ABSTRACT
The pentameric FERRY Rab5 effector complex is a molecular link between mRNA and early endosomes in mRNA intracellular distribution. Here, we determine the cryo-EM structure of human FERRY. It reveals a unique clamp-like architecture that bears no resemblance to any known structure of Rab effectors. A combination of functional and mutational studies reveals that while the Fy-2 C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils and Fy-5 concur to bind mRNA. Mutations causing truncations of Fy-2 in patients with neurological disorders impair Rab5 binding or FERRY complex assembly. Thus, Fy-2 serves as a binding hub connecting all five complex subunits and mediating the binding to mRNA and early endosomes via Rab5. Our study provides mechanistic insights into long-distance mRNA transport and demonstrates that the particular architecture of FERRY is closely linked to a previously undescribed mode of RNA binding, involving coiled-coil domains.
Subject(s)
Vesicular Transport Proteins , rab5 GTP-Binding Proteins , Humans , Vesicular Transport Proteins/metabolism , rab5 GTP-Binding Proteins/genetics , rab5 GTP-Binding Proteins/analysis , rab5 GTP-Binding Proteins/metabolism , Endosomes/genetics , Endosomes/metabolismABSTRACT
The evolutionarily conserved Target of Rapamycin (TOR) complex-2 (TORC2) is an essential regulator of plasma membrane homeostasis in budding yeast (Saccharomyces cerevisiae). In this yeast, TORC2 phosphorylates and activates the effector protein kinase Ypk1 and its paralog Ypk2. These protein kinases, in turn, carry out all the crucial functions of TORC2 by phosphorylating and thereby controlling the activity of at least a dozen downstream substrates. A previously uncharacterized interplay between the Rab5 GTPases and TORC2 signaling was uncovered through analysis of a newly suspected Ypk1 target. Muk1, one of two guanine nucleotide exchange factors for the Rab5 GTPases, was found to be a physiologically relevant Ypk1 substrate; and, genetic analysis indicates that Ypk1-mediated phosphorylation activates the guanine nucleotide exchange activity of Muk1. Second, it was demonstrated both in vivo and in vitro that the GTP-bound state of the Rab5 GTPase Vps21/Ypt51 physically associates with TORC2 and acts as a direct positive effector required for full TORC2 activity. These interrelationships provide a self-reinforcing control circuit for sustained up-regulation of TORC2-Ypk1 signaling. In this overview, we summarize the experimental basis of these findings, their implications, and speculate as to the molecular basis for Rab5-mediated TORC2 activation.
Subject(s)
Mechanistic Target of Rapamycin Complex 2/metabolism , Saccharomyces cerevisiae/metabolism , rab5 GTP-Binding Proteins/physiology , Glycogen Synthase Kinase 3/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Guanine Nucleotide Exchange Factors/physiology , Humans , Mechanistic Target of Rapamycin Complex 2/analysis , Models, Molecular , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/physiology , Signal Transduction , Up-Regulation , rab5 GTP-Binding Proteins/analysis , rab5 GTP-Binding Proteins/metabolismABSTRACT
Rab family protein Rab5a has been implicated in cancer progression. To date, its expression pattern in human pancreatic cancer has not been investigated. This study aims to examine clinical significance, biological role, and potential mechanism of action of mRab5a in human pancreatic cancer. We analyzed Rab5a protein in cancer tissue of 111 cases of pancreatic cancer using immunohistochemistry. The results show that Rab5a overexpression correlates with high T stage, positive nodal status, and advanced TNM stage. We performed knockdown of Rab5a through transfection of Rab5a-specific siRNA in the Capan-2 cell line, which shows high endogenous expression, and of Rab5a plasmid in the CFPAC-1 cell line, which shows low endogenous expression. Rab5a knockdown inhibited cell proliferation and invasion while its overexpression promoted cell proliferation and invasion. In addition, overexpression of Rab5a induced resistance to 5-FU and gemcitabine while its knockdown reduced resistance to 5-FU and gemcitabine. Furthermore, our results show that Rab5a overexpression upregulates Wnt signaling and expression of Wnt target genes including c-myc and MMP7. Blocking Wnt signaling abolished the effects of Rab5a on Wnt targets and on cancer cell proliferation. In summary, our results show that Rab5a is overexpressed in pancreatic cancer and promotes aggressive biological behavior through regulation of the Wnt/ß-catenin signaling pathway.
