs were also localized in some endosomes, lysosomes, and cytoplasmic vesicles of hepatocytes. We conclude that the HSPGs recognized by this antibody have a restricted distribution in rat liver: they are largely confined to the sinusoidal plasmalemmal domain and to biosynthetic and endocytic compartments of hepatocytes.

Meeting report – Arf and Rab family G proteins

Publisher: The Company of Biologists

Date: 12-2013

DOI: 10.1242/JCS.143610

Abstract: A FASEB Summer Research Conference entitled ‘Arf and Rab family G proteins’ was held in July 2013 at Snowmass Village, Snowmass, Colorado. Arfs and Rabs are two families of GTPases that control membrane trafficking in eukaryotic cells, and increasing evidence indicates that their functions are tightly coordinated. Because many workers in this field have focused on only one family, this meeting was designed to integrate our understanding of the two families. The conference was organized by Elizabeth Sztul (University of Alabama, Birmingham, USA) and Jim Casanova (University of Virginia, Charlottesville, USA), and provided an opportunity for ∼90 scientists to communicate their work and discuss future directions for the field. The talks highlighted the structural, functional and regulatory properties of Arf and Rab GTPases and the need to develop coordinated approaches to investigate them. Here, we present the major themes that emerged from the meeting.

Vesicle budding on Golgi membranes: Regulation by G proteins and myosin motors

Publisher: Elsevier BV

Date: 08-1998

DOI: 10.1016/S0167-4889(98)00055-X

Abstract: One of the main functions of the Golgi complex is to generate transport vesicles for the post-Golgi trafficking of proteins in secretory pathways. Many different populations of vesicles are distinguished by unique sets of structural and regulatory proteins which participate in vesicle budding and fusion. Monomeric and heterotrimeric G proteins regulate vesicle budding and secretory traffic into and out of the Golgi complex. An inventory of G protein alpha subunits associated with Golgi membranes highlights their erse involvement and potential for coupling Golgi trafficking, through various signal transduction pathways, to cell growth or other more specialized cell functions. Cytoskeletal proteins are now also known to associate specifically with the Golgi complex and Golgi-derived vesicles. Amongst these, conventional and unconventional myosins are recruited to vesicle membranes. Several roles in vesicle budding and vesicle trafficking can be proposed for these actin-based motors.

Automated Analysis of Cell Surface Ruffling: Ruffle Quantification Macro.

Publisher: Bio-Protocol, LLC

Date: 2020

DOI: 10.21769/BIOPROTOC.3494

Rodent blood-stage Plasmodium survive in dendritic cells that infect naive mice

Publisher: Proceedings of the National Academy of Sciences

Date: 20-06-2011

DOI: 10.1073/PNAS.1108579108

Abstract: Plasmodium spp. parasites cause malaria in 300 to 500 million in iduals each year. Disease occurs during the blood-stage of the parasite's life cycle, where the parasite is thought to replicate exclusively within erythrocytes. Infected in iduals can also suffer relapses after several years, from Plasmodium vivax and Plasmodium ovale surviving in hepatocytes. Plasmodium falciparum and Plasmodium malariae can also persist after the original bout of infection has apparently cleared in the blood, suggesting that host cells other than erythrocytes (but not hepatocytes) may harbor these blood-stage parasites, thereby assisting their escape from host immunity. Using blood stage transgenic Plasmodium berghei -expressing GFP (PbGFP) to track parasites in host cells, we found that the parasite had a tropism for CD317 + dendritic cells. Other studies using confocal microscopy, in vitro cultures, and cell transfer studies showed that blood-stage parasites could infect, survive, and replicate within CD317 + dendritic cells, and that small numbers of these cells released parasites infectious for erythrocytes in vivo. These data have identified a unique survival strategy for blood-stage Plasmodium , which has significant implications for understanding the escape of Plasmodium spp. from immune-surveillance and for vaccine development.

