Ubiquitin-specific protease TRE17/USP6 promotes tumor cell invasion through the regulation of glycoprotein CD147 intracellular trafficking.

Disordered expression and distribution of plasma membrane proteins at the cell surface leads to diverse malignant phenotypes in tumors, including cell invasion. The ubiquitin-specific protease TRE17/USP6, an oncogene identified in Ewing sarcoma, is highly expressed in several cancers and locally aggressive tumor-like lesions. We have previously demonstrated that TRE17 regulates the trafficking of plasma membrane proteins that enter cells via clathrin-independent endocytosis (CIE); TRE17 prevents CIE cargo proteins from being targeted to lysosomes for degradation by deubiquitylating them. However, functional insights into the effects of TRE17-mediated CIE cargo trafficking on cell invasion remain unknown. Here, we show that increased expression of TRE17 enhances invasiveness of the human sarcoma cell line HT-1080 by elevating the cell surface levels of the membrane glycoprotein CD147, which plays a central role in tumor progression. We demonstrate overexpression of TRE17 decreases ubiquitylated CD147, which is accompanied by suppression of CD147 transport to lysosomes, resulting in the stabilization and increase of cell surface-localized CD147. On the other hand, we show knockdown of TRE17 decreases cell surface CD147, which is coupled with reduced production of matrix metalloproteinases, the enzymes responsible for extracellular matrix degradation. Furthermore, we demonstrate that inhibition of CD147 by a specific inhibitor alleviated the TRE17-promoted tumor cell invasion. We therefore propose a model for the pathogenesis of TRE17-driven tumors in which TRE17 increases CD147 at the cell surface by preventing its lysosomal degradation, which in turn enhances matrix metalloproteinase synthesis and matrix degradation, thereby promoting tumor cell invasion.

Recent advances in understanding the molecular basis of melanogenesis in melanocytes.

Melanin pigments are responsible for human skin and hair color, and they protect the body from harmful ultraviolet light. The black and brown melanin pigments are synthesized in specialized lysosome-related organelles called melanosomes in melanocytes. Mature melanosomes are transported within melanocytes and transferred to adjacent keratinocytes, which constitute the principal part of human skin. The melanosomes are then deposited inside the keratinocytes and darken the skin (a process called tanning). Owing to their dark color, melanosomes can be seen easily with an ordinary light microscope, and melanosome research dates back approximately 150 years; since then, biochemical studies aimed at isolating and purifying melanosomes have been conducted. Moreover, in the last two decades, hundreds of molecules involved in regulating melanosomal functions have been identified by analyses of the genes of coat-color mutant animals and patients with genetic diseases characterized by pigment abnormalities, such as hypopigmentation. In recent years, dynamic analyses by more precise microscopic observations have revealed specific functions of a variety of molecules involved in melanogenesis. This review article focuses on the latest findings with regard to the steps (or mechanisms) involved in melanosome formation and transport of mature melanosomes within epidermal melanocytes. Finally, we will touch on current topics in melanosome research, particularly on the "melanosome transfer" and "post-transfer" steps, and discuss future directions in pigment research.

TBC1D24 regulates recycling of clathrin-independent cargo proteins mediated by tubular recycling endosomes.

Many plasma membrane proteins enter cells by clathrin-independent endocytosis (CIE). Rab family small GTPases play pivotal roles in CIE and following intracellular trafficking of cargo proteins. Here, we provide evidence that TBC1D24, which contains an atypical Rab GAP domain, facilitates formation of tubular recycling endosomes (TREs) that are a hallmark of the CIE cargo trafficking pathway in HeLa cells. Overexpression of TBC1D24 in HeLa cells dramatically increased TREs loaded with CIE cargo proteins, while deletion of TBC1D24 impaired TRE formation and delayed the recycling of CIE cargo proteins back to the plasma membrane. We also found that TBC1D24 binds to Rab22A, through which TBC1D24 regulates TRE-mediated CIE cargo recycling. These findings provide insight into regulatory mechanisms for CIE cargo trafficking.

Unveiling the interaction between the molecular motor Myosin Vc and the small GTPase Rab3A.