Subject(s)
Biomarkers, Tumor/analysis , Carcinoma, Pancreatic Ductal/pathology , Pancreatic Neoplasms/pathology , rab5 GTP-Binding Proteins/biosynthesis , Blotting, Western , Disease Progression , Gene Knockdown Techniques , Humans , Immunohistochemistry , Prognosis , Real-Time Polymerase Chain Reaction , Wnt Signaling Pathway/physiology , rab5 GTP-Binding Proteins/analysis , Pancreatic NeoplasmsABSTRACT
Upon Mycobacterium tuberculosis infection, macrophages may undergo apoptosis, which has been considered an innate immune response. The pathways underlying the removal of dead cells in homeostatic apoptosis have been extensively studied, but little is known regarding how cells that undergo apoptotic death during mycobacterial infection are removed. This study shows that macrophages induced to undergo apoptosis with mycobacteria cell wall proteins are engulfed by J-774A.1 monocytic cells through the mannose receptor. This demonstration was achieved through assays in which phagocytosis was inhibited with a blocking anti-mannose receptor antibody and with mannose receptor competitor sugars. Moreover, elimination of the mannose receptor by a specific siRNA significantly diminished the expression of the mannose receptor and the phagocytosis of apoptotic cells. As shown by immunofluorescence, engulfed apoptotic bodies are initially located in Rab5-positive phagosomes, which mature to express the phagolysosome marker LAMP1. The phagocytosis of dead cells triggered an anti-inflammatory response with the production of TGF-ß and IL-10 but not of the proinflammatory cytokines IL-12 and TNF-α. This study documents the previously unreported participation of the mannose receptor in the removal of apoptotic cells in the setting of tuberculosis (TB) infection. The results challenge the idea that apoptotic cell phagocytosis in TB has an immunogenic effect.
Subject(s)
Apoptosis , Cell Wall/immunology , Lectins, C-Type/physiology , Macrophages/immunology , Mannose-Binding Lectins/physiology , Monocytes/immunology , Mycobacterium smegmatis/immunology , Phagocytosis , Receptors, Cell Surface/physiology , Animals , Cell Line, Tumor , Extracellular Vesicles/ultrastructure , Fluorescent Antibody Technique , Interleukin-10/metabolism , Interleukin-12/metabolism , Lectins, C-Type/genetics , Lysosomal Membrane Proteins/genetics , Lysosomal Membrane Proteins/metabolism , Macrophages/cytology , Macrophages/microbiology , Mannose Receptor , Mannose-Binding Lectins/genetics , Mice , Mycobacterium smegmatis/growth & development , Phagosomes/immunology , Phagosomes/ultrastructure , RNA, Small Interfering , Receptors, Cell Surface/genetics , Transforming Growth Factor beta/metabolism , Tumor Necrosis Factor-alpha/metabolism , rab5 GTP-Binding Proteins/analysisABSTRACT
BACKGROUND: Transmembrane protein 106B (TMEM106B) has been identified as a risk factor for frontotemporal lobar degeneration, which is the second most common form of progressive dementia in people under 65 years of age. Mutations in charged multivesicular body protein 2B (CHMP2B), which is involved in endosomal protein trafficking, have been found in chromosome 3-linked frontotemporal dementia. Despite the number of studies on both CHMP2B and TMEM106B in the endolysosomal pathway, little is known about the relationship between CHMP2B and TMEM106B in the endosomal/autophagy pathway. RESULTS: This study found that endogenous TMEM106B was partially sequestered in CHMP2B-positive structures, suggesting its possible involvement in endosomal sorting complexes required for transport (ESCRT)-associated pathways. The role of single nucleotide polymorphisms of TMEM106B (T185, S185, or S134N) in the ESCRT-associated pathways were characterized. The T185 and S185 variants were more localized to Rab5-/Rab7-positive endosomes compared with S134N, while all of the variants were more localized to Rab7-positive endosomes compared to Rab5-positive endosomes. T185 was more associated with CHMP2B compared to S185. Autophagic flux was slightly reduced in the T185-expressing cells compared to the control or S185-expressing cells. Moreover, T185 slightly enhanced the accumulation of EGFR, impairments in autophagic flux, and neurotoxicity that were caused by CHMP2B(Intron5) compared to S185-expressing cells. CONCLUSIONS: These findings suggest that the T185 variant functions as a risk factor in neurodegeneration with endolysosomal defects. This study provides a better understanding of pathogenic functions of TMEM106B, which is a risk factor for the progression of neurodegenerative diseases that are associated with endosomal defects in the aged brain.