A heterotrimeric G protein, G alpha i-3, on Golgi membranes regulates the secretion of a heparan sulfate proteoglycan in LLC-PK1 epithelial cells

Publisher: Rockefeller University Press

Date: 15-09-1991

DOI: 10.1083/JCB.114.6.1113

Abstract: A heterotrimeric G alpha i subunit, alpha i-3, is localized on Golgi membranes in LLC-PK1 and NRK epithelial cells where it colocalizes with mannosidase II by immunofluorescence. The alpha i-3 was found to be localized on the cytoplasmic face of Golgi cisternae and it was distributed across the whole Golgi stack. The alpha i-3 subunit is found on isolated rat liver Golgi membranes by Western blotting and G alpha i-3 on the Golgi apparatus is ADP ribosylated by pertussis toxin. LLC-PK1 cells were stably transfected with G alpha i-3 on an MT-1, inducible promoter in order to overexpress alpha i-3 on Golgi membranes. The intracellular processing and constitutive secretion of the basement membrane heparan sulfate proteoglycan (HSPG) was measured in LLC-PK1 cells. Overexpression of alpha i-3 on Golgi membranes in transfected cells retarded the secretion of HSPG and accumulated precursors in the medial-trans-Golgi. This effect was reversed by treatment of cells with pertussis toxin which results in ADP-ribosylation and functional uncoupling of G alpha i-3 on Golgi membranes. These results provide evidence for a novel role for the pertussis toxin sensitive G alpha i-3 protein in Golgi trafficking of a constitutively secreted protein in epithelial cells.

Mammalian GRIP domain proteins differ in their membrane binding properties and are recruited to distinct domains of the TGN

Publisher: The Company of Biologists

Date: 15-11-2004

DOI: 10.1242/JCS.01497

Abstract: The four mammalian golgins, p230/golgin-245, golgin-97, GCC88 and GCC185 are targeted to trans-Golgi network (TGN) membranes by their C-terminal GRIP domain in a G-protein-dependent process. The Arf-like GTPase, Arl1, has been shown to mediate TGN recruitment of p230/golgin245 and golgin-97 by interaction with their GRIP domains however, it is not known whether all the TGN golgins bind to Arl1 and whether they are all recruited to the same or different TGN domains. Here we demonstrate differences in membrane binding properties and TGN domain recruitment of the mammalian GRIP domain proteins. Overexpression of full-length GCC185 resulted in the appearance of small punctate structures dispersed in the cytoplasm of transfected cells that were identified as membrane tubular structures by immunoelectron microscopy. The cytoplasmic GCC185-labelled structures were enriched for membrane binding determinants of GCC185 GRIP, whereas the three other mammalian GRIP family members did not colocalize with the GCC185-labelled structures. These GCC185-labelled structures included the TGN resident protein α2,6 sialyltransferase and excluded the recycling TGN protein, TGN46. The Golgi stack was unaffected by overexpression of GCC185. Overexpression of both full-length GCC185 and GCC88 showed distinct and nonoverlapping structures. We also show that the GRIP domains of GCC185 and GCC88 differ in membrane binding properties from each other and, in contrast to p230/golgin-245 and golgin-97, do not interact with Arl1 in vivo. Collectively these results show that GCC88, GCC185 and p230/golgin245 are recruited to functionally distinct domains of the TGN and are likely to be important for the maintenance of TGN subdomain structure, a critical feature for mediating protein sorting and membrane transport.

RP105 Engages Phosphatidylinositol 3-Kinase p110δ To Facilitate the Trafficking and Secretion of Cytokines in Macrophages during Mycobacterial Infection

Publisher: The American Association of Immunologists

Date: 15-10-2015

DOI: 10.4049/JIMMUNOL.1500017

Abstract: Cytokines are key regulators of adequate immune responses to infection with Mycobacterium tuberculosis. We demonstrate that the p110δ catalytic subunit of PI3K acts as a downstream effector of the TLR family member RP105 (CD180) in promoting mycobacteria-induced cytokine production by macrophages. Our data show that the significantly reduced release of TNF and IL-6 by RP105−/− macrophages during mycobacterial infection was not accompanied by diminished mRNA or protein expression. Mycobacteria induced comparable activation of NF-κB and p38 MAPK signaling in wild-type (WT) and RP105−/− macrophages. In contrast, mycobacteria-induced phosphorylation of Akt was abrogated in RP105−/− macrophages. The p110δ-specific inhibitor, Cal-101, and small interfering RNA–mediated knockdown of p110δ diminished mycobacteria-induced TNF secretion by WT but not RP105−/− macrophages. Such interference with p110δ activity led to reduced surface-expressed TNF in WT but not RP105−/− macrophages, while leaving TNF mRNA and protein expression unaffected. Activity of Bruton’s tyrosine kinase was required for RP105-mediated activation of Akt phosphorylation and TNF release by mycobacteria-infected macrophages. These data unveil a novel innate immune signaling axis that orchestrates key cytokine responses of macrophages and provide molecular insight into the functions of RP105 as an innate immune receptor for mycobacteria.

