Regulation of intracellular membrane trafficking and cell dynamics by syntaxin-6

Intracellular membrane trafficking along endocytic and secretory transport pathways plays a critical role in diverse cellular functions including both developmental and pathological processes. Briefly, proteins and lipids destined for transport to distinct locations are collectively assembled into vesicles and delivered to their target site by vesicular fusion. SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins are required for these events, during which v-SNAREs (vesicle SNAREs) interact with t-SNAREs (target SNAREs) to allow transfer of cargo from donor vesicle to target membrane. Recently, the t-SNARE family member, syntaxin-6, has been shown to play an important role in the transport of proteins that are key to diverse cellular dynamic processes. In this paper, we briefly discuss the specific role of SNAREs in various mammalian cell types and comprehensively review the various roles of the Golgi- and endosome-localized t-SNARE, syntaxin-6, in membrane trafficking during physiological as well as pathological conditions.     

PIP2 signaling,an integrator of cell polarity and vesicle trafficking in directionally migrating cells

Cell migration is a fundamental cellular process required for embryonic development to wound healing and also plays a key role in tumor metastasis and atherosclerosis. Migration is regulated at multiple strata, from cytoskeletal reorganization to vesicle trafficking. In migrating cells, signaling pathways are integrated with vesicle trafficking machineries in a highly coordinated fashion to accomplish the recruitment and trafficking of the trans-membrane proteins toward the leading edge. Different signaling molecules regulate cell migration in different physio-pathological contexts, among them, phosphatidylinositol-4,5-biphosphate (PIP2) is an integral component of the plasma membrane and pleiotropic lipid signaling molecule modulating diverse biological processes, including actin cytoskeletal dynamics and vesicle trafficking required for cell migration. In this commentary, we provide a brief overview of our current understandings on the phosphoinositide signaling and its implication in regulation of cell polarity and vesicle trafficking in migrating cells. In addition, we highlight the coordinated role of PIPKIγi2, a focal adhesion-targeted enzyme that synthesizes PIP2, and the exocyst complex, a PIP2-effector, in the trafficking of E-cadherin in epithelial cells and integrins in migrating cancer cells.     

PIP2 signaling,an integrator of cell polarity and vesicle trafficking in directionally migrating cells

Cell migration is a fundamental cellular process required for embryonic development to wound healing and also plays a key role in tumor metastasis and atherosclerosis. Migration is regulated at multiple strata, from cytoskeletal reorganization to vesicle trafficking. In migrating cells, signaling pathways are integrated with vesicle trafficking machineries in a highly coordinated fashion to accomplish the recruitment and trafficking of the trans-membrane proteins toward the leading edge. Different signaling molecules regulate cell migration in different physio-pathological contexts, among them, phosphatidylinositol-4,5-biphosphate (PIP2) is an integral component of the plasma membrane and pleiotropic lipid signaling molecule modulating diverse biological processes, including actin cytoskeletal dynamics and vesicle trafficking required for cell migration. In this commentary, we provide a brief overview of our current understandings on the phosphoinositide signaling and its implication in regulation of cell polarity and vesicle trafficking in migrating cells. In addition, we highlight the coordinated role of PIPKIγi2, a focal adhesion-targeted enzyme that synthesizes PIP2, and the exocyst complex, a PIP2-effector, in the trafficking of E-cadherin in epithelial cells and integrins in migrating cancer cells.     

Dominant-negative alleles of 14-3-3 proteins cause defects in actin organization and vesicle targeting in the yeast Saccharomyces cerevisiae

14-3-3 Proteins are thought to function as adapters in signaling complexes [1,2], thereby participating in cellular processes including vesicle trafficking and exocytosis [3,4]. To delineate further the function of 14-3-3 proteins during vesicle trafficking, we generated dominant-negative alleles of the two 14-3-3 homologues, Bmh1p and Bmh2p, in budding yeast and analyzed their phenotype in respect to exocytosis. Cells overexpressing the carboxy-terminal region of Bmh2p failed to polarize vesicular transport although bulk exocytosis remained unaffected and showed a disrupted actin cytoskeleton. Our data suggest that 14-3-3 proteins may act primarily on the actin cytoskeleton to regulate vesicle targeting.     