Vertebrates usually have three class V myosin paralogues (MyoV) to control membrane trafficking in the actin-rich cell cortex, but their functional overlapping or differentiation through cargoes selectivity is yet only partially understood. In this work, we reveal that the globular tail domain of MyoVc binds to the active form of small GTPase Rab3A with nanomolar affinity, a feature shared with MyoVa but not with MyoVb. Using molecular docking analyses guided by chemical cross-linking restraints, we propose a model to explain how Rab3A selectively recognizes MyoVa and MyoVc via a distinct binding site from that used by Rab11A. The MyoVa/c binding interface involves multiple residues from both lobules (I and II) and the short helix at the α2-α3 link region, which is conserved between MyoVa and MyoVc, but not in MyoVb. This motif is also responsible for the selective binding of RILPL2 by MyoVa and potentially MyoVc. Together, these findings support the selective recruitment of MyoVa and MyoVc to exocytic pathways via Rab3A and expand our knowledge about the functional evolution of class V myosins. SIGNIFICANCE: Hormone secretion, neurotransmitter release, and cytoplasm membrane recycling are examples of processes that rely on the interaction of molecular motors and Rab GTPases to regulate the intracellular trafficking and tethering of vesicles. Defects in these proteins may cause neurological impairment, immunodeficiency, and other severe disorders, being fatal in some cases. Despite their crucial roles, little is known about how these molecular motors are selectively recruited by specific members of the large family of Rab GTPases. In this study, we unveil the interaction between the actin-based molecular motor Myosin Vc and the small GTPase Rab3A, a key coordinator of vesicle trafficking and exocytosis in mammalian cells. Moreover, we propose a model for their recognition and demonstrate that Rab3A specifically binds to the globular tail of Myosins Va and Vc, but not of Myosin Vb, advancing our knowledge about the molecular basis for the selective recruitment of class V myosins by Rab GTPases.

Cytoplasmic control of Rab family small GTPases through BAG6.

Rab family small GTPases are master regulators of distinct steps of intracellular vesicle trafficking in eukaryotic cells. GDP-bound cytoplasmic forms of Rab proteins are prone to aggregation due to the exposure of hydrophobic groups but the machinery that determines the fate of Rab species in the cytosol has not been elucidated in detail. In this study, we find that BAG6 (BAT3/Scythe) predominantly recognizes a cryptic portion of GDP-associated Rab8a, while its major GTP-bound active form is not recognized. The hydrophobic residues of the Switch I region of Rab8a are essential for its interaction with BAG6 and the degradation of GDP-Rab8a via the ubiquitin-proteasome system. BAG6 prevents the excess accumulation of inactive Rab8a, whose accumulation impairs intracellular membrane trafficking. BAG6 binds not only Rab8a but also a functionally distinct set of Rab family proteins, and is also required for the correct distribution of Golgi and endosomal markers. From these observations, we suggest that Rab proteins represent a novel set of substrates for BAG6, and the BAG6-mediated pathway is associated with the regulation of membrane vesicle trafficking events in mammalian cells.

SNARE dynamics during melanosome maturation.

Historically, studies on the maturation and intracellular transport of melanosomes in melanocytes have greatly contributed to elucidating the general mechanisms of intracellular transport in many different types of mammalian cells. During melanosome maturation, melanosome cargoes including melanogenic enzymes (e.g. tyrosinase) are transported from endosomes to immature melanosomes by membrane trafficking, which must require a membrane fusion process likely regulated by SNAREs [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptors]. In the present study, we review the literature concerning the expression and function of SNAREs (e.g. v-SNARE vesicle-associated membrane protein 7 and t-SNAREs syntaxin-3/13 and synaptosomal-associated protein-23) in melanocytes, especially in regard to the fusion process in which melanosome cargoes are finally delivered to immature melanosomes. We also describe the recent discovery of the SNARE recycling system on mature melanosomes in melanocytes. Such SNARE dynamics, especially the SNARE recycling system, on melanosomes will be useful in understanding as yet unidentified SNARE dynamics on other organelles.

LPA1/3 signaling mediates tumor lymphangiogenesis through promoting CRT expression in prostate cancer.

