Insulin stimulates membrane fusion and GLUT4 accumulation in clathrin coats on adipocyte plasma membranes

Total internal reflection fluorescence (TIRF) microscopy reveals highly mobile structures containing enhanced green fluorescent protein-tagged glucose transporter 4 (GLUT4) within a zone about 100 nm beneath the plasma membrane of 3T3-L1 adipocytes. We developed a computer program (Fusion Assistant) that enables direct analysis of the docking/fusion kinetics of hundreds of exocytic fusion events. Insulin stimulation increases the fusion frequency of exocytic GLUT4 vesicles by approximately 4-fold, increasing GLUT4 content in the plasma membrane. Remarkably, insulin signaling modulates the kinetics of the fusion process, decreasing the vesicle tethering/docking duration prior to membrane fusion. In contrast, the kinetics of GLUT4 molecules spreading out in the plasma membrane from exocytic fusion sites is unchanged by insulin. As GLUT4 accumulates in the plasma membrane, it is also immobilized in punctate structures on the cell surface. A previous report suggested these structures are exocytic fusion sites (Lizunov et al., J. Cell Biol. 169:481-489, 2005). However, two-color TIRF microscopy using fluorescent proteins fused to clathrin light chain or GLUT4 reveals these structures are clathrin-coated patches. Taken together, these data show that insulin signaling accelerates the transition from docking of GLUT4-containing vesicles to their fusion with the plasma membrane and promotes GLUT4 accumulation in clathrin-based endocytic structures on the plasma membrane.     

Structural Mechanisms for Regulation of Membrane Traffic by Rab GTPases

In all eukaryotic organisms, Rab GTPases function as critical regulators of membrane traffic, organelle biogenesis and maturation, and related cellular processes. The numerous Rab proteins have distinctive yet overlapping subcellular distributions throughout the endomembrane system. Intensive investigation has clarified the underlying molecular and structural mechanisms for several ubiquitous Rab proteins that control membrane traffic between tubular-vesicular organelles in the exocytic, endocytic and recycling pathways. In this review, we focus on structural insights that inform our current understanding of the organization of the Rab family as well as the mechanisms for membrane targeting and activation, interaction with effectors, deactivation and specificity determination.     

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.     

Temporal and spatial coordination of exocytosis and endocytosis

In secretory cells, exocytosis and compensatory endocytosis are tightly coupled membrane trafficking processes that control the surface area and composition of the plasma membrane. While exocytic and endocytic processes have been studied independently in great detail, at present there is much interest in understanding the mode of their coupling. This review discusses emerging insights into the coupling of these processes, both in the chemical synapses of neurons and in non-neuronal cells.     

Roles of the TRAPP-II Complex and the Exocyst in Membrane Deposition during Fission Yeast Cytokinesis

The cleavage-furrow tip adjacent to the actomyosin contractile ring is believed to be the predominant site for plasma-membrane insertion through exocyst-tethered vesicles during cytokinesis. Here we found that most secretory vesicles are delivered by myosin-V on linear actin cables in fission yeast cytokinesis. Surprisingly, by tracking individual exocytic and endocytic events, we found that vesicles with new membrane are deposited to the cleavage furrow relatively evenly during contractile-ring constriction, but the rim of the cleavage furrow is the main site for endocytosis. Fusion of vesicles with the plasma membrane requires vesicle tethers. Our data suggest that the transport particle protein II (TRAPP-II) complex and Rab11 GTPase Ypt3 help to tether secretory vesicles or tubulovesicular structures along the cleavage furrow while the exocyst tethers vesicles at the rim of the division plane. We conclude that the exocyst and TRAPP-II complex have distinct localizations at the division site, but both are important for membrane expansion and exocytosis during cytokinesis.     

Membrane Diffusion Occurs by Continuous-Time Random Walk Sustained by Vesicular Trafficking

Diffusion in cellular membranes is regulated by processes that occur over a range of spatial and temporal scales. These processes include membrane fluidity, interprotein and interlipid interactions, interactions with membrane microdomains, interactions with the underlying cytoskeleton, and cellular processes that result in net membrane movement. The complex, non-Brownian diffusion that results from these processes has been difficult to characterize, and moreover, the impact of factors such as membrane recycling on membrane diffusion remains largely unexplored. We have used a careful statistical analysis of single-particle tracking data of the single-pass plasma membrane protein CD93 to show that the diffusion of this protein is well described by a continuous-time random walk in parallel with an aging process mediated by membrane corrals. The overall result is an evolution in the diffusion of CD93: proteins initially diffuse freely on the cell surface but over time become increasingly trapped within diffusion-limiting membrane corrals. Stable populations of freely diffusing and corralled CD93 are maintained by an endocytic/exocytic process in which corralled CD93 is selectively endocytosed, whereas freely diffusing CD93 is replenished by exocytosis of newly synthesized and recycled CD93. This trafficking not only maintained CD93 diffusivity but also maintained the heterogeneous distribution of CD93 in the plasma membrane. These results provide insight into the nature of the biological and biophysical processes that can lead to significantly non-Brownian diffusion of membrane proteins and demonstrate that ongoing membrane recycling is critical to maintaining steady-state diffusion and distribution of proteins in the plasma membrane.     