Subject(s)
Endosomal Sorting Complexes Required for Transport/metabolism , Endosomes/physiology , Frontotemporal Dementia/metabolism , Membrane Proteins/physiology , Nerve Tissue Proteins/physiology , Neurons/metabolism , Polymorphism, Single Nucleotide , Protein Transport/physiology , Animals , Autophagy , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/embryology , Endosomal Sorting Complexes Required for Transport/genetics , Endosomal Sorting Complexes Required for Transport/physiology , Endosomes/chemistry , Exons/genetics , Frontotemporal Dementia/genetics , Frontotemporal Dementia/pathology , HEK293 Cells , Humans , Immunoprecipitation , Introns/genetics , Lysosomes/metabolism , Membrane Proteins/genetics , Mice , Nerve Degeneration , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/pathology , Protein Interaction Maps , Proteolysis , Recombinant Fusion Proteins/metabolism , rab GTP-Binding Proteins/analysis , rab5 GTP-Binding Proteins/analysis , rab7 GTP-Binding ProteinsABSTRACT
Rab5A is reported to correlate with cancer development and progression. The purpose of this study is to explore the association between Rab5A expression and the clinical characteristics of colorectal cancer (CRC). Data containing three independent investigations from Oncomine database demonstrated that Rab5A is overexpression in CRC compared with normal tissue, similar result was also found in 32 matched CRC tissue samples by qPCR. The protein expression of Rab5A was examined in 390 CRC specimens and the results showed that high expression of Rab5A was significantly correlated with tumor size (P = 0.008), serum CEA (P = 0.002), liver metastasis (P = 0.014) and clinical stage (P = 0.010). Kaplan-Meier method suggested that overexpression of Rab5A protein expression had shorter overall survival times in CRC patients (P < 0.001). Multivariate Cox regression analysis confirmed Rab5A expression, tumor size and clinical stage as independent prognostic factor in CRC. In conclusion, the data indicated that higher expression of Rab5A was observed in CRC tissues and Rab5A may be identified as a useful predictor of metastasis and prognosis for CRC.
Subject(s)
Biomarkers, Tumor/analysis , Colorectal Neoplasms/enzymology , rab5 GTP-Binding Proteins/analysis , Aged , Biomarkers, Tumor/genetics , Chi-Square Distribution , Colorectal Neoplasms/genetics , Colorectal Neoplasms/mortality , Colorectal Neoplasms/pathology , Colorectal Neoplasms/therapy , Databases, Factual , Female , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Kaplan-Meier Estimate , Male , Middle Aged , Multivariate Analysis , Neoplasm Metastasis , Neoplasm Staging , Proportional Hazards Models , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Risk Factors , Tissue Array Analysis , Treatment Outcome , Tumor Burden , Up-Regulation , rab5 GTP-Binding Proteins/geneticsABSTRACT
Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and α-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.