Fusion, Fission, and Secretion During Phagocytosis

Publisher: American Physiological Society

Date: 12-2007

DOI: 10.1152/PHYSIOL.00028.2007

Abstract: Phagocytosis is essential for the elimination of pathogens and for clearance of apoptotic bodies. The ingestion process entails extensive remodeling of the cellular membranes, particularly when large and/or multiple particles are engulfed. The membrane fusion and fission events that accompany phagocytosis are described. The coordinated sequence of membrane trafficking events required for phagocytosis involves multiple organelles and also serves other cellular functions, such as cytokine secretion.

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

DOI: 10.1371/JOURNAL.PONE.0046242

The ins and outs of E-cadherin trafficking

Publisher: Elsevier BV

Date: 08-2004

DOI: 10.1016/J.TCB.2004.07.007

Small GTPases in trafficking - a family approach: Introducing a rolling series focused on groups or families of small GTPases in trafficking

Publisher: Informa UK Limited

Date: 02-01-2016

DOI: 10.1080/21592799.2016.1178036

Phosphoinositide 3-kinase δ regulates membrane fission of Golgi carriers for selective cytokine secretion

Publisher: Rockefeller University Press

Date: 13-09-2010

DOI: 10.1083/JCB.201001028

Abstract: Phosphoinositide 3-kinase (PI3K) p110 isoforms are membrane lipid kinases classically involved in signal transduction. Lipopolysaccharide (LPS)-activated macrophages constitutively and abundantly secrete proinflammatory cytokines including tumor necrosis factor-α (TNF). Loss of function of the p110δ isoform of PI3K using inhibitors, RNA-mediated knockdown, or genetic inactivation in mice abolishes TNF trafficking and secretion, trapping TNF in tubular carriers at the trans-Golgi network (TGN). Kinase-active p110δ localizes to the Golgi complex in LPS-activated macrophages, and TNF is loaded into p230-labeled tubules, which cannot undergo fission when p110δ is inactivated. Similar blocks in fission of these tubules and in TNF secretion result from inhibition of the guanosine triphosphatase dynamin 2. These findings demonstrate a new function for p110δ as part of the membrane fission machinery required at the TGN for the selective trafficking and secretion of cytokines in macrophages.

A Leep1 into migration and macropinocytosis

Publisher: Rockefeller University Press

Date: 15-06-2021

DOI: 10.1083/JCB.202105141

Abstract: Actin organization underpins conserved functions at the leading edge of cells. In this issue, Yang et al. (2021. J. Cell Biol.0.1083/jcb.202010096) characterize Leep1 as a bi-functional regulator of migration and macropinocytosis through PIP3 and the Scar/WAVE complex.

SCIMP is a universal Toll-like receptor adaptor in macrophages

Publisher: Oxford University Press (OUP)

Date: 29-08-2019

DOI: 10.1002/JLB.2MA0819-138RR

Abstract: In innate immune cells, pathogens and danger signals activate TLRs, unleashing potent and tailored inflammatory responses. Previously, we reported that an immune-specific transmembrane adaptor, SLP adaptor and CSK interacting membrane protein (SCIMP), interacts with TLR4 via direct binding to its cytoplasmic TIR domain. SCIMP scaffolds a Src family kinase, Lyn, for TLR4 phosphorylation and activation. Consequently, SCIMP is able to direct selective production of the proinflammatory cytokines IL-6 and IL-12p40 downstream of TLR4 in macrophages. Here, we set out to investigate whether SCIMP also acts as an adaptor for other TLR family members. We report here that SCIMP is phosphorylated and activated in response to agonists of multiple TLRs, including TLR2, TLR3, TLR4, and TLR9. SCIMP also interacts with TLRs that are known to signal from both the cell surface and endosomal compartments. In so doing, this transmembrane adaptor presents Lyn, along with other effectors such as Grb2, Csk, and SLP65, to multiple TLRs during cellular activation. CRISPR-mediated knockout or silencing of SCIMP in macrophages alters TLR signaling outputs and the production of IL-6 and IL-12p40 downstream of multiple TLRs, and upon challenge with live bacteria. Furthermore, the selectivity in cytokine responses is preserved downstream of TLR3, with inducible expression of Il-12p40 and IL-6, but not IFNβ, being SCIMP dependent. SCIMP is thus a universal TLR adaptor for scaffolding the Lyn tyrosine kinase and its effectors to enable responses against a wide range of danger signals.

