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.     

SNARE membrane trafficking dynamics in vivo

The ER/Golgi soluble NSF attachment protein receptor (SNARE) membrin, rsec22b, and rbet1 are enriched in approximately 1-micrometer cytoplasmic structures that lie very close to the ER. These appear to be ER exit sites since secretory cargo concentrates in and exits from these structures. rsec22b and rbet1 fused to fluorescent proteins are enriched at approximately 1-micrometer ER exit sites that remained more or less stationary, but periodically emitted streaks of fluorescence that traveled generally in the direction of the Golgi complex. These exit sites were reused and subsequent tubules or streams of vesicles followed similar trajectories. Fluorescent membrin- enriched approximately 1-micrometer peripheral structures were more mobile and appeared to translocate through the cytoplasm back and forth, between the periphery and the Golgi area. These mobile structures could serve to collect secretory cargo by fusing with ER-derived vesicles and ferrying the cargo to the Golgi. The post-Golgi SNAREs, syntaxin 6 and syntaxin 13, when fused to fluorescent proteins each displayed characteristic patterns of movement. However, syntaxin 13 was the only SNARE whose life cycle appeared to involve interactions with the plasma membrane. These studies reveal the in vivo spatiotemporal dynamics of SNARE proteins and provide new insight into their roles in membrane trafficking.     

Separation of membrane trafficking and actin remodeling functions of ARF6 with an effector domain mutant

The ADP-ribosylation factor 6 (ARF6) GTPase has a dual function in cells, regulating membrane traffic and organizing cortical actin. ARF6 activation is required for recycling of the endosomal membrane back to the plasma membrane (PM) and also for ruffling at the PM induced by Rac. Additionally, ARF6 at the PM induces the formation of actin-containing protrusions. To identify sequences in ARF6 that are necessary for these distinct functions, we examined the behavior of a chimeric protein of ARF1 and ARF6. The 1-6 chimera (with the amino half of ARF1 and the carboxyl half of ARF6) localized like ARF6 in HeLa cells and moved between the endosome and PM, but it did not form protrusions, an ARF6 effector function. Two residues in the amino-terminal half of ARF6, Q37 and S38, when substituted into the 1-6 chimera allowed protrusion formation, whereas removal of these residues from ARF6 resulted in an inability to form protrusions. Interestingly, expression of 1-6 in cells selectively inhibited protrusions induced by wild-type ARF6 but had no effect on ARF6-regulated membrane movement or Rac-induced ruffling. Thus, we have uncoupled two functions of ARF6, one involved in membrane trafficking, which is necessary for Rac ruffling, and another involved in protrusion formation.     

Probing phosphoinositide functions in signaling and membrane trafficking

The inositol phospholipids (PIs) comprise a family of eight species with different combinations of phosphate groups arranged around the inositol ring. PIs are among the most versatile signaling molecules known, with key roles in receptor-mediated signal transduction, actin remodeling and membrane trafficking. Recent studies have identified effector proteins and specific lipid-binding domains through which PIs signal. These lipid-binding domains can be used as probes to further our understanding of the spatial and temporal control of individual PI species. New layers of complexity revealed by the use of such probes include the occurrence of PIs at intracellular locations, the identification of phosphatidylinositol signaling hotspots and the presence of non-membrane pools of PIs in cell nuclei.     

Cytoskeletal regulation: coordinating actin and microtubule dynamics in membrane trafficking

The Arp2/3 complex is essential for actin nucleation and filament elongation in a variety of intracellular processes. This functional versatility is exerted through the regulation of its activity by nucleation-promoting factors (NPFs). The discovery of a new NPF, WHAMM, reveals unexpected connections between the actin and microtubule cytoskeletons and membrane dynamics during ER-to-Golgi transport.     

Probing intracellular vesicle trafficking and membrane remodelling by cryo-EM

Protein transport between the membranous compartments of the eukaryotic cells is mediated by the constant fission and fusion of the membrane-bounded vesicles from a donor to an acceptor membrane. While there are many membrane remodelling complexes in eukaryotes, COPII, COPI, and clathrin-coated vesicles are the three principal classes of coat protein complexes that participate in vesicle trafficking in the endocytic and secretory pathways. These vesicle-coat proteins perform two key functions: deforming lipid bilayers into vesicles and encasing selective cargoes. The three trafficking complexes share some commonalities in their structural features but differ in their coat structures, mechanisms of cargo sorting, vesicle formation, and scission. While the structures of many of the proteins involved in vesicle formation have been determined in isolation by X-ray crystallography, elucidating the proteins' structures together with the membrane is better suited for cryogenic electron microscopy (cryo-EM). In recent years, advances in cryo-EM have led to solving the structures and mechanisms of several vesicle trafficking complexes and associated proteins.     