Subject(s)
Cytoskeleton/ultrastructure , Endocytosis , Imaging, Three-Dimensional/methods , Microscopy, Fluorescence/methods , Organelles/ultrastructure , Actinin/analysis , Actins/analysis , Animals , Cell Line , Clathrin/analysis , Clathrin-Coated Vesicles/chemistry , Clathrin-Coated Vesicles/ultrastructure , Coated Pits, Cell-Membrane/chemistry , Coated Pits, Cell-Membrane/ultrastructure , Cytoskeleton/chemistry , Cytoskeleton/metabolism , Endosomes/chemistry , Endosomes/ultrastructure , Golgi Apparatus/ultrastructure , Image Processing, Computer-Assisted , Imaging, Three-Dimensional/instrumentation , Microscopy, Fluorescence/instrumentation , Mitochondria/chemistry , Mitochondria/ultrastructure , Organelles/chemistry , Organelles/metabolism , rab5 GTP-Binding Proteins/analysisABSTRACT
Phagocytosis by cells of the innate immune system, such as macrophages, and the subsequent successful maturation of the phagosome, is key for the clearance of pathogens. The fungal pathogen Cryptococcus neoformans is known to overcome killing by host phagocytes and both replicate within these cells and also escape via a non-lytic process termed vomocytosis. Here we demonstrate that, during intracellular growth, cryptococci modify phagolysosome maturation. Live cryptococci, but not heat-killed pathogens or inert targets, induce the premature removal of the early phagosome markers Rab5 and Rab11. In addition, significant acidification of the phagosome, calcium flux and protease activity is hindered, thus rendering the phagosome permissive for cryptococcal proliferation. Interestingly, several attenuated cryptococcal mutants retain this ability to subvert phagosomal maturation, suggesting that hitherto unidentified pathogen mechanisms regulate this process.
Subject(s)
Cryptococcus neoformans/physiology , Host-Pathogen Interactions , Immune Evasion , Macrophages/immunology , Macrophages/microbiology , Phagosomes/metabolism , Phagosomes/microbiology , Animals , Calcium/metabolism , Cell Line , Cryptococcus neoformans/growth & development , Cryptococcus neoformans/immunology , Hydrogen-Ion Concentration , Macrophages/chemistry , Mice , Peptide Hydrolases/metabolism , Phagosomes/chemistry , rab GTP-Binding Proteins/analysis , rab5 GTP-Binding Proteins/analysisABSTRACT
UNLABELLED: Hepatitis C virus (HCV) NS5A is essential for viral genome replication within cytoplasmic replication complexes and virus assembly at the lipid droplet (LD) surface, although its definitive functions are poorly understood. We developed approaches to investigate NS5A dynamics during a productive infection. We report here that NS5A motility and efficient HCV RNA replication require the microtubule network and the cytoplasmic motor dynein and demonstrate that both motile and relatively static NS5A-positive foci are enriched with host factors VAP-A and Rab5A. Pulse-chase imaging revealed that newly synthesized NS5A foci are small and distinct from aged foci, while further studies using a unique dual fluorescently tagged infectious HCV chimera showed a relatively stable association of NS5A foci with core-capped LDs. These results reveal new details about the dynamics and maturation of NS5A and the nature of potential sites of convergence of HCV replication and assembly pathways. IMPORTANCE: Hepatitis C virus (HCV) is a major cause of serious liver disease worldwide. An improved understanding of the HCV replication cycle will enable development of novel and improved antiviral strategies. Here we have developed complementary fluorescent labeling and imaging approaches to investigate the localization, traffic and interactions of the HCV NS5A protein in living, virus-producing cells. These studies reveal new details as to the traffic, composition and biogenesis of NS5A foci and the nature of their association with putative sites of virus assembly.