A real-time view of life within 100 nm of the plasma membrane

The plasma membrane is a two-dimensional compartment that relays most biological signals sent or received by a cell. Signalling involves membrane receptors and their associated enzyme cascades as well as organelles such as exocytic and endocytic vesicles. Advances in light microscope design, new organelle-specific vital stains and fluorescent proteins have renewed the interest in evanescent field fluorescence microscopy, a method uniquely suited to image the plasma membrane with its associated organelles and macromolecules in living cells. The method shows even the smallest vesicles made by cells, and can image the dynamics of single protein molecules.     

The endocytic protein machinery as an actin-driven membrane-remodeling machine

In clathrin-mediated endocytosis, a principal membrane trafficking route of all eukaryotic cells, forces are applied to invaginate the plasma membrane and form endocytic vesicles. These forces are provided by specific endocytic proteins and the polymerizing actin cytoskeleton. One of the best-studied endocytic systems is endocytosis in yeast, known for its simplicity, experimental amenability, and overall similarity to human endocytosis. Importantly, the yeast endocytic protein machinery generates and transmits tremendous force to bend the plasma membrane, making this system beneficial for mechanistic studies of cellular force-driven membrane reshaping. This review summarizes important protein players, molecular functions, applied forces, and open questions and perspectives of this robust, actin-powered membrane-remodeling protein machine.     

Recycling of intact dense core vesicles in neurites of NGF-treated PC12 cells

Exocytic fusion in neuroendocrine cells does not always result in complete release of the peptide contents from dense core vesicles (DCVs). In this study, we use fluorescence imaging and immunoelectron microscopy to examine the retention, endocytosis and recycling of chromogranin B in DCVs of NGF-treated PC12 cells. Our results indicate that DCVs retained and retrieved an intact core that was available for subsequent exocytic release. The endocytic process was inhibited by cyclosporine A or by substitution of extracellular Ca(2+) with Ba(2+) and the total recycling time was less than 5 min.     

Lipid determinants of endocytosis and exocytosis in budding yeast

Cells maintain physicochemical characteristics of membranes in order to allow for proper function of membrane-associated cellular processes, such as endocytosis and exocytosis. To investigate the interplay between membrane properties and biological processes, we applied lipid engineering approaches that allowed for systematic manipulation of fatty acid unsaturation and sterol biosynthesis, the main regulators of membrane fluidity. In combination with electrophysiological membrane capacitance measurements, we were able to study the dependence of the endo- and exocytic activity of Saccharomyces cerevisiae on membrane lipid composition in vivo. We found that a strong decrease in the cell's total ergosterol content leads to a severely reduced frequency of vesicle fission (endocytosis), whereas the exocytic activity remained largely unaffected. In contrast, increased lipid saturation lowered both endocytic and the exocytic activity, with the former being more severely affected. We were able to correlate the decreased ratio of endocytic/exocytic frequencies (f_endo/f_exo) upon lipid perturbation with the growth of yeast protoplasts, which is based on a surface enlargement resulting from a net excess of exocytic over endocytic flux. Experiments using clathrin-deficient mutants confirm a correlation between reduced endocytic activity and increased size of intact walled cells, as well as accelerated protoplast growth. These data show that lipid composition is intimately tied to membrane trafficking in yeast cells and suggest that endocytosis is particularly dependent on the lipid-defined properties of cell membrane.     

Intracellular Salmonella enterica redirect exocytic transport processes in a Salmonella pathogenicity island 2-dependent manner

During intracellular life, Salmonella enterica proliferate within a specialized membrane compartment, the Salmonella-containing vacuole (SCV), and interfere with the microtubule cytoskeleton and cellular transport. To characterize the interaction of intracellular Salmonella with host cell transport processes, we utilized various model systems to follow microtubule-dependent transport. The vesicular stomatitis virus glycoprotein (VSVG) is a commonly used marker to follow protein transport from the Golgi to the plasma membrane. Using a VSVG-GFP fusion protein, we observed that virulent intracellular Salmonella alter exocytotic transport and recruit exocytotic transport vesicles to the SCV. This virulence function was dependent on the function of the type III secretion system encoded by Salmonella Pathogenicity Island 2 (SPI2) and more specifically on a subset of SPI2 effector proteins. Furthermore, the Golgi to plasma membrane traffic of the shingolipid C(5)-ceramide was redirected to the SCV by virulent Salmonella. We propose that Salmonella modulates the biogenesis of the SCV by deviating this compartment from the default endocytic pathway to an organelle that interacts with the exocytic pathway. This observation might reveal a novel element of the intracellular survival and replication strategy of Salmonella.     