Targeting and signaling of Rho of plants guanosine triphosphatases require synergistic interaction between guanine nucleotide inhibitor and vesicular trafficking

Most eukaryotic cells are polarized. Common toolbox regulating cell polarization includes Rho guanosine triphosphatases (GTPases), in which spatiotemporal activation is regulated by a plethora of regulators. Rho of plants (ROPs) are the only Rho GTPases in plants. Although vesicular trafficking was hinted in the regulation of ROPs, it was unclear where vesicle‐carried ROP starts, whether it is dynamically regulated, and which components participate in vesicle‐mediated ROP targeting. In addition, although vesicle trafficking and guanine nucleotide inhibitor (GDI) pathways in Rho signaling have been extensively studied in yeast, it is unknown whether the two pathways interplay. Unclear are also cellular and developmental consequences of their interaction in multicellular organisms. Here, we show that the dynamic targeting of ROP through vesicles requires coat protein complex II and ADP‐ribosylation factor 1‐mediated post‐Golgi trafficking. Trafficking of vesicle‐carried ROPs between the plasma membrane and the trans‐Golgi network is mediated through adaptor protein 1 and sterol‐mediated endocytosis. Finally, we show that GDI and vesicle trafficking synergistically regulate cell polarization and ROP targeting, suggesting that the establishment and maintenance of cell polarity is regulated by an evolutionarily conserved mechanism.     

Rho proteins: linking signaling with membrane trafficking

Rho proteins are well known for their effects on the actin cytoskeleton, and are activated in response to a variety of extracellular stimuli. Several Rho family members are localized to vesicular compartments, and increasing evidence suggests that they play important roles in the trafficking of vesicles on both endocytic and exocytic pathways. In particular, RhoA, RhoB, RhoD, Rac and Cdc42 have been shown to affect various steps of membrane trafficking. The underlying molecular basis for these effects of Rho proteins are incompletely understood, but in the case of Cdc42 it appears that it can drive vesicle movement through Arp2/3 complex-mediated actin polymerization at the surface of the vesicle. This is similar to what is believed to happen when Rac and Cdc42 stimulate actin polymerization at the plasma membrane. Rho proteins may also affect membrane trafficking by altering phosphatidylinositide composition of membrane compartments, or through interactions with microtubules.     

Actin in membrane trafficking

Actin cytoskeleton remodeling provides the forces required for a variety of cellular processes based on membrane dynamics, such as endocytosis, exocytosis, and vesicular trafficking at the Golgi. All these events are coordinated by networks of associated proteins, and some of them are functionally connected with cell migration. The site and the duration of actin polymerization, in connection with vesicle budding and fusion, are tightly controlled by both small GTPases and the large GTPase dynamin. Recent advances in the understanding of the mechanisms coupling actin dynamics with membrane trafficking at the cell surface have been brought by the combined studies of actin polymerizing factors and of the endocytic/exocytic machinery.     

Proteomic profiling of the prechylomicron transport vesicle involved in the assembly and secretion of apoB‐48‐containing chylomicrons in the intestinal enterocytes

Intracellular assembly of chylomicrons (CM) occurs in intestinal enterocytes through a series of complex vesicular interactions. CM are transported from the ER to the Golgi using a specialized vesicular compartment called the prechylomicron transport vesicle (PCTV). In this study, PCTVs were isolated from the enteric ER of the Syrian Golden hamster, and characterized using 2‐DE and MS. Proteomic profiles of PCTV‐associated proteins were developed with the intention of identifying proteins involved in the formation, transport, lipidation, and assembly of CM particles. Positively identified proteins included those involved in lipoprotein assembly, namely microsomal triglyceride transfer protein and apolipoprotein B‐48, as well as proteins involved in vesicular transport, such as Sar1 and vesicle‐associated membrane protein 7. Other groups of proteins found were chaperones, intracellular vesicular trafficking proteins, fatty acid‐binding proteins, and lipid‐related proteins. These findings have increased our understanding of the transport vesicle involved in the intracellular assembly and transport of CM and can provide insight into potential cellular factors responsible for dysregulation of intestinal CM production.     

