Human Vam6p promotes lysosome clustering and fusion in vivo

Regulated fusion of mammalian lysosomes is critical to their ability to acquire both internalized and biosynthetic materials. Here, we report the identification of a novel human protein, hVam6p, that promotes lysosome clustering and fusion in vivo. Although hVam6p exhibits homology to the Saccharomyces cerevisiae vacuolar protein sorting gene product Vam6p/Vps39p, the presence of a citron homology (CNH) domain at the NH2 terminus is unique to the human protein. Overexpression of hVam6p results in massive clustering and fusion of lysosomes and late endosomes into large (2–3 μm) juxtanuclear structures. This effect is reminiscent of that caused by expression of a constitutively activated Rab7. However, hVam6p exerts its effect even in the presence of a dominant-negative Rab7, suggesting that it functions either downstream of, or in parallel to, Rab7. Data from gradient fractionation, two-hybrid, and coimmunoprecipitation analyses suggest that hVam6p is a homooligomer, and that its self-assembly is mediated by a clathrin heavy chain repeat domain in the middle of the protein. Both the CNH and clathrin heavy chain repeat domains are required for induction of lysosome clustering and fusion. This study implicates hVam6p as a mammalian tethering/docking factor characterized with intrinsic ability to promote lysosome fusion in vivo

Cooperation of MICAL-L1, syndapin2, and phosphatidic acid in tubular recycling endosome biogenesis.

Endocytic transport necessitates the generation of membrane tubules and their subsequent fission to transport vesicles for sorting of cargo molecules. The endocytic recycling compartment, an array of tubular and vesicular membranes decorated by the Eps15 homology domain protein, EHD1, is responsible for receptor and lipid recycling to the plasma membrane. It has been proposed that EHD dimers bind and bend membranes, thus generating recycling endosome (RE) tubules. However, recent studies show that molecules interacting with CasL-Like1 (MICAL-L1), a second, recently identified RE tubule marker, recruits EHD1 to preexisting tubules. The mechanisms and events supporting the generation of tubular recycling endosomes were unclear. Here, we propose a mechanism for the biogenesis of RE tubules. We demonstrate that MICAL-L1 and the BAR-domain protein syndapin2 bind to phosphatidic acid, which we identify as a novel lipid component of RE. Our studies demonstrate that direct interactions between these two proteins stabilize their association with membranes, allowing for nucleation of tubules by syndapin2. Indeed, the presence of phosphatidic acid in liposomes enhances the ability of syndapin2 to tubulate membranes in vitro. Overall our results highlight a new role for phosphatidic acid in endocytic recycling and provide new insights into the mechanisms by which tubular REs are generated.journal articleresearch support, n.i.h., extramuralresearch support, non-u.s. gov't2013 Jun2013 04 17importe

Hyaluronidase Hyal1 Increases Tumor Cell Proliferation and Motility through Accelerated Vesicle Trafficking

Background: Hyal1 is a turnover enzyme for hyaluronan that accelerates metastatic cancer by increasing cell motility. Results: Hyal1-overexpressing cells have a higher rate of endocytosis that impacts cargo internalization and recycling. Conclusion: The higher rate of vesicle trafficking increases motility receptor function and nutrient uptake. Significance: This novel mechanism implicates Hyal1 trafficking in multiple signaling events during tumor progression

Hyaluronidase Hyal1 Increases Tumor Cell Proliferation and Motility through Accelerated Vesicle Trafficking

Background: Hyal1 is a turnover enzyme for hyaluronan that accelerates metastatic cancer by increasing cell motility. Results: Hyal1-overexpressing cells have a higher rate of endocytosis that impacts cargo internalization and recycling. Conclusion: The higher rate of vesicle trafficking increases motility receptor function and nutrient uptake. Significance: This novel mechanism implicates Hyal1 trafficking in multiple signaling events during tumor progression

Amyloid precursor-like protein 2 (APLP2) affects the actin cytoskeleton and increases pancreatic cancer growth and metastasis.

Amyloid precursor-like protein 2 (APLP2) is aberrantly expressed in pancreatic cancer. Here we showed that APLP2 is increased in pancreatic cancer metastases, particularly in metastatic lesions found in the diaphragm and intestine. Examination of matched human primary tumor-liver metastasis pairs showed that 38.1% of the patients had positive APLP2 expression in both the primary tumor and the corresponding liver metastasis. Stable knock-down of APLP2 expression (with inducible shRNA) in pancreatic cancer cells reduced the ability of these cells to migrate and invade. Loss of APLP2 decreased cortical actin and increased intracellular actin filaments in pancreatic cancer cells. Down-regulation of APLP2 decreased the weight and metastasis of orthotopically transplanted pancreatic tumors in nude mice

Receptor-mediated internalization promotes increased endosome size and number in a RAB4- and RAB5-dependent manner