Lysophosphatidic acid (LPA) is a bioactive lipid growth factor which is present in high levels in serum and platelets. LPA binds to its specific G-protein-coupled receptors, including LPA1 to LPA6, thereby regulating various physiological functions, including cancer growth, angiogenesis, and lymphangiogenesis. Our previous study showed that LPA promotes the expression of the lymphangiogenic factor vascular endothelial growth factor (VEGF)-C in prostate cancer (PCa) cells. Interestingly, LPA has been shown to regulate the expression of calreticulin (CRT), a multifunctional chaperone protein, but the roles of CRT in PCa progression remain unclear. Here we investigated the involvement of CRT in LPA-mediated VEGF-C expression and lymphangiogenesis in PCa. Knockdown of CRT significantly reduced LPA-induced VEGF-C expression in PC-3 cells. Moreover, LPA promoted CRT expression through LPA receptors LPA1 and LPA3, reactive oxygen species (ROS) production, and phosphorylation of eukaryotic translation initiation factor 2α (eIF2α). Tumor-xenografted mouse experiments further showed that CRT knockdown suppressed tumor growth and lymphangiogenesis. Notably, clinical evidence indicated that the LPA-producing enzyme autotaxin (ATX) is related to CRT and that CRT level is highly associated with lymphatic vessel density and VEGF-C expression. Interestingly, the pharmacological antagonist of LPA receptors significantly reduced the lymphatic vessel density in tumor and lymph node metastasis in tumor-bearing nude mice. Together, our results demonstrated that CRT is critical in PCa progression through the mediation of LPA-induced VEGF-C expression, implying that targeting the LPA signaling axis is a potential therapeutic strategy for PCa.

Physiological function of phospholipase D2 in anti-tumor immunity: regulation of CD8+ T lymphocyte proliferation.

Two major phospholipase D (PLD) isozymes in mammals, PLD1 and PLD2, hydrolyze the membrane phospholipid phosphatidylcholine to choline and the lipid messenger phosphatidic acid. Although their roles in cancer cells have been well studied, their functions in tumor microenvironment have not yet been clarified. Here, we demonstrate that PLD2 in cytotoxic CD8+ T cells plays a crucial role in anti-tumor immunity by regulating their cell proliferation. We found that growth of tumors formed by subcutaneously transplanted cancer cells is enhanced in Pld2-knockout mice. Interestingly, this phenotype was found to be at least in part attributable to the ablation of Pld2 from bone marrow cells. The number of CD8+ T cells, which induce cancer cell death, significantly decreased in the tumor produced in Pld2-knockout mice. In addition, CD3/CD28-stimulated proliferation of primary cultured splenic CD8+ T cells is markedly suppressed by Pld2 ablation. Finally, CD3/CD28-dependent activation of Erk1/2 and Ras is inhibited in Pld2-deleted CD8+ T cells. Collectively, these results indicate that PLD2 in CD8+ T cells plays a key role in their proliferation through activation of the Ras/Erk signaling pathway, thereby regulating anti-tumor immunity.

The RAB2B-GARIL5 Complex Promotes Cytosolic DNA-Induced Innate Immune Responses.

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that induces the IFN antiviral response. However, the regulatory mechanisms that mediate cGAS-triggered signaling have not been fully explored. Here, we show the involvement of a small GTPase, RAB2B, and its effector protein, Golgi-associated RAB2B interactor-like 5 (GARIL5), in the cGAS-mediated IFN response. RAB2B-deficiency affects the IFN response induced by cytosolic DNA. Consistent with this, RAB2B deficiency enhances replication of vaccinia virus, a DNA virus. After DNA stimulation, RAB2B colocalizes with stimulator of interferon genes (STING), the downstream signal mediator of cGAS, on the Golgi apparatus. The GTP-binding activity of RAB2B is required for its localization on the Golgi apparatus and for recruitment of GARIL5. GARIL5 deficiency also affects the IFN response induced by cytosolic DNA and enhances replication of vaccinia virus. These findings indicate that the RAB2B-GARIL5 complex promotes IFN responses against DNA viruses by regulating the cGAS-STING signaling axis.

Arf6 in lymphatic endothelial cells regulates lymphangiogenesis by controlling directional cell migration.

The small GTPase Arf6 plays pivotal roles in a wide variety of cellular events such as endocytosis, exocytosis, and actin cytoskeleton reorganization. However, the physiological functions of Arf6 at the whole animal level have not yet been thoroughly understood. Here, we show that Arf6 regulates developmental and tumor lymphangiogenesis in mice. Lymphatic endothelial cell (LEC)-specific Arf6 conditional knockout (LEC-Arf6 cKO) mouse embryos exhibit severe skin edema and impairment in the formation of lymphatic vessel network at the mid-gestation stage. Knockdown of Arf6 in human LECs inhibits in vitro capillary tube formation and directed cell migration induced by vascular endothelial growth factor-C (VEGF-C) by inhibiting VEGF-C-induced internalization of ß1 integrin. Finally, we found that LEC-Arf6 cKO mice transplanted with B16 melanoma cells attenuated tumor lymphangiogenesis and progression. Collectively, these results demonstrate that Arf6 in LECs plays a crucial role in physiological and pathological lymphangiogenesis.