参与植物膜泡运输的蛋白质家族及功能研究进展

真核细胞的内吞和分泌途径中蛋白质和脂类的运输主要由膜泡运输介导。参与膜泡运输的蛋白质家族包括SNARE蛋白家族、RAB蛋白家族、被膜蛋白复合体、Sec1蛋白家族、Arf蛋白家族。这些蛋白质家族在进化中高度保守,并且在植物中已经鉴定了许多哺乳动物和酵母蛋白的同源物。近年来一些研究发现这些蛋白质不仅仅调节植物细胞的膜泡运输,还影响植物的许多生理活动和功能,例如向重性生长、胞质分裂、激素极性运输、气孔运动以及抗病性等。现主要阐述迄今在植物中研究这五类蛋白质家族功能的最新进展。     

The role of ADP-ribosylation factor and SAR1 in vesicular trafficking in plants

Ras-like small GTP binding proteins regulate a wide variety of intracellular signalling and vesicular trafficking pathways in eukaryotic cells including plant cells. They share a common structure that operates as a molecular switch by cycling between active GTP-bound and inactive GDP-bound conformational states. The active GTP-bound state is regulated by guanine nucleotide exchange factors (GEF), which promote the exchange of GDP for GTP. The inactive GDP-bound state is promoted by GTPase-activating proteins (GAPs) which accelerate GTP hydrolysis by orders of magnitude. Two types of small GTP-binding proteins, ADP-ribosylation factor (Arf) and secretion-associated and Ras-related (Sar), are major regulators of vesicle biogenesis in intracellular traffic and are founding members of a growing family that also includes Arf-related proteins (Arp) and Arf-like (Arl) proteins. The most widely involved small GTPase in vesicular trafficking is probably Arf1, which not only controls assembly of COPI- and AP1, AP3, and AP4/clathrin-coated vesicles but also recruits other proteins to membranes, including some that may be components of further coats. Recent molecular, structural and biochemical studies have provided a wealth of detail of the interactions between Arf and the proteins that regulate its activity as well as providing clues for the types of effector molecules which are controlled by Arf. Sar1 functions as a molecular switch to control the assembly of protein coats (COPII) that direct vesicle budding from ER. The crystallographic analysis of Sar1 reveals a number of structurally unique features that dictate its function in COPII vesicle formation. In this review, I will summarize the current knowledge of Arf and Sar regulation in vesicular trafficking in mammalian and yeast cells and will highlight recent advances in identifying the elements involved in vesicle formation in plant cells. Additionally, I will briefly discuss the similarities and dissimilarities of vesicle traffic in plant, mammalian and yeast cells.     

The role of ADP-ribosylation factor and SAR1 in vesicular trafficking in plants

Ras-like small GTP binding proteins regulate a wide variety of intracellular signalling and vesicular trafficking pathways in eukaryotic cells including plant cells. They share a common structure that operates as a molecular switch by cycling between active GTP-bound and inactive GDP-bound conformational states. The active GTP-bound state is regulated by guanine nucleotide exchange factors (GEF), which promote the exchange of GDP for GTP. The inactive GDP-bound state is promoted by GTPase-activating proteins (GAPs) which accelerate GTP hydrolysis by orders of magnitude. Two types of small GTP-binding proteins, ADP-ribosylation factor (Arf) and secretion-associated and Ras-related (Sar), are major regulators of vesicle biogenesis in intracellular traffic and are founding members of a growing family that also includes Arf-related proteins (Arp) and Arf-like (Arl) proteins. The most widely involved small GTPase in vesicular trafficking is probably Arf1, which not only controls assembly of COPI- and AP1, AP3, and AP4/clathrin-coated vesicles but also recruits other proteins to membranes, including some that may be components of further coats. Recent molecular, structural and biochemical studies have provided a wealth of detail of the interactions between Arf and the proteins that regulate its activity as well as providing clues for the types of effector molecules which are controlled by Arf. Sar1 functions as a molecular switch to control the assembly of protein coats (COPII) that direct vesicle budding from ER. The crystallographic analysis of Sar1 reveals a number of structurally unique features that dictate its function in COPII vesicle formation. In this review, I will summarize the current knowledge of Arf and Sar regulation in vesicular trafficking in mammalian and yeast cells and will highlight recent advances in identifying the elements involved in vesicle formation in plant cells. Additionally, I will briefly discuss the similarities and dissimilarities of vesicle traffic in plant, mammalian and yeast cells.     