SNAREing immunity: the role of SNAREs in the immune system

Publisher: Springer Science and Business Media LLC

Date: 12-2006

DOI: 10.1038/NRI1980

Abstract: The trafficking of molecules and membranes within cells is a prerequisite for all aspects of cellular immune functions, including the delivery and recycling of cell-surface proteins, secretion of immune mediators, ingestion of pathogens and activation of lymphocytes. SNARE (soluble-N-ethylmaleimide-sensitive-factor accessory-protein receptor)-family members mediate membrane fusion during all steps of trafficking, and function in almost all aspects of innate and adaptive immune responses. Here, we provide an overview of the roles of SNAREs in immune cells, offering insight into one level at which precision and tight regulation are instilled on immune responses.

Screening of human primary melanocytes of defined melanocortin-1 receptor genotype: pigmentation marker, ultrastructural and UV-survival studies.

Publisher: Wiley

Date: 13-05-2003

DOI: 10.1034/J.1600-0749.2003.00033.X

Abstract: Recent population studies have demonstrated an association with the red-hair and fair-skin phenotype with variant alleles of the melanocortin-1 receptor (MC1R) which result in amino acid substitutions within the coding region leading to an altered receptor activity. In particular, Arg151Cys, Arg160Trp and Asp294His were the most commonly associated variants seen in the south-east Queensland population with at least one of these alleles found in 93% of those with red hair. In order to study the in idual effects of these variants on melanocyte biology and melanocytic pigmentation, we established a series of human melanocyte strains genotyped for the MC1R receptor which included wild-type consensus, variant heterozygotes, compound heterozygotes and homozygotes for Arg151Cys, Arg160Trp, Val60Leu and Val92Met alleles. These strains ranged from darkly pigmented to amelanotic, with all strains of consensus sequence having dark pigmentation. UV sensitivity was found not to be associated with either MC1R genotype or the level of pigmentation with a range of sensitivities seen across all genotypes. Ultrastructural analysis demonstrated that while consensus strains contained stage IV melanosomes in their terminal dendrites, Arg151Cys and Arg160Trp homozygote strains contained only stage II melanosomes. This was despite being able to show expression of tyrosinase and tyrosinase-related protein-1 markers, although at reduced levels and an ability to convert exogenous 3,4-dihydroxyphenyl-alanine (DOPA) to melanin in these strains.

Cytokine Secretion Is Distinct from Secretion of Cytotoxic Granules in NK Cells

Publisher: The American Association of Immunologists

Date: 05-2010

DOI: 10.4049/JIMMUNOL.0803954

Abstract: NK cells are renowned for their ability to kill virally infected or transformed host cells by release of cytotoxic granules containing granzymes and perforin. NK cells also have important regulatory capabilities chiefly mediated by secretion of cytokines, such as IFN-γ and TNF. The secretory pathway for the release of cytokines in NK cells is unknown. In this study, we show localization and trafficking of IFN-γ and TNF in human NK cells in compartments and vesicles that do not overlap with perforin or other late endosome granule markers. Cytokines in post-Golgi compartments colocalized with markers of the recycling endosome (RE). REs are functionally required for cytokine release because inactivation of REs or mutation of RE-associated proteins Rab11 and vesicle-associated membrane protein-3 blocked cytokine surface delivery and release. In contrast, REs are not needed for release of perforin from preformed granules but may be involved at earlier stages of granule maturation. These findings suggest a new role for REs in orchestrating secretion in NK cells. We show that the cytokines IFN-γ and TNF are trafficked and secreted via a different pathway than perforin. Although perforin granules are released in a polarized fashion at lytic synapses, distinct carriers transport both IFN-γ and TNF to points all over the cell surface, including within the synapse, for nonpolarized release.

Macropinocytosis: Insights from immunology and cancer.