Mechanisms of synaptic plasticity: from membrane to intracellular AMPAR trafficking

Brief periods of repetitive neural firing onto adjacent neurons can lead to changes in synaptic plasticity, that is, changes in the make-up of macromolecular complexes located at synapses. This process includes the regulated trafficking of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) to synaptic membranes. Little is known, however, about how the AMPARs are regulated before they are shuttled to the membrane. Greger et al. have found that the length of the cytoplasmic tails of constituent subunits of a given AMPAR is determined by editing [at a glutamine (Q) or an arginine (R) codon] near their C termini. Tail length, in turn, dictates whether AMPARs will be retained or quickly released from the endoplasmic reticulum.     

Thrombospondins in the heart: potential functions in cardiac remodeling

Cardiac remodeling after myocardial injury involves inflammation, angiogenesis, left ventricular hypertrophy and matrix remodeling. Thrombospondins (TSPs) belong to the group of matricellular proteins, which are non-structural extracellular matrix proteins that modulate cell–matrix interactions and cell function in injured tissues or tumors. They interact with different matrix and membrane-bound proteins due to their diverse functional domains. That the expression of TSPs strongly increases during cardiac stress or injury indicates an important role for them during cardiac remodeling. Recently, the protective properties of TSP expression against heart failure have been acknowledged. The current review will focus on the biological role of TSPs in the ischemic and hypertensive heart, and will describe the functional consequences of TSP polymorphisms in cardiac disease.     

Differential functions of Hrs and ESCRT proteins in endocytic membrane trafficking

A ubiquitin-binding endosomal protein machinery is responsible for sorting endocytosed membrane proteins into intraluminal vesicles of multivesicular endosomes (MVEs) for subsequent degradation in lysosomes. The Hrs-STAM complex and endosomal sorting complex required for transport (ESCRT)-I, -II and -III are central components of this machinery. Here, we have performed a systematic analysis of their importance in four trafficking pathways through endosomes. Neither Hrs, Tsg101 (ESCRT-I), Vps22/EAP30 (ESCRT-II), nor Vps24/CHMP3 (ESCRT-III) was required for ligand-mediated internalization of epidermal growth factor (EGF) receptors (EGFRs) or for recycling of cation-independent mannose 6-phosphate receptors (CI-M6PRs) from endosomes to the trans-Golgi network (TGN). In contrast, both Hrs and ESCRT subunits were equally required for degradation of both endocytosed EGF and EGFR. Whereas depletion of Hrs or Tsg101 caused enhanced recycling of endocytosed EGFRs, this was not the case with depletion of Vps22 or Vps24. Depletion of Vps24 instead caused a strong increase in the levels of CI-M6PRs and a dramatic redistribution of the Golgi and the TGN. These results indicate that, although Hrs-STAM and ESCRT-I, -II and -III have a common function in degradative protein sorting, they play differential roles in other trafficking pathways, probably reflecting their functions at distinct stages of the endocytic pathway.     

Epithelial cell cytoskeleton and intracellular trafficking V. Confluence of membrane trafficking and motility in epithelial cell models

Migration of epithelial cells occurs in a variety of important biological processes including tissue morphogenesis, wound healing, and the metastasis of epithelial tumors. In some instances, the cells remain attached to each other and migrate together as a sheet, maintaining epithelial integrity. In others (e.g., metastasis), junctional complexes are disrupted and cells migrate individually. In both cases, motility involves the extension of membranous protrusions (filopodia and lamellipodia) in the direction of movement and the transient assembly and disassembly of integrin-mediated adhesions with the extracellular matrix. The driving force for these events is provided by regulated changes in the organization of the actin cytoskeleton, which are thought to be coordinated with alterations in intracellular membrane traffic. In this themes article, I review current hypotheses about how these processes are integrated and attempt to identify fruitful areas for future research.     

PIST regulates the intracellular trafficking and plasma membrane expression of Cadherin 23

Background  

The atypical cadherin protein cadherin 23 (CDH23) is crucial for proper function of retinal photoreceptors and inner ear hair cells. As we obtain more and more information about the specific roles of cadherin 23 in photoreceptors and hair cells, the regulatory mechanisms responsible for the transport of this protein to the plasma membrane are largely unknown.     

GTPases in intracellular trafficking: an overview

Small GTPases that belong to the ras sub-families of Rab, Arf, and Rho, and the large GTPase dynamin, regulate intracellular trafficking. This issue of Seminars of Cell and Developmental Biology highlights topics regarding mechanisms by which these GTPases regulate the different steps of vesicular transport: vesicle formation, scission, targeting and fusion. In addition, the emerging roles of GTPases in coordination of individual transport steps as well as coordination of intracellular trafficking with other cellular processes are reviewed. Finally, common structures and mechanisms underlying the function of the ras-like GTPases and the importance of their function to human health and disease are discussed.     