Regulation of HGF-induced hepatocyte proliferation by the small GTPase Arf6 through the PIP2-producing enzyme PIP5K1A.

HGF and its receptor c-Met are critical molecules in various biological processes. Others and we have previously shown that the small GTPase Arf6 plays a pivotal role in HGF signaling in hepatocytes. However, the molecular mechanism of how Arf6 regulates HGF signaling is unclear. Here, we show that Arf6 plays an important role in HGF-stimulated hepatocyte proliferation and liver regeneration through the phosphatidylinositol 4,5-bisphosphate (PIP2)-producing enzyme PIP5K1A. We find that knockdown of Arf6 and PIP5K1A in HepG2 cells inhibits HGF-stimulated proliferation, Akt activation, and generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and its precursor PIP2. Interestingly, PIP5K1A is recruited to c-Met upon HGF stimulation in an Arf6 activity-dependent manner. Finally, we show that hepatocyte proliferation and liver regeneration after partial hepatectomy are suppressed in Pip5k1a knockout mice. These results provide insight into the molecular mechanism for HGF-stimulated hepatocyte proliferation and liver regeneration: Arf6 recruits PIP5K1A to c-Met and activates it upon HGF stimulation to produce PIP2 and subsequently PIP3, which in turn activates Akt to promote hepatocyte proliferation, thereby accelerating liver regeneration after liver injury.

The small G protein Arf6 expressed in keratinocytes by HGF stimulation is a regulator for skin wound healing.

The earlier step of cutaneous wound healing process, re-epithelialization of the wounded skin, is triggered by a variety of growth factors. However, molecular mechanisms through which growth factors trigger skin wound healing are less understood. Here, we demonstrate that hepatocyte growth factor (HGF)/c-Met signaling-induced expression of the small G protein Arf6 mRNA in keratinocytes is essential for the skin wound healing. Arf6 mRNA expression was dramatically induced in keratinocytes at the wounded skin, which was specifically suppressed by the c-Met inhibitor. Wound healing of the skin was significantly delayed in keratinocyte-specific Arf6 conditional knockout mice. Furthermore, Arf6 deletion from keratinocytes remarkably suppressed HGF-stimulated cell migration and peripheral membrane ruffle formation, but did not affect skin morphology and proliferation/differentiation of keratinocytes. These results are consistent with the notion that Arf6 expressed in skin keratinocytes through the HGF/c-Met signaling pathway in response to skin wounding plays an important role in skin wound healing by regulating membrane dynamics-based motogenic cellular function of keratinocytes.

Rab32 subfamily small GTPases: pleiotropic Rabs in endosomal trafficking.

Rab small GTPases, well-known regulators of membrane trafficking pathways in eukaryotic cells, comprise approximately 60 different members in mammals. During the past decade, our understanding of the functions of mammalian Rab32 subfamily members (Rab32 and Rab38) have deepened, especially on the biogenesis of lysosome-related organelles, such as melanosomes, and the protection mechanisms against several pathogenic microbial infections. Endosome-mediated membrane trafficking by Rab32 subfamily members plays pivotal roles in these events. In this review, we provide an overview of the regulatory mechanisms of mammalian Rab32-family members in endosomal trafficking, especially focusing on their GEF, GAP and effector molecules, and describe the latest findings on physiological and pathological functions regulated by these molecules.

C9orf72 and RAB7L1 regulate vesicle trafficking in amyotrophic lateral sclerosis and frontotemporal dementia.

A non-coding hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), however, the precise molecular mechanism by which the C9orf72 hexanucleotide repeat expansion directs C9ALS/FTD pathogenesis remains unclear. Here, we report a novel disease mechanism arising due to the interaction of C9ORF72 with the RAB7L1 GTPase to regulate vesicle trafficking. Endogenous interaction between C9ORF72 and RAB7L1 was confirmed in human SH-SY5Y neuroblastoma cells. The C9orf72 hexanucleotide repeat expansion led to haploinsufficiency resulting in severely defective intracellular and extracellular vesicle trafficking and a dysfunctional trans-Golgi network phenotype in patient-derived fibroblasts and induced pluripotent stem cell-derived motor neurons. Genetic ablation of RAB7L1or C9orf72 in SH-SY5Y cells recapitulated the findings in C9ALS/FTD fibroblasts and induced pluripotent stem cell neurons. When C9ORF72 was overexpressed or antisense oligonucleotides were targeted to the C9orf72 hexanucleotide repeat expansion to upregulate normal variant 1 transcript levels, the defective vesicle trafficking and dysfunctional trans-Golgi network phenotypes were reversed, suggesting that both loss- and gain-of-function mechanisms play a role in disease pathogenesis. In conclusion, we have identified a novel mechanism for C9ALS/FTD pathogenesis highlighting the molecular regulation of intracellular and extracellular vesicle trafficking as an important pathway in C9ALS/FTD pathogenesis.