Publisher: Elsevier BV

Date: 08-2020

DOI: 10.1016/J.CEB.2020.06.005

Membrane localization of the pertussis toxin-sensitive G-protein subunits alpha i-2 and alpha i-3 and expression of a metallothionein-alpha i-2 fusion gene in LLC-PK1 cells

Publisher: Proceedings of the National Academy of Sciences

Date: 06-1990

DOI: 10.1073/PNAS.87.12.4635

Abstract: The renal epithelial cell line LLC-PK1 has topographically distinct regulatory roles for the alpha subunits of pertussis toxin-sensitive guanine nucleotide regulatory proteins (alpha i subunit) these include the inhibition of adenylyl cyclase at the basolateral membrane and the stimulation of Na+ channel activity at the apical membrane. We now report that LLC-PK1 cells contain two members of the alpha i protein family, alpha i-2 and alpha i-3, which have distinct cellular locations consistent with their erse functional roles. By using specific alpha i antibodies and immunofluorescence, the alpha i-2 subunit was found to be localized to the basolateral membrane, whereas the alpha i-3 subunit was concentrated in the Golgi and was also detectable at low levels on apical membranes in some cells. Induction of a chimeric mouse metallothionein 1-rat or canine alpha i-2 gene stably transfected into the LLC-PK1 cells produced an increase in the content of the alpha i-2 subunit, which was targeted only to the basolateral membrane. These findings suggest that alpha i subunit specificity for effectors may be achieved in polarized renal epithelial cells by their geographic segregation to different cellular membranes. The LLC-PK1 cell stably transfected with the metallothionein-alpha i-2 fusion gene will provide a model for the study of guanine nucleotide regulatory protein function in epithelia.

A life in pictures—Marilyn Gist Farquhar

Publisher: Rockefeller University Press

Date: 22-01-2020

DOI: 10.1083/JCB.202001010

Abstract: Marilyn Gist Farquhar is remembered for her contributions over seven decades as a pioneering microscopist, an inspiring researcher, mentor, and eminent leader of cell biology.

Contextual Binding of p120 ctn to E-cadherin at the Basolateral Plasma Membrane in Polarized Epithelia

Publisher: Elsevier BV

Date: 10-2003

DOI: 10.1074/JBC.M305525200

EGF induces macropinocytosis and SNX1-modulated recycling of E-cadherin

Publisher: The Company of Biologists

Date: 15-05-2007

DOI: 10.1242/JCS.000653

Abstract: In epithelia, junction proteins are endocytosed for modulation of cell-cell adhesion and cell polarity. In response to growth factors, the cell-cell adhesion protein E-cadherin is internalized from the cell surface with degradation or recycling as potential fates. However, the cellular machinery involved in cadherin internalization and recycling remains controversial. Here we investigated EGF-induced E-cadherin internalization. EGF stimulation of MCF-7 cells resulted in Rac1-modulated macropinocytosis of the E-cadherin-catenin complex into endosomal compartments that colocalized with EEA1 and the sorting nexin, SNX1. Depletion of cellular SNX1 levels by siRNA resulted in increased intracellular accumulation and turnover of E-cadherin internalized from the cell surface in response to EGF. Moreover, SNX1 was also required for efficient recycling of internalized E-cadherin and re-establishment of epithelial adhesion. Together, these findings demonstrate a role for SNX1 in retrieval of E-cadherin from a degradative endosomal pathway and in membrane trafficking pathways that regulate E-cadherin recycling.