Implications of receptor-mediated endocytosis and intracellular trafficking dynamics in the development of antibody drug conjugates

The use of antibody-drug conjugates (ADCs) as a therapeutic platform to treat cancer has recently gained substantial momentum. This therapeutic modality has the potential to increase the efficacy and reduce the systemic toxicity associated with current therapeutic regimens. The efficacy of ADCs, however, relies on the proper exploitation of intracellular sorting dynamics of the antigen as well as the specificity, selectivity and pharmacokinetic properties of the antibody itself. Our understanding of endocytosis and endosomal trafficking of receptors has appreciably increased in recent years, as improvements in the assays used to study these events have resolved many of the molecular mechanisms regulating these processes. As a result, we now have the knowledge necessary to exploit these pathways efficiently to improve the efficacy of antibody-based therapy. This review discusses some recent studies that have explored how endo/lysosomal dynamics can affect the efficacy of engineered therapeutic antibodies, including ADCs.     

Regulation of phospholipase D activity, membrane targeting and intracellular trafficking by phosphoinositides

Generation of PA (phosphatidic acid) by PLD (phospholipase D)-catalysed hydrolysis of phosphatidylcholine plays a pivotal role in cellular signalling pathways that regulate organization of the actin cytoskeleton, vesicular transport and exocytosis and stimulation of cell growth and survival. PLD regulation and function are intimately linked with phosphoinositide metabolism. Phosphatidyl 4-phosphate 5-kinase is stimulated by PA in vitro and this enzyme is the downstream effector of a significant subset of PLD signalling pathways. Yeast and mammalian PLDs are potently and specifically activated by the product of this kinase, PtdIns(4,5)P2, through interactions mediated by a polybasic motif within the catalytic core of the enzyme. Integrity of this motif is critical for agonist activation of mammalian PLD and for PLD function in secretion, sporulation and exocytosis in vivo. Although dispensable for catalysis in vitro, these PLD enzymes also contain N-terminal PH (pleckstrin) and PX (phox) homology domains. Binding studies using recombinantly expressed PLD fragments indicate that the PH and PX domains also interact specifically with distinct phosphoinositide ligands. Both the PX and PH domains are important for PLD function by controlling the dynamic association of the enzyme with the plasma membrane and its intracellular trafficking by the endocytic pathway. These results identify two distinct modes of regulation of PLD by phosphoinositides: stimulation of catalysis mediated by the polybasic domain and dynamic regulation of membrane targeting mediated primarily by the PH and PX domains.     

Spatiotemporal dynamics of actin remodeling and endomembrane trafficking in alveolar epithelial type I cell wound healing

Alveolar epithelial type I cell (ATI) wounding is prevalent in ventilator-injured lungs and likely contributes to pathogenesis of "barotrauma" and "biotrauma." In experimental models most wounded alveolar cells repair plasma membrane (PM) defects and survive insults. Considering the force balance between edge energy at the PM wound margins and adhesive interactions of the lipid bilayer with the underlying cytoskeleton (CSK), we tested the hypothesis that subcortical actin depolymerization is a key facilitator of PM repair. Using real-time fluorescence imaging of primary rat ATI transfected with a live cell actin-green fluorescent protein construct (Lifeact-GFP) and loaded with N-rhodamine phosphatidylethanolamine (PE), we examined the spatial and temporal coordination between cytoskeletal remodeling and PM repair following micropuncture. Membrane integrity was inferred from the fluorescence intensity profiles of the cytosolic label calcein AM. Wounding led to rapid depolymerization of the actin CSK near the wound site, concurrent with accumulation of endomembrane-derived N-rhodamine PE. Both responses were sustained until PM integrity was reestablished, which typically occurs between ～10 and 40 s after micropuncture. Only thereafter did the actin CSK near the wound begin to repolymerize, while the rate of endomembrane lipid accumulation decreased. Between 60 and 90 s after successful PM repair, after translocation of the actin nucleation factor cortactin, a dense actin fiber network formed. In cells that did not survive micropuncture injury, actin remodeling did not occur. These novel results highlight the importance of actin remodeling in ATI cell repair and suggest molecular targets for modulating the repair process.     

Spatiotemporal dynamics of membrane remodeling and fusion proteins during endocytic transport

Organelles of the endolysosomal system undergo multiple fission and fusion events to combine sorting of selected proteins to the vacuole with endosomal recycling. This sorting requires a consecutive remodeling of the organelle surface in the course of endosomal maturation. Here we dissect the remodeling and fusion machinery on endosomes during the process of endocytosis. We traced selected GFP-tagged endosomal proteins relative to exogenously added fluorescently labeled α-factor on its way from the plasma membrane to the vacuole. Our data reveal that the machinery of endosomal fusion and ESCRT proteins has similar temporal localization on endosomes, whereas they precede the retromer cargo recognition complex. Neither deletion of retromer nor the fusion machinery with the vacuole affects this maturation process, although the kinetics seems to be delayed due to ESCRT deletion. Of importance, in strains lacking the active Rab7-like Ypt7 or the vacuolar SNARE fusion machinery, α-factor still proceeds to late endosomes with the same kinetics. This indicates that endosomal maturation is mainly controlled by the early endosomal fusion and remodeling machinery but not the downstream Rab Ypt7 or the SNARE machinery. Our data thus provide important further understanding of endosomal biogenesis in the context of cargo sorting.