ACAP3 regulates neurite outgrowth through its GAP activity specific to Arf6 in mouse hippocampal neurons.

ACAP3 (ArfGAP with coiled-coil, ankyrin repeat and pleckstrin homology domains 3) belongs to the ACAP family of GAPs (GTPase-activating proteins) for the small GTPase Arf (ADP-ribosylation factor). However, its specificity to Arf isoforms and physiological functions remain unclear. In the present study, we demonstrate that ACAP3 plays an important role in neurite outgrowth of mouse hippocampal neurons through its GAP activity specific to Arf6. In primary cultured mouse hippocampal neurons, knockdown of ACAP3 abrogated neurite outgrowth, which was rescued by ectopically expressed wild-type ACAP3, but not by its GAP activity-deficient mutant. Ectopically expressed ACAP3 in HEK (human embryonic kidney)-293T cells showed the GAP activity specific to Arf6. In support of this observation, the level of GTP-bound Arf6 was significantly increased by knockdown of ACAP3 in hippocampal neurons. In addition, knockdown and knockout of Arf6 in mouse hippocampal neurons suppressed neurite outgrowth. These results demonstrate that ACAP3 positively regulates neurite outgrowth through its GAP activity specific to Arf6. Furthermore, neurite outgrowth suppressed by ACAP3 knockdown was rescued by expression of a fast cycle mutant of Arf6 that spontaneously exchanges guanine nucleotides on Arf6, but not by that of wild-type, GTP- or GDP-locked mutant Arf6. Thus cycling between active and inactive forms of Arf6, which is precisely regulated by ACAP3 in concert with a guanine-nucleotide-exchange factor(s), seems to be required for neurite outgrowth of hippocampal neurons.

A Varp-Binding Protein, RACK1, Regulates Dendrite Outgrowth through Stabilization of Varp Protein in Mouse Melanocytes.

Varp (VPS9-ankyrin repeat protein) in melanocytes is thought to function as a key player in the pigmentation of mammals. Varp regulates two different melanocyte functions: (i) transport of melanogenic enzymes to melanosomes by functioning as a Rab32/38 effector and (ii) promotion of dendrite outgrowth by functioning as a Rab21-guanine nucleotide exchange factor. The Varp protein level has recently been shown to be negatively regulated by proteasomal degradation through interaction of the ankyrin repeat 2 (ANKR2) domain of Varp with Rab40C. However, the molecular mechanisms by which Varp escapes from Rab40C and retains its own expression level remain completely unknown. Here, we identified RACK1 (receptor of activated protein kinase C 1) as a Varp-ANKR2 binding partner and investigated its involvement in Varp stabilization in mouse melanocytes. The results showed that knockdown of endogenous RACK1 in melanocytes caused dramatic reduction of the Varp protein level and inhibition of dendrite outgrowth, and intriguingly, overexpression of RACK1 inhibited the interaction between Varp and Rab40C and counteracted the negative effect of Rab40C on dendrite outgrowth. These findings indicated that RACK1 competes with Rab40C for binding to the ANKR2 domain of Varp and regulates dendrite outgrowth through stabilization of Varp in mouse melanocytes.

Pathological functions of the small GTPase Arf6 in cancer progression: Tumor angiogenesis and metastasis.

Although several lines of evidence have shown that the small GTPase ADP-ribosylation factor 6 (Arf6) plays pivotal roles in cancer progression of several types of cancers, little is known about the functions of Arf6 in tumor microenvironment. We demonstrated that Arf6 in vascular endothelial cells (VECs) plays a crucial role in tumor angiogenesis and growth using endothelial cell-specific Arf6 conditional knockout mice into which B16 melanoma and Lewis lung carcinoma cells were implanted. It was also found that Arf6 in VECs positively regulates hepatocyte growth factor (HGF)-induced ß1 integrin recycling, which is a critical event for tumor angiogenesis by promoting cell migration. Importantly, pharmacological inhibition of HGF-induced Arf6 activation significantly suppresses tumor angiogenesis and growth in mice, suggesting that Arf6 signaling would be a potential target for anti-angiogenic therapy. In this manuscript, we summarize the multiple roles of Arf6 in cancer progression, particularly in cancer cell invasion/metastasis and our recent findings on tumor angiogenesis, and discuss a possible approach to develop innovative anti-cancer drugs.