Cytokine secretion in macrophages: SNAREs, Rabs, and membrane trafficking

Publisher: Frontiers Media SA

Date: 27-10-2014

DOI: 10.3389/FIMMU.2014.00538

Endocytosis of uncleaved tumor necrosis factor-alpha in macrophages

Publisher: Elsevier BV

Date: 2001

DOI: 10.1038/LABINVEST.3780216

Abstract: Activated monocytes and macrophages secrete the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). TNF-alpha is produced as a 26 kd transmembrane protein that is cleaved to release a 17 kd soluble protein. TNF-alpha in both forms is biologically active. The intracellular trafficking of membrane-associated TNF-alpha in lipopolysaccharide-activated mouse macrophages was assessed after treatment with the metalloprotease inhibitor BB-3103, which prevents the cleavage of pro-TNF-alpha. Immunoprecipitation and immunofluorescence studies showed sustained expression of cell-associated TNF-alpha in the presence of the inhibitor. Cell immunoreactivity and surface biotinylation revealed that uncleaved TNF-alpha accumulated on the cell surface and was endocytosed, appearing in intracellular vesicles. Perturbation of post-Golgi traffic blocked the surface expression of 26 kd TNF-alpha. Tracking a bolus of TNF-alpha over time in cycloheximide-treated cells confirmed that uncleaved TNF-alpha is first transported to the cell surface and subsequently endocytosed. Vesicular structures immunoreactive for TNF-alpha were identified as endosomes by double labeling. The secretory and membrane-associated endocytic trafficking of TNF-alpha provides a mechanism for modulating the quantity of biologically active 26 kd TNF-alpha expressed on macrophages, allowing regulation of paracrine and autocrine responses.

Entry of cholera toxin into polarized human intestinal epithelial cells. Identification of an early brefeldin A sensitive event required for A1-peptide generation

Publisher: American Society for Clinical Investigation

Date: 12-1993

DOI: 10.1172/JCI116917

University Of Queensland

Location: Australia

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Monash University

Location: Australia

View Organisation

Membrane Dynamics In Protein Trafficking

Start Date: 1997

End Date: 1999

Funder: National Health and Medical Research Council

REGULATION OF PROTEOGLYCAN SECRETION

Start Date: 1990

End Date: 1995

Funder: National Institute of Diabetes and Digestive and Kidney Diseases

LPS-regulated SNAREs And Control Of Cytokine Secretion In Macrophages.

Start Date: 2004

End Date: 2006

Funder: National Health and Medical Research Council

Regulations Of G Protein Signalling On The Golgi Complex

Start Date: 2001

End Date: 2005

Funder: National Health and Medical Research Council

G Protein Signalling On The Golgi Complex

Start Date: 1998

End Date: 2000

Funder: National Health and Medical Research Council

G Proteins Regulate Secretion

Start Date: 1995

End Date: 1997

Funder: National Health and Medical Research Council

Research Fellowship - Grant ID:401609

Start Date: 2006

End Date: 2010

Funder: National Health and Medical Research Council

CELLULAR BIOLOGY OF RENAL FUNCTION AND DISEASE

Start Date: 1987

End Date: 2013

Funder: National Institute of Diabetes and Digestive and Kidney Diseases

Cellular Regulation Of Receptor Signalling And Cytokine Responses

Start Date: 2016

End Date: 2019

Funder: National Health and Medical Research Council

A New Master Adaptor Protein For Toll-like Receptor Signalling

Start Date: 2016

End Date: 2019

Funder: National Health and Medical Research Council

Macrophage Polarisation And Control Of Pulmonary Inflammation.

Start Date: 2018

End Date: 2021

Funder: National Health and Medical Research Council

Regulators Of G Protein Signalling On The Golgi Complex

Start Date: 2001

End Date: 2005

Funder: National Health and Medical Research Council

Trafficking And Regulation Of Inflammatory Cytokines

Start Date: 2016

End Date: 2005

Funder: National Health and Medical Research Council

GTPase Regulation Of Cellular Pathways In Inflammation And Cancer

Start Date: 2016

End Date: 2005

Funder: National Health and Medical Research Council

Protein Trafficking In Inflammation And Disease

Start Date: 2011

End Date: 2016

Funder: National Health and Medical Research Council

High Speed Fluorescence Imaging Coupled With Total Internal Reflection Microscopy And Fluorescence Recovery After Photobleaching System

Start Date: 2008

End Date: 2008

Funder: Australian Research Council

Inhibition Of Pro-inflammatory Cytokine Secretion- A New Route To Therapeutics Of Chronic Inflammatory Disease

Start Date: 2009

End Date: 2012

Funder: Australian Research Council

The Molecular Basis Of Macropinocytosis In Mammalian Cells: The Composition Of Endosome Proteins And Their Function

Start Date: 2007

End Date: 2009

Funder: Australian Research Council

Innate Immune Signalling In Mycobacterium Tuberculosis Infection

Start Date: 2017

End Date: 2019

Funder: National Health and Medical Research Council

A Novel Mechanism For IL-1β Secretion

Start Date: 2017

End Date: 2019

Funder: National Health and Medical Research Council

Cytokine Trafficking And Secretion In Macrophages

Start Date: 2003

End Date: 2008

Funder: National Institute of Diabetes and Digestive and Kidney Diseases

Parallel Genetic And Cellular Analysis Of Melanogensis: A New Paradigm To Study Variation In Pigmentation

Start Date: 2004

End Date: 2006

Funder: Australian Research Council

Polarized Trafficking Of E-cadherin In Epithelial Cells.