Retromer-Mediated Protein Sorting and Vesicular Trafficking

Retromer is an evolutionarily conserved multimeric protein complex that mediates intracellular transport of various vesicular cargoes and functions in a wide variety of cellular processes including polarized trafficking,developmental signaling and lysosome biogenesis.Through its interaction with the Rab GTPases and their effectors,membrane lipids,molecular motors,the endocytic machinery and actin nucleation promoting factors,retromer regulates sorting and trafficking of transmembrane proteins from endosomes to the trans-Golgi network(TGN) and the plasma membrane.In this review.I highlight recent progress in the understanding of relromer-medialed protein sorting and vesicle trafficking and discuss how retromer contributes to a diverse set of developmental,physiological and pathological processes.     

Golgi inCOGnito: From vesicle tethering to human disease

The Conserved Oligomeric Golgi (COG) complex, a multi-subunit vesicle tethering complex of the CATCHR (Complexes Associated with Tethering Containing Helical Rods) family, controls several aspects of cellular homeostasis by orchestrating retrograde vesicle traffic within the Golgi. The COG complex interacts with all key players regulating intra-Golgi trafficking, namely SNAREs, SNARE-interacting proteins, Rabs, coiled-coil tethers, and vesicular coats. In cells, COG deficiencies result in the accumulation of non-tethered COG-complex dependent (CCD) vesicles, dramatic morphological and functional abnormalities of the Golgi and endosomes, severe defects in N- and O- glycosylation, Golgi retrograde trafficking, sorting and protein secretion. In humans, COG mutations lead to severe multi-systemic diseases known as COG-Congenital Disorders of Glycosylation (COG-CDG). In this report, we review the current knowledge of the COG complex and analyze COG-related trafficking and glycosylation defects in COG-CDG patients.     

Analysis of phosphoinositides in protein trafficking

Phosphoinositides are key regulators of vesicle-mediated protein trafficking. Their roles include recruiting vesicle coat and effector proteins to the site of budding and promoting vesicle fusion. The intracellular levels of phosphoinositides and their localization to intracellular membranes are critical to their functions. An analytical procedure was developed that optimizes the recovery of radiolabeled cellular phosphoinositides. Quantitative analyses of yeast cellular phosphoinositides indicated that this approach is useful for examining the intracellular membrane phosphoinositide compositions related to trafficking phenomena. The approach will also enable investigators to determine whole-plant phosphoinositide compositions that have been difficult to achieve in the past. These analytical advances should be generally applicable to studies of phosphoinositide dynamics related to membrane trafficking in yeast, plant, and animal cells.     

Proteomics on brefeldin A-treated Arabidopsis roots reveals profilin 2 as a new protein involved in the cross-talk between vesicular trafficking and the actin cytoskeleton

The growing importance of vesicular trafficking and cytoskeleton dynamic reorganization during plant development requires the exploitation of novel experimental approaches. Several genetic and cell biological studies have used diverse pharmaceutical drugs that inhibit vesicular trafficking and secretion to study these phenomena. Here, proteomic and cell biology approaches were applied to study effects of brefeldin A (BFA), an inhibitor of vesicle recycling and secretion, in Arabidopsis roots. The main aim of this study was to obtain an overview of proteins affected by BFA, but especially to identify new proteins involved in the vesicular trafficking and its cross-talk to the actin cytoskeleton. The results showed that BFA altered vesicular trafficking and caused the formation of BFA-compartments which was accompanied by differential expression of several proteins in root cells. Some of the BFA-up-regulated proteins belong to the class of the vesicular trafficking proteins, such as V-ATPase and reversibly glycosylated polypeptide, while others, such as profilin 2 and elongation factor 1 alpha, are rather involved in the remodeling of the actin cytoskeleton. Upregulation of profilin 2 by BFA was verified by immunoblot and live imaging at subcellular level. The latter approach also revealed that profilin 2 accumulated in BFA-compartments which was accompanied by remodeling of the actin cytoskeleton in BFA-treated root cells. Thus, profilin 2 seems to be involved in the cross-talk between vesicular trafficking and the actin cytoskeleton, in a BFA-dependent manner.     