Independent secretion of proteoglycans and collagens in chick chondrocyte cultures during acute ascorbic acid treatment.

1. Liver plasma membranes originating from the sinusoidal, lateral and canalicular surface domains of hepatocytes were covalently labelled with sulpho-N-hydroxysuccinamide-biotin. After solubilization in Triton X-114, treatment with a phosphatidylinositol-specific phospholipase C (PI-PLC), two-phase partitioning and 125I-streptavidin labelling of the proteins resolved by PAGE, six major polypeptides (molecular masses 110, 85, 70, 55, 38 and 35 kDa) were shown to be anchored in bile canalicular membrane vesicles by a glycosyl-phosphatidylinositol (G-PI) 'tail'. 2. Permeabilized 'early' and 'late' endocytic vesicles isolated from liver were also examined. Two polypeptides (110 and 35 kDa) were shown to be anchored by a G-PI tail in 'late' endocytic vesicles. 3. Analysis of marker enzymes in bile-canalicular vesicles treated with PI-PLC showed that 5'-nucleotidase and alkaline phosphatase, but not leucine aminopeptidase and ecto-Ca2(+)-ATPase activities were released from the membrane. A low release and recovery of alkaline phosphodiesterase activity was noted. The cleavage from the membrane of 5'-nucleotidase as a 70 kDa polypeptide was confirmed by Western blotting using an antibody to this enzyme. 4. Antibodies raised to proteins released from bile-canalicular vesicles by PI-PLC treatment, and purified by partitioning in aqueous and Triton X-114 phases, localized to the bile canaliculi in thin liver sections. Antibodies to proteins not hydrolysed by this treatment stained by immunofluorescence the sinusoidal and canalicular surface regions of hepatocytes. 5. Antibodies generated to proteins cleaved by PI-PLC treatment of canalicular vesicles were shown to identify, by Western blotting, a major 110 kDa polypeptide in these vesicles. Two polypeptides (55 and 38 kDa) were detected in MDCK and HepG-2 cultured cells. 6. Since two of the six G-PI-anchored proteins targeted to the bile-canalicular plasma membrane were also detected in 'late' endocytic vesicles, the results suggest that a junction where exocytic and endocytic traffic routes meet occurs in a 'late' endocytic compartment.     

A quantitative imaging-based screen reveals the exocyst as a network hub connecting endocytosis and exocytosis

The coupling of endocytosis and exocytosis underlies fundamental biological processes ranging from fertilization to neuronal activity and cellular polarity. However, the mechanisms governing the spatial organization of endocytosis and exocytosis require clarification. Using a quantitative imaging-based screen in budding yeast, we identified 89 mutants displaying defects in the localization of either one or both pathways. High-resolution single-vesicle tracking revealed that the endocytic and exocytic mutants she4∆ and bud6∆ alter post-Golgi vesicle dynamics in opposite ways. The endocytic and exocytic pathways display strong interdependence during polarity establishment while being more independent during polarity maintenance. Systems analysis identified the exocyst complex as a key network hub, rich in genetic interactions with endocytic and exocytic components. Exocyst mutants displayed altered endocytic and post-Golgi vesicle dynamics and interspersed endocytic and exocytic domains compared with control cells. These data are consistent with an important role for the exocyst in coordinating endocytosis and exocytosis.     

Clathrin: Its Role in Receptor-Mediated Vesicular Transport and Specialized Functions in Neurons

Abstract

Clathrin constitutes the coat of vesicles involved in three receptor-mediated intracellular transport pathways; the export of aggregated material from the trans-Golgi network for regulated secretion, the transfer of lysosomal hydrolases from the trans-Golgi network to lysosomes and receptor-mediated endocytosis at the plasma membrane. The clathrin subunits and the other major coat constituents, the adaptor polypeptides, interact in specific ways to build the characteristic polygonal clathrin lattice and to attach the coat to integral membrane receptors. Both clathrin coat assembly and disassembly on the cytoplasmic side of the membrane are multistep processes that are regulated by the coat constituents themselves and by cytosolic proteins and factors. Neurons represent a cell type with distinct morphology and special demands on exocytic and endocytic pathways that requires neuron-specific constituents and modifications of clathrin-coated vesicles.     

