Exocytosis of catecholamine (CA)-containing and CA-free granules in chromaffin cells

Recent evidence suggests that endocytosis in neuroendocrine cells and neurons can be tightly coupled to exocytosis, allowing rapid retrieval from the plasma membrane of fused vesicles for future use. This can be a much faster mechanism for membrane recycling than classical clathrin-mediated endocytosis. During a fast exo-endocytotic cycle, the vesicle membrane does not fully collapse into the plasma membrane; nevertheless, it releases the vesicular contents through the fusion pore. Once the vesicle is depleted of transmitter, its membrane is recovered without renouncing its identity. In this report, we show that chromaffin cells contain catecholamine-free granules that retain their ability to fuse with the plasma membrane. These catecholamine-free granules represent 7% of the total population of fused vesicles, but they contributed to 47% of the fusion events when the cells were treated with reserpine for several hours. We propose that rat chromaffin granules that transiently fuse with the plasma membrane preserve their exocytotic machinery, allowing another round of exocytosis.     

ATG16L1 meets ATG9 in recycling endosomes

Autophagosomes are formed by double-membraned structures, which engulf portions of cytoplasm. Autophagosomes ultimately fuse with lysosomes, where their contents are degraded. The origin of the autophagosome membrane may involve different sources, such as mitochondria, Golgi, endoplasmic reticulum, plasma membrane, and recycling endosomes. We recently observed that ATG9 localizes on the plasma membrane in clathrin-coated structures and is internalized following a classical endocytic pathway through early and then recycling endosomes. By contrast, ATG16L1 is also internalized by clathrin-mediated endocytosis but via different clathrin-coated pits, and appears to follow a different route to the recycling endosomes. The R-SNARE VAMP3 mediates the coalescence of the 2 different pools of vesicles (containing ATG16L1 or ATG9) in recycling endosomes. The heterotypic fusion between ATG16L1- and ATG9-containing vesicles strongly correlates with subsequent autophagosome formation. Thus, ATG9 and ATG16L1 both traffic from the plasma membrane to autophagic precursor structures and provide 2 routes from the plasma membrane to autophagosomes.     

Commentary: ATG16L1 meets ATG9 in recycling endosomes: Additional roles for the plasma membrane and endocytosis in autophagosome biogenesis

Autophagosomes are formed by double-membraned structures, which engulf portions of cytoplasm. Autophagosomes ultimately fuse with lysosomes, where their contents are degraded. The origin of the autophagosome membrane may involve different sources, such as mitochondria, Golgi, endoplasmic reticulum, plasma membrane, and recycling endosomes. We recently observed that ATG9 localizes on the plasma membrane in clathrin-coated structures and is internalized following a classical endocytic pathway through early and then recycling endosomes. By contrast, ATG16L1 is also internalized by clathrin-mediated endocytosis but via different clathrin-coated pits, and appears to follow a different route to the recycling endosomes. The R-SNARE VAMP3 mediates the coalescence of the 2 different pools of vesicles (containing ATG16L1 or ATG9) in recycling endosomes. The heterotypic fusion between ATG16L1- and ATG9-containing vesicles strongly correlates with subsequent autophagosome formation. Thus, ATG9 and ATG16L1 both traffic from the plasma membrane to autophagic precursor structures and provide 2 routes from the plasma membrane to autophagosomes.     

From sorting endosomes to exocytosis: association of Rab4 and Rab11 GTPases with the Fc receptor, FcRn, during recycling

A longstanding question in cell biology is how is the routing of intracellular organelles within cells regulated? Although data support the involvement of Rab4 and Rab11 GTPases in the recycling pathway, the function of Rab11 in particular is uncertain. Here we have analyzed the association of these two Rab GTPases with the Fc receptor, FcRn, during intracellular trafficking. This Fc receptor is both functionally and structurally distinct from the classical Fcgamma receptors and transports immunoglobulin G (IgG) within cells. FcRn is therefore a recycling receptor that sorts bound IgG from unbound IgG in sorting endosomes. In the current study we have used dual color total internal reflection fluorescence microscopy (TIRFM) and wide-field imaging of live cells to analyze the events in human endothelial cells that are involved in the trafficking of FcRn positive (FcRn(+)) recycling compartments from sorting endosomes to exocytic sites at the plasma membrane. Our data are consistent with the following model for this pathway: FcRn leaves sorting endosomes in Rab4(+)Rab11(+) or Rab11(+) compartments. For Rab4(+)Rab11(+) compartments, Rab4 depletion occurs by segregation of the two Rab proteins into discrete domains that can separate. The Rab11(+)FcRn(+) vesicle or tubule subsequently fuses with the plasma membrane in an exocytic event. In contrast to Rab11, Rab4 is not involved in exocytosis.     

Coupling exo- and endocytosis: An essential role for PIP2 at the synapse

Chemical synapses are specialist points of contact between two neurons, where information transfer takes place. Communication occurs through the release of neurotransmitter substances from small synaptic vesicles in the presynaptic terminal, which fuse with the presynaptic plasma membrane in response to neuronal stimulation. However, as neurons in the central nervous system typically only possess ~ 200 vesicles, high levels of release would quickly lead to a depletion in the number of vesicles, as well