Toward the systems biology of vesicle transport

Systems biology aims to study complex biological processes, such as intracellular traffic, as a whole. Systematic genome-wide assays have the potential to identify the transport machinery, delineate pathways and uncover the molecular components of physiological processes that influence trafficking. A goal of this approach is to create predictive models of intracellular trafficking pathways that reflect these relationships. In this review, we highlight current genome-wide technologies of particular relevance to vesicle transport and describe recent applications of these technologies in the framework of systems biology. Systems approaches hold great promise for placing trafficking pathways in their cellular contexts.     

RhoB and the mammalian Diaphanous-related formin mDia2 in endosome trafficking

Rho GTPases and the dynamic assembly and disassembly of actin filaments have been shown to have critical roles in both the internalization and trafficking of growth factor receptors. While all three mammalian Diaphanous-related (mDia1/2/3) formin GTPase effector proteins have been localized on endosomes, a role for their actin nucleation, filament elongation, and/or bundling remains poorly understood in the context of intracellular trafficking. In a study of a functional relationship between RhoB, a GTPase known to associate with both early- and late-endosomes, and the formin mDia2, we show that 1) RhoB and mDia2 interact on endosomes; 2) GTPase activity-the ability to hydrolyze GTP to GDP-is required for the ability of RhoB to govern endosome dynamics; and 3) the actin dynamics controlled by RhoB and mDia2 is necessary for vesicle trafficking. These studies further suggest that Rho GTPases significantly influence the activity of mDia family formins in driving cellular membrane remodeling through the regulation of actin dynamics.     

Redirection of host vesicle trafficking pathways by intracellular parasites

Bacterial and protozooan intracellular parasites have evolved diverse mechanisms for evasion of host cellular defenses associated with adaptations for survival in distinct intracellular compartments. As the reagents identifying discrete steps in vesicle maturation and trafficking have become increasingly available, it has become clear that the vacuoles occupied by intracellular parasites are much more diverse than had been previously appreciated. Many parasites induce selective fusion competence with the vacuoles they occupy, without affecting vesicular trafficking elsewhere in the cell. A likely means of controlling vesicular interactions is modification of the parasitophorous vacuole membrane by the insertion of parasite-specific proteins. A rapidly expanding class of bacterial proteins that modify the vacuolar membrane are the chlamydial inclusion membrane proteins. Although the functions of most of these proteins remain to be defined, the majority are expressed early in the infectious process, suggesting that modification of the vacuole is critical to the outcome of the host–parasite interaction.     

Phosphatidic acid plays key roles regulating plant development and stress responses

Phospholipids, including phosphatidic acid(PA),phosphatidylcholine(PC), phosphatidylethanolamine(PE),phosphatidylglycerol(PG), phosphatidylserine(PS) and phosphoinositides, have emerged as an important class of cellular messenger molecules in various cellular and physiological processes, of which PA attracts much attention of researchers. In addition to its effect on stimulating vesicle trafficking, many studies have demonstrated that PA plays a crucial role in various signaling pathways by binding target proteins and regulating their activity and subcellular localization. Here, we summarize the functional mechanisms and target proteins underlying PA-mediated regulation of cellular signaling, development, hormonal responses, and stress responses in plants.     

Glycosylation Is Dispensable for Sorting of Synaptotagmin 1 but Is Critical for Targeting of SV2 and Synaptophysin to Recycling Synaptic Vesicles

Glycosylation is a major form of post-translational modification of synaptic vesicle membrane proteins. For example, the three major synaptic vesicle glycoproteins, synaptotagmin 1, synaptophysin, and SV2, represent ∼30% of the total copy number of vesicle proteins. Previous studies suggested that glycosylation is required for the vesicular targeting of synaptotagmin 1, but the role of glycosylation of synaptophysin and SV2 has not been explored in detail. In this study, we analyzed all glycosylation sites on synaptotagmin 1, synaptophysin, and SV2A via mutagenesis and optical imaging of pHluorin-tagged proteins in cultured neurons from knock-out mice lacking each protein. Surprisingly, these experiments revealed that glycosylation is completely dispensable for the sorting of synaptotagmin 1 to SVs whereas the N-glycans on SV2A are only partially dispensable. In contrast, N-glycan addition is essential for the synaptic localization and function of synaptophysin. Thus, glycosylation plays distinct roles in the trafficking of each of the three major synaptic vesicle glycoproteins.