RUTBC1 Functions as a GTPase-activating Protein for Rab32/38 and Regulates Melanogenic Enzyme Trafficking in Melanocytes.

Two cell type-specific Rab proteins, Rab32 and Rab38 (Rab32/38), have been proposed as regulating the trafficking of melanogenic enzymes, including tyrosinase and tyrosinase-related protein 1 (Tyrp1), to melanosomes in melanocytes. Like other GTPases, Rab32/38 function as switch molecules that cycle between a GDP-bound inactive form and a GTP-bound active form; the cycle is thought to be regulated by an activating enzyme, guanine nucleotide exchange factor (GEF), and an inactivating enzyme, GTPase-activating protein (GAP), which stimulates the GTPase activity of Rab32/38. Although BLOC-3 has already been identified as a Rab32/38-specific GEF that regulates the trafficking of tyrosinase and Tyrp1, no physiological GAP for Rab32/38 in melanocytes has ever been identified, and it has remained unclear whether Rab32/38 is involved in the trafficking of dopachrome tautomerase, another melanogenic enzyme, in mouse melanocytes. In this study we investigated RUTBC1, which was originally characterized as a Rab9-binding protein and GAP for Rab32 and Rab33B in vitro, and the results demonstrated that RUTBC1 functions as a physiological GAP for Rab32/38 in the trafficking of all three melanogenic enzymes in mouse melanocytes. The results of this study also demonstrated the involvement of Rab9A in the regulation of the RUTBC1 localization and in the trafficking of all three melanogenic enzymes. We discovered that either excess activation or inactivation of Rab32/38 achieved by manipulating RUTBC1 inhibits the trafficking of all three melanogenic enzymes. These results collectively indicate that proper spatiotemporal regulation of Rab32/38 is essential for the trafficking of all three melanogenic enzymes in mouse melanocytes.

Activation of the Small G Protein Arf6 by Dynamin2 through Guanine Nucleotide Exchange Factors in Endocytosis.

The small G protein Arf6 and the GTPase dynamin2 (Dyn2) play key roles in clathrin-mediated endocytosis (CME). However, their functional relationship remains obscure. Here, we show that Arf6 functions as a downstream molecule of Dyn2 in CME. Wild type of Dyn2 overexpressed in HeLa cells markedly activates Arf6, while a GTPase-lacking Dyn2 mutant does not. Of the Arf6-specific guanine nucleotide exchange factors, EFA6A, EFA6B, and EFA6D specifically interact with Dyn2. Furthermore, overexpression of dominant negative mutants or knockdown of EFA6B and EFA6D significantly inhibit Dyn2-induced Arf6 activation. Finally, overexpression of the binding region peptide of EFA6B for Dyn2 or knockdown of EFA6B and EFA6D significantly suppresses clathrin-mediated transferrin uptake. These results provide evidence for a novel Arf6 activation mechanism by Dyn2 through EFA6B and EFA6D in CME in a manner dependent upon the GTPase activity of Dyn2.

Measurement of Rab35 activity with the GTP-Rab35 trapper RBD35.

Small GTPase Rab35 functions as a molecular switch for membrane trafficking, specifically for endocytic recycling, by cycling between a GTP-bound active form and a GDP-bound inactive form. Although Rab35 has been shown to regulate various cellular processes, including cytokinesis, cell migration, and neurite outgrowth, its precise roles in these processes are not fully understood. Since a molecular tool that could be used to measure Rab35 activity would be useful for identifying the mechanisms by which Rab35 mediates membrane trafficking, we recently used a RUN domain-containing region of RUSC2 to develop an active Rab35 trapper, and we named it RBD35 (Rab-binding domain specific for Rab35). Because RBD35 specifically interacts with the GTP-bound active form of Rab35 and does not interact with any of the other 59 Rab proteins identified in humans and mice, RBD35 is a useful tool for measuring the level of active Rab35 by pull-down assays and for inhibiting the function of Rab35 by overexpression. In this chapter, we describe the assay procedures for analyzing Rab35 with RBD35.