Start Date: 2007

End Date: 2008

Funder: National Health and Medical Research Council

E-Cadherin Endocytosis In Morphogenesis: Recycling And Growth Factor Induced Uptake.

Start Date: 2007

End Date: 2008

Funder: National Health and Medical Research Council

Trafficking Of E-cadherin In Epithelial Cells.

Start Date: 2003

End Date: 2005

Funder: National Health and Medical Research Council

Combined Genetic And Cellular Analysis Of Melanisation To Study Variation In Human Pigmentation

Start Date: 2007

End Date: 2009

Funder: Australian Research Council

SNARE-mediated Perforin And Cytokine Release In Natural Killer Cells

Start Date: 2011

End Date: 2013

Funder: Australian Research Council

Molecular, Genetic And Cellular Analysis Of Melanisation In Human Pigmentation

Start Date: 2010

End Date: 2012

Funder: Australian Research Council

Cholesterol And Hydroxycholesterol Shaping Phagocytosis

Start Date: 2014

End Date: 2016

Funder: Australian Research Council

Recycling Endosomes Governing Cell Polarity And Cytokine Secretion.

Start Date: 2009

End Date: 2014

Funder: National Health and Medical Research Council

Modulation Of Endosomes For Pathogen Invasion

Start Date: 2009

End Date: 2011

Funder: National Health and Medical Research Council

SNARE-mediated Protein Trafficking In Macrophages

Start Date: 2008

End Date: 2009

Funder: National Health and Medical Research Council

Cytokine Secretion: A Model For Protein Trafficking.

Start Date: 2001

End Date: 2002

Funder: National Health and Medical Research Council

Recycling Of E-cadherin: Implications For Dynamic Cell Adhesion

Start Date: 2001

End Date: 2002

Funder: National Health and Medical Research Council

Membrane Fusion In Macrophage Function

Start Date: 2010

End Date: 2013

Funder: National Health and Medical Research Council

Regulating The Secretion Of Inflammatory Cytokines

Start Date: 2010

End Date: 2013

Funder: National Health and Medical Research Council

Machine Learning For Organelle Selection & Feature Detection In Live Cells

Start Date: 2018

End Date: 2020

Funder: Australian Research Council

Lattice Light Sheet Microscopy For Imaging Biology In Real Space And Time

Start Date: 2017

End Date: 2017

Funder: Australian Research Council

Fighting Infection: Exploiting Host-pathogen Interactions

Start Date: 2010

End Date: 2015

Funder: National Health and Medical Research Council

Discovery Projects - Grant ID: DP230100675

Start Date: 08-2023

End Date: 08-2026

Amount: $565,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0991919

Start Date: 05-2010

End Date: 05-2013

Amount: $336,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP180101910

Start Date: 2018

End Date: 12-2021

Amount: $656,020.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP210100101

Start Date: 01-2023

End Date: 01-2026

Amount: $711,535.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP230100504

Start Date: 05-2023

End Date: 05-2026

Amount: $640,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP1094964

Start Date: 2010

End Date: 12-2013

Amount: $429,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP110103920

Start Date: 2011

End Date: 12-2014

Amount: $315,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE170100206

Start Date: 05-2017

End Date: 12-2017

Amount: $550,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0771169

Start Date: 2007

End Date: 12-2009

Amount: $495,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0771706

Start Date: 2007

End Date: 12-2009

Amount: $263,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0451738

Start Date: 2004

End Date: 12-2006

Amount: $255,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0882864

Start Date: 2008

End Date: 01-2009

Amount: $260,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP140101461

Start Date: 2014

End Date: 12-2016

Amount: $420,000.00

Funder: Australian Research Council

ARC Centres Of Excellence - Grant ID: CE230100021

Start Date: 12-2023

End Date: 12-2030

Amount: $35,000,000.00

Funder: Australian Research Council