The GTPase-activating protein GRAF1 regulates the CLIC/GEEC endocytic pathway

Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK.
Current Biology (Impact Factor: 9.57). 12/2008; 18(22):1802-8. DOI: 10.1016/j.cub.2008.10.044
Source: PubMed


Clathrin-independent endocytosis is an umbrella term for a variety of endocytic pathways that internalize numerous cargoes independently of the canonical coat protein Clathrin [1, 2]. Electron-microscopy studies have defined the pleiomorphic CLathrin-Independent Carriers (CLICs) and GPI-Enriched Endocytic Compartments (GEECs) as related major players in such uptake [3, 4]. This CLIC/GEEC pathway relies upon cellular signaling and activation through small G proteins, but mechanistic insight into the biogenesis of its tubular and tubulovesicular carriers is lacking. Here we show that the Rho-GAP-domain-containing protein GRAF1 marks, and is indispensable for, a major Clathrin-independent endocytic pathway. This pathway is characterized by its ability to internalize bacterial exotoxins, GPI-linked proteins, and extracellular fluid. We show that GRAF1 localizes to PtdIns(4,5)P2-enriched, tubular, and punctate lipid structures via N-terminal BAR and PH domains. These membrane carriers are relatively devoid of caveolin1 and flotillin1 but are associated with activity of the small G protein Cdc42. This study provides the first specific noncargo marker for CLIC/GEEC endocytic membranes and demonstrates how GRAF1 can coordinate small G protein signaling and membrane remodeling to facilitate internalization of CLIC/GEEC pathway cargoes.

Download full-text


Available from: Katia Cortese
  • Source
    • "CLDN18 protein was observed in the plasma membrane of epithelial cells lining the gastric pit region and at the base of the gastric glands as previously reported in normal human stomach specimens (Sahin et al., 2008; Figure 4A). ARHGAP26 was previously detected on pleiomorphic tubular and punctate membrane structures in HeLa cells (Lundmark et al., 2008). We observed ARHGAP26 in normal stomach on vesicular structures "
    [Show abstract] [Hide abstract]
    ABSTRACT: Genome rearrangements, a hallmark of cancer, can result in gene fusions with oncogenic properties. Using DNA paired-end-tag (DNA-PET) whole-genome sequencing, we analyzed 15 gastric cancers (GCs) from Southeast Asians. Rearrangements were enriched in open chromatin and shaped by chromatin structure. We identified seven rearrangement hot spots and 136 gene fusions. In three out of 100 GC cases, we found recurrent fusions between CLDN18, a tight junction gene, and ARHGAP26, a gene encoding a RHOA inhibitor. Epithelial cell lines expressing CLDN18-ARHGAP26 displayed a dramatic loss of epithelial phenotype and long protrusions indicative of epithelial-mesenchymal transition (EMT). Fusion-positive cell lines showed impaired barrier properties, reduced cell-cell and cell-extracellular matrix adhesion, retarded wound healing, and inhibition of RHOA. Gain of invasion was seen in cancer cell lines expressing the fusion. Thus, CLDN18-ARHGAP26 mediates epithelial disintegration, possibly leading to stomach H(+) leakage, and the fusion might contribute to invasiveness once a cell is transformed. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Jul 2015 · Cell Reports
  • Source
    • "This is not surprising given that GRAF1a will dimerize through its membrane interaction BAR domain while still presenting hydrophobic insertion sequences on each molecule. However, the BAR-PH domain of GRAF1 can also alter local membrane curvature because it induces liposome tubulation in vitro (Lundmark et al., 2008). The presence of a curvature effector on LDs would be consistent with an effect on droplet fusion, as this might well prime an area of the surface, making it fusogenic, as is observed with synaptotagmin during vesicle fusion (Martens et al., 2007). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Lipid droplets are found in all cell types. Normally present at low levels in the brain, they accumulate in tumours and are associated with neurodegenerative diseases. However, little is known about the mechanisms controlling their homeostasis in the brain. We have found that the longest GRAF1 isoform, GRAF1a, is enriched in the brains of neonates. Endogenous GRAF1a is found on lipid droplets in oleic-acid fed primary glial cells. Exclusive localization requires a GRAF1a-specific hydrophobic segment and two membrane-binding regions, a BAR and a PH domain. Overexpression of GRAF1a promotes lipid droplet clustering, inhibits droplet mobility and severely perturbs lipolysis following the chase of fatty acid-overloaded cells. Under these conditions, GRAF1a concentrates at the interface between lipid droplets. Although GRAF1 knockout mice do not show any gross abnormal phenotype, the total lipid droplet volume that accumulates in GRAF1(-/-) primary glia upon incubation with fatty acids is reduced compared to GRAF1(+/+) cells. These results provide additional insights into the mechanisms contributing to lipid droplet growth in non-adipocyte cells, and suggest that proteins with membrane sculpting BAR domains play a role in droplet homeostasis.
    Full-text · Article · Sep 2014 · Journal of Cell Science
  • Source
    • "Nevertheless, these arguments all highlight the complexities in studying vesicular uptake. It is also worth mentioning that there are several other clathrin-independent endocytic pathways, such as the recently described CLIC/GLEEC pathway (78, 152). The extent to which these other pathways may be involved remains to be determined. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Extracellular vesicles (EVs) are small vesicles released by donor cells that can be taken up by recipient cells. Despite their discovery decades ago, it has only recently become apparent that EVs play an important role in cell-to-cell communication. EVs can carry a range of nucleic acids and proteins which can have a significant impact on the phenotype of the recipient. For this phenotypic effect to occur, EVs need to fuse with target cell membranes, either directly with the plasma membrane or with the endosomal membrane after endocytic uptake. EVs are of therapeutic interest because they are deregulated in diseases such as cancer and they could be harnessed to deliver drugs to target cells. It is therefore important to understand the molecular mechanisms by which EVs are taken up into cells. This comprehensive review summarizes current knowledge of EV uptake mechanisms. Cells appear to take up EVs by a variety of endocytic pathways, including clathrin-dependent endocytosis, and clathrin-independent pathways such as caveolin-mediated uptake, macropinocytosis, phagocytosis, and lipid raft–mediated internalization. Indeed, it seems likely that a heterogeneous population of EVs may gain entry into a cell via more than one route. The uptake mechanism used by a given EV may depend on proteins and glycoproteins found on the surface of both the vesicle and the target cell. Further research is needed to understand the precise rules that underpin EV entry into cells.
    Full-text · Article · Aug 2014
Show more