Subject(s)
Hepacivirus/immunology , Viral Nonstructural Proteins/analysis , Virus Assembly , Virus Replication , Cell Line , Dyneins/metabolism , Hepatocytes/chemistry , Hepatocytes/virology , Humans , Microtubules/metabolism , Vesicular Transport Proteins/analysis , rab5 GTP-Binding Proteins/analysisABSTRACT
Usher syndrome is a genetically heterogeneous disorder characterized by hearing and balance dysfunction and progressive retinitis pigmentosa. Mouse models carrying mutations for the nine Usher-associated genes have splayed stereocilia, and some show delayed maturation of ribbon synapses suggesting these proteins may play different roles in terminal differentiation of auditory hair cells. The presence of the Usher proteins at the basal and apical aspects of the neurosensory epithelia suggests the existence of regulated trafficking through specific transport proteins and routes. Immature mouse cochleae and UB/OC-1 cells were used in this work to address whether specific variants of PCDH15 and VLGR1 are being selectively transported to opposite poles of the hair cells. Confocal colocalization studies between apical and basal vesicular markers and the different PCDH15 and VLGR1 variants along with sucrose density gradients and the use of vesicle trafficking inhibitors show the existence of Usher protein complexes in at least two vesicular subpools. The apically trafficked pool colocalized with the early endosomal vesicle marker, rab5, while the basally trafficked pool associated with membrane microdomains and SNAP25. Moreover, coimmunoprecipitation experiments between SNAP25 and VLGR1 show a physical interaction of these two proteins in organ of Corti and brain. Collectively, these findings establish the existence of a differential vesicular trafficking mechanism for specific Usher protein variants in mouse cochlear hair cells, with the apical variants playing a potential role in endosomal recycling and stereocilia development/maintenance, and the basolateral variants involved in vesicle docking and/or fusion through SNAP25-mediated interactions.
Subject(s)
Cadherins/metabolism , Cell Polarity/physiology , Hair Cells, Auditory/ultrastructure , Protein Precursors/metabolism , Receptors, G-Protein-Coupled/metabolism , Transport Vesicles/physiology , ADP-Ribosylation Factor 1/analysis , Animals , Brain Chemistry , Cadherin Related Proteins , Cadherins/biosynthesis , Cadherins/genetics , Cell Compartmentation , Cell Differentiation , Disease Models, Animal , Gene Knockdown Techniques , Hair Cells, Auditory/metabolism , Immunoprecipitation , Mice , Mice, Neurologic Mutants , Mutation , Organ of Corti/chemistry , Organ of Corti/ultrastructure , Protein Interaction Mapping , Protein Precursors/biosynthesis , Protein Precursors/genetics , Protein Transport/drug effects , RNA Interference , Receptors, G-Protein-Coupled/biosynthesis , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/genetics , Structure-Activity Relationship , Synaptosomal-Associated Protein 25/analysis , Synaptosomal-Associated Protein 25/chemistry , Synaptosomal-Associated Protein 25/metabolism , Transport Vesicles/chemistry , Usher Syndromes/metabolism , rab5 GTP-Binding Proteins/analysisABSTRACT
Several membrane trafficking proteins have been shown to participate in spindle assembly and stability during mitosis. Despite the fact that the role of some of them has been clarified, the requirement for these molecules in mitosis is still poorly understood.
Subject(s)
Mitosis , rab5 GTP-Binding Proteins/metabolism , Animals , Chromosomal Proteins, Non-Histone/analysis , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes/metabolism , Humans , Kinetochores/metabolism , Microfilament Proteins/analysis , Microfilament Proteins/metabolism , rab5 GTP-Binding Proteins/analysisABSTRACT
Rab20, a member of the Rab GTPase family, is known to be involved in membrane trafficking, however its implication in FcγR-mediated phagocytosis is unclear. We examined the spatiotemporal localization of Rab20 during phagocytosis of IgG-opsonized erythrocytes (IgG-Es) in RAW264 macrophages. By the live-cell imaging of fluorescent protein-fused Rab20, it was shown that Rab20 was transiently associated with the phagosomal membranes. During the early stage of phagosome formation, Rab20 was not localized on the membranes of phagocytic cups, but was gradually recruited to the newly formed phagosomes. Although Rab20 was colocalized with Rab5 to some extent, the association of Rab20 with the phagosomes persisted even after the loss of Rab5 from the phagosomal membranes. Then, Rab20 was colocalized with Rab7 and Lamp1, late endosomal/lysosomal markers, on the internalized phagosomes. Moreover, our analysis of Rab20 mutant expression revealed that the maturation of phagosomes was significantly delayed in cells expressing the GDP-bound mutant Rab20-T19N. These data suggest that Rab20 is an important component of phagosome and regulates the phagosome maturation during FcγR-mediated phagocytosis.