Resident CAPS on dense-core vesicles docks and primes vesicles for fusion

The Ca²⁺-dependent exocytosis of dense-core vesicles in neuroendocrine cells requires a priming step during which SNARE protein complexes assemble. CAPS (aka CADPS) is one of several factors required for vesicle priming; however, the localization and dynamics of CAPS at sites of exocytosis in live neuroendocrine cells has not been determined. We imaged CAPS before, during, and after single-vesicle fusion events in PC12 cells by TIRF micro­scopy. In addition to being a resident on cytoplasmic dense-core vesicles, CAPS was present in clusters of approximately nine molecules near the plasma membrane that corresponded to docked/tethered vesicles. CAPS accompanied vesicles to the plasma membrane and was present at all vesicle exocytic events. The knockdown of CAPS by shRNA eliminated the VAMP-2–dependent docking and evoked exocytosis of fusion-competent vesicles. A CAPS(ΔC135) protein that does not localize to vesicles failed to rescue vesicle docking and evoked exocytosis in CAPS-depleted cells, showing that CAPS residence on vesicles is essential. Our results indicate that dense-core vesicles carry CAPS to sites of exocytosis, where CAPS promotes vesicle docking and fusion competence, probably by initiating SNARE complex assembly.     

Detecting protein-phospholipid interactions. Epidermal growth factor-induced activation of phospholipase D1b in situ

Phospholipase D (PLD) proteins have been identified in secretory and endocytic vesicles, consistent with their proposed role in regulating membrane traffic. However, their sites of catalytic action remain obscure. We have developed here a novel, analytical approach to monitor PLD activation in intact cells employing lifetime imaging microscopy to measure fluorescence resonance energy transfer between protein and membrane phospholipid. Verification and application of this technique demonstrates a dispersed endosomal, epidermal growth factor-induced activation of the PLD1b isoform. Application of this approach will facilitate the spatial resolution of many protein-phospholipid interactions that are key events in the regulation of cellular processes.     

Membrane traffic and synaptic cross-talk during host cell entry byTrypanosoma cruzi

It is widely accepted that Trypanosoma cruzi can exploit the natural exocytic response of the host to cell damage, utilizing host cell lysosomes as important effectors. It is, though, increasingly clear that the parasite also exploits endocytic mechanisms which allow for incorporation of plasma membrane into the parasitophorous vacuole. Further, that these endocytic mechanisms are involved in cross‐talk with the exocytic machinery, in the recycling of vesicles and in the manipulation of the cytoskeleton. Here we review the mechanisms by which T. cruzi exploits features of the exocytic and endocytic pathways in epithelial and endothelial cells and the evidence for cross‐talk between these pathways.     

Coupling actin dynamics and membrane dynamics during endocytosis.

A convergence of cellular, genetic and biochemical studies supports the hypothesis that the actin cytoskeleton is coupled to endocytic processes, but the roles played by actin filaments during endocytosis are not yet clear. Recent studies have identified several proteins that may functionally link the endocytic machinery with actin filament dynamics. Three of these proteins, Abp1p, Pan1p and cortactin, are activators of actin assembly nucleated by the Arp2/3 complex, a key regulator of actin assembly in vivo. Two others, intersectin and syndapin, bind N-WASp, a potent activator of actin assembly via the Arp2/3 complex. All of these proteins also bind components of the endocytic machinery, and thus, could coordinately regulate actin assembly and trafficking events. Hip1R, an F-actin-binding protein that associates with clathrin-coated vesicles, may physically link endocytic vesicles to actin filaments. The GTPase dynamin is implicated in modulating actin filaments at specialized actin-rich structures of the cell cortex, suggesting that dynamin may regulate the organization of cortical actin filaments as well as regulate actin dynamics during endocytosis. Finally, myosin VI may generate actin-dependent forces for membrane invagination or vesicle movement during the early stages of endocytosis.     

Synaptophysin is targeted to similar microvesicles in CHO and PC12 cells.

Synaptophysin, an integral membrane protein of small synaptic vesicles, was expressed by transfection in fibroblastic CHO-K1 cells. The properties and localization of synaptophysin were compared between transfected CHO-K1 cells and native neuroendocrine PC12 cells. Both cell types similarly glycosylate synaptophysin and sort it into indistinguishable microvesicles. These become labeled by endocytic markers and are primarily concentrated below the plasmalemma and at the area of the Golgi complex and the centrosomes. A small pool of synaptophysin is transiently found on the plasma membrane. In CHO-K1 cells synaptophysin co-localizes with transferrin that has been internalized by receptor-mediated endocytosis. These findings suggest that synaptophysin in transfected CHO-K1 cells and neuroendocrine PC12 cells is directed into a pathway of recycling microvesicles which, in CHO cells, is shown to coincide with that of the transferrin receptor. They further indicate that fibroblasts have the ability to sort a synaptic vesicle membrane protein. Our results suggest a pathway for the evolution of small synaptic vesicles from a constitutively recycling organelle which is normally present in all cells.