Despite their significance in receptor-mediated internalization and continued signal transduction in cells, early/sorting endosomes (EE/SE) remain incompletely characterized, with many outstanding questions that surround the dynamics of their size and number. While several studies have reported increases in EE/SE size and number resulting from endocytic events, few studies have addressed such dynamics in a methodological and quantitative manner. Herein we apply quantitative fluorescence microscopy to measure the size and number of EE/SE upon internalization of two different ligands: transferrin and epidermal growth factor. Additionally, we used siRNA knock-down to determine the involvement of 5 different endosomal RAB proteins (RAB4, RAB5, RAB8A, RAB10 and RAB11A) in EE/SE dynamics. Our study provides new information on the dynamics of endosomes during endocytosis, an important reference for researchers studying receptor-mediated internalization and endocytic events

Eps15 Homology Domain Protein 4 (EHD4) is required for Eps15 Homology Domain Protein 1 (EHD1)-mediated endosomal recruitment and fission.

Upon internalization, receptors are trafficked to sorting endosomes (SE) where they undergo sorting and are then packaged into budding vesicles that undergo fission and transport within the cell. Eps15 Homology Domain Protein 1 (EHD1), the best-characterized member of the Eps15 Homology Domain Protein (EHD) family, has been implicated in catalyzing the fission process that releases endosome-derived vesicles for recycling to the plasma membrane. Indeed, recent studies suggest that upon receptor-mediated internalization, EHD1 is recruited from the cytoplasm to endosomal membranes where it catalyzes vesicular fission. However, the mechanism by which this recruitment occurs remains unknown. Herein, we demonstrate that the EHD1 paralog, EHD4, is required for the recruitment of EHD1 to SE. We show that EHD4 preferentially dimerizes with EHD1, and knock-down of EHD4 expression by siRNA, shRNA or by CRISPR/Cas9 gene-editing leads to impaired EHD1 SE-recruitment and enlarged SE. Moreover, we demonstrate that at least 3 different asparagine-proline-phenylalanine (NPF) motif-containing EHD binding partners, Rabenosyn-5, Syndapin2 and MICAL-L1, are required for the recruitment of EHD1 to SE. Indeed, knock-down of any of these SE-localized EHD interaction partners leads to enlarged SE, presumably due to impaired endosomal fission. Overall, we identify a novel mechanistic role for EHD4 in recruitment of EHD1 to SE, thus positioning EHD4 as an essential component of the EHD1-fission machinery at SE

Characterization of a Nonclathrin Endocytic Pathway: Membrane Cargo and Lipid Requirements

Clathrin-independent endocytosis internalizes plasma membrane proteins that lack cytoplasmic sequences recognized by clathrin adaptor proteins. There is evidence for different clathrin-independent pathways but whether they share common features has not been systematically tested. Here, we examined whether CD59, an endogenous glycosylphosphatidyl inositol-anchored protein (GPI-AP), and major histocompatibility protein class I (MHCI), an endogenous, integral membrane protein, entered cells through a common mechanism and followed a similar itinerary. At early times of internalization, CD59 and MHCI were found in the same Arf6-associated endosomes before joining clathrin cargo proteins such as transferrin in common sorting endosomes. CD59 and MHCI, but not transferrin, also were observed in the Arf6-associated tubular recycling membranes. Endocytosis of CD59 and MHCI required free membrane cholesterol because it was inhibited by filipin binding to the cell surface. Expression of active Arf6 stimulated endocytosis of GPI-APs and MHCI to the same extent and led to their accumulation in Arf6 endosomes that labeled intensely with filipin. This blocked delivery of GPI-APs and MHCI to early sorting endosomes and to lysosomes for degradation. Endocytosis of transferrin was not affected by any of these treatments. These observations suggest common mechanisms for endocytosis without clathrin

Scratching the surface: Actin’ and other roles for the C-terminal Eps15 homology domain protein, EHD2

The C-terminal Eps15 homology domaincontaining (EHD) proteins participate in multiple aspects of endocytic membrane trafficking. Of the four mammalian EHD proteins, EHD2 appears to be the most disparate, both in terms of sequence homology, and in subcellular localization/function. Since its initial description as a plasma membrane-associated protein, the precise function of EHD2 has remained enigmatic. Various reports have suggested roles for EHD2 at the plasma membrane, within the endocytic transport system, and even in the nucleus. For example, EHD2 facilitates membrane fusion/repair in muscle cells. Recently the focus has shifted to the role of EHD2 in regulating caveolae. Indeed, EHD2 is highly expressed in tissues rich in caveolae, including fat, muscle and blood vessels. This review highlights cumulative evidence linking EHD2 to actin-rich structures at the plasma membrane, where the plasma membraneassociated phospholipid phosphatidylinositol 4,5- bisphosphate controls EHD2 recruitment. Herein we examine the key pathways where EHD2 might function, and address its potential involvement in these processes