Subject(s)
Macrophages/cytology , Phagocytosis , Phagosomes/immunology , Receptors, IgG/immunology , rab GTP-Binding Proteins/analysis , rab GTP-Binding Proteins/immunology , Animals , Cell Line , Erythrocytes/immunology , Immunoglobulin G/immunology , Lysosomal Membrane Proteins/analysis , Lysosomal Membrane Proteins/immunology , Macrophages/immunology , Macrophages/ultrastructure , Mice , Mutation , Phagosomes/ultrastructure , Sheep , rab GTP-Binding Proteins/genetics , rab5 GTP-Binding Proteins/analysis , rab5 GTP-Binding Proteins/immunology , rab7 GTP-Binding ProteinsABSTRACT
A key regulator of receptor-mediated endocytosis, Rab5, plays a pivotal role in cargo receptor internalization, endosomal maturation, and transduction and degradation of internalized signaling molecules and recycling cargo receptor. Stressful conditions within cells lead to increased Rab5 activation, and increasing evidence correlates Rab5 activity abnormalities with certain diseases. Current antibody-based imaging methods cannot distinguish active Rab5 from total Rab5 population and provide dynamic information on magnitude and duration of Rab5 activation in cellular events and pathogenesis. We report here novel molecular imaging probes that specifically target GTP-bound Rab5 associated with the early endosome membrane in live cells and fixed mouse brain tissues. Our Rab5 activity fluorescent biosensor (RAFB) contains the Rab5 binding domain of the Rab5 effector Rabaptin 5, a fluorophore (a quantum dot or fluorescent dye) and a cell-penetrating peptide for live-cell delivery. The quantum dot conjugated RAFB was able to image the elevated Rab5 activity in both the cortex and hippocampi tissues of a Ts65Dn mouse. A prequenched RAFB based on fluorescence resonance energy transfer (FRET) can image cytosolic active Rab5 in single live cells. This novel method should enable imaging of the biological process in which Rab5 activity is regulated in various cellular systems.
Subject(s)
Biosensing Techniques , Fluorescein-5-isothiocyanate/analogs & derivatives , Peptides/metabolism , rab5 GTP-Binding Proteins/analysis , rab5 GTP-Binding Proteins/metabolism , Animals , Binding Sites , Brain/metabolism , Cell-Penetrating Peptides/metabolism , Cerebral Cortex/metabolism , Cytoplasm/metabolism , Endosomes/metabolism , Fluorescein-5-isothiocyanate/chemistry , Fluorescein-5-isothiocyanate/metabolism , Fluorescence Resonance Energy Transfer , HeLa Cells , Hippocampus/metabolism , Humans , Mice , Peptides/chemistry , Protein Engineering , Quantum Dots , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolism , rab5 GTP-Binding Proteins/geneticsABSTRACT
This study examined the role of Rab5a GTPase in regulating hCG-induced internalization and trafficking of the hCG-LH receptor complex in transfected 293T cells. Coexpression of wild-type Rab5a (WT) or constitutively active Rab5a (Q79L) with LHR significantly increased hCG-induced LHR internalization. Conversely, coexpression of dominant negative Rab5a (S34N) with LHR reduced internalization. Confocal microscopy showed LHR colocalizing with Rab5a (WT) and Rab5a (Q79L) in punctuate structures. Coexpression of Rab5a (WT) and Rab5a (Q79L) with LHR significantly increased colocalization of LHR in early endosomes. Conversely, dominant negative Rab5a (S34N) decreased this colocalization. While Rab5a stimulated internalization of LHR, it significantly decreased LHR recycling to the cell surface and increased degradation. Dominant negative Rab5a (S34N) increased LHR recycling and decreased degradation. These results suggest that Rab5a plays a role in LHR trafficking by facilitating internalization and fusion to early endosomes, increasing the degradation of internalized receptor resulting in a reduction in LHR recycling.
Subject(s)
Chorionic Gonadotropin/metabolism , Endocytosis , Receptors, LH/metabolism , rab5 GTP-Binding Proteins/physiology , Cell Line , Chorionic Gonadotropin/analysis , Endosomes/metabolism , Humans , Microscopy, Confocal , Receptors, LH/analysis , Transfection , rab5 GTP-Binding Proteins/analysis , rab5 GTP-Binding Proteins/metabolismABSTRACT
Multiple label immunoelectron microscopy localizes and detects multiple antigens in cells and tissues. In double labeling, two kinds of primary antibodies from different animal species are used after being mixed in a single solution. To distinguish the different antigens, secondary antibodies should be labeled with colloidal gold particles of different diameter. Generally, the secondary antibody that is used for detecting the antigen with lower distribution density is labeled with smaller-sized gold particles. In this chapter, double-label immunoelectron microscopy of gelatin-embedded cultured cells using the cryosectioning technique is described.
Subject(s)
Immunohistochemistry/methods , Microscopy, Immunoelectron/methods , Staining and Labeling/methods , Antibodies/chemistry , Antibodies/immunology , Antigens/analysis , Antigens/immunology , Caveolin 1/analysis , Caveolin 1/immunology , Cells, Cultured , Cryoultramicrotomy , Endothelial Cells/cytology , Endothelial Cells/immunology , Endothelial Cells/ultrastructure , Gelatin/chemistry , Gold Colloid/chemistry , Gold Colloid/immunology , Humans , Particle Size , Tissue Embedding , rab5 GTP-Binding Proteins/analysis , rab5 GTP-Binding Proteins/immunologyABSTRACT
Activated receptor tyrosine kinases recruit many signaling proteins to activate downstream cell proliferation and survival pathways, including phosphatidylinositol 3-kinase (PI3K) consisting of a p85 regulatory protein and a p110 catalytic protein. We have recently shown the p85alpha protein also has in vitro GTPase activating protein (GAP) activity towards Rab5 and Rab4, small GTPases that regulate vesicle trafficking events for activated receptors. Expression of a GAP-defective mutant, p85R274A, resulted in sustained levels of activated platelet-derived growth factor receptors (PDGFRs) and enhanced downstream signaling. In this report we have characterized Rab5- and Rab4-mediated PDGFR trafficking in cells expressing wild type p85 and GAP-defective mutant p85R274A. Wild type p85 overexpressing cells had slower PDGFR trafficking consistent with enhanced GAP activity deactivating Rab5 and Rab4 to block their vesicle trafficking functions. Mutant p85R274A expression increased the internalization rate of PDGFRs, a Rab5-dependent process, without preventing PDGFR ubiquitination. Immunofluorescence studies further demonstrated that p85R274A-expressing cells showed Rab5 accumulation at intracellular locations. Pull-down and FRAP (fluorescence recovery after photobleaching) experiments indicate this is likely membrane-associated Rab5-GTP, sustained due to decreased p85 GAP activity for the p85R274A mutant. These cells also had substantial amounts of activated PDGFRs in Rab4-positive recycling endosomes, a compartment that usually contains primarily deactivated/dephosphorylated receptors. Our results suggest that the PDGFR-associated GAP activity of p85 regulates both Rab5 and Rab4 functions in cells to influence the movement of activated PDGFR through endosomal compartments. Disruption of this regulation by p85R274A expression impacts PDGFR phosphorylation/dephosphorylation, degradation kinetics and downstream signaling by altering the time receptors spend in specific intracellular endosomal compartments. These results demonstrate that the p85alpha protein is an important regulator of Rab-mediated PDGFR trafficking, which significantly impacts receptor signaling and degradation.
Subject(s)
GTPase-Activating Proteins/metabolism , Receptors, Platelet-Derived Growth Factor/metabolism , rab4 GTP-Binding Proteins/metabolism , rab5 GTP-Binding Proteins/metabolism , Animals , Cell Membrane/chemistry , Dogs , GTPase-Activating Proteins/genetics , Mice , NIH 3T3 Cells , Point Mutation , Protein Transport , Ubiquitination , rab5 GTP-Binding Proteins/analysisABSTRACT
The Bunyaviridae constitute a large family of enveloped animal viruses, many members of which cause serious diseases. However, early bunyavirus-host cell interactions and entry mechanisms remain largely uncharacterized. Investigating Uukuniemi virus, a bunyavirus of the genus Phlebovirus, we found that virus attachment to the cell surface was specific but inefficient, with 25% of bound viruses being endocytosed within 10 min, mainly via noncoated vesicles. The viruses entered Rab5a+ early endosomes and, subsequently, Rab7a+ and LAMP-1+ late endosomes. Acid-activated penetration, occurring 20-40 min after internalization, required maturation of early to late endosomes. The pH threshold for viral membrane fusion was 5.4, and entry was sensitive to temperatures below 25 degrees C. Together, our results indicate that Uukuniemi virus penetrates host cells by acid-activated membrane fusion from late endosomal compartments. This study also highlights the importance of the degradative branch of the endocytic pathway in facilitating entry of late-penetrating viruses.
Subject(s)
Uukuniemi virus/physiology , Virus Internalization , Animals , Cell Line , Endocytosis , Endosomes/chemistry , Endosomes/virology , Humans , Hydrogen-Ion Concentration , Lysosomal-Associated Membrane Protein 1/analysis , Microscopy, Electron, Transmission , Microscopy, Fluorescence , rab GTP-Binding Proteins/analysis , rab5 GTP-Binding Proteins/analysis , rab7 GTP-Binding ProteinsABSTRACT
Western blots are widely used for analysis of the expression levels of specific proteins. Blotting is conducted after sodium dodecyl sulfate or native polyacrylamide gel electrophoresis without staining the gel. However, when it is necessary to analyze the gel, duplicate polyacrylamide gels (one of which is stained) usually must be prepared, leading to the consumption of precious sample. Thus, we have developed a convenient and efficient Western blot method using a stained gel. This simple modification should be beneficial for the analysis of samples that are limited in quantity and/or samples for which the stained gel serves as the loading control.
Subject(s)
Blotting, Western/methods , Electrophoresis, Polyacrylamide Gel/methods , rab5 GTP-Binding Proteins/analysis , Coloring Agents , Organometallic Compounds , Recombinant Fusion Proteins/analysisABSTRACT
Vav proteins are guanine nucleotide exchange factors for Rho GTPases that regulate cell adhesion, motility, spreading and proliferation in response to growth factor signalling. In this work, we show that Vav2 expression delayed epidermal growth factor receptor (EGFR) internalization and degradation, and enhanced EGFR, ERK and Akt phosphorylations. This effect of Vav2 on EGFR degradation is dependent on its guanine nucleotide exchange function. Knockdown of Vav2 in HeLa cells enhanced EGFR degradation and reduced cell proliferation. epidermal growth factor stimulation led to co-localization of Vav2 with EGFR and Rab5 in endosomes. We further show that the effect of Vav2 on EGFR stability is modulated by its interaction with two endosome-associated proteins and require RhoA function. Thus, in this work, we report for the first time that Vav2 can regulate growth factors receptor signalling by slowing receptor internalization and degradation through its interaction with endosome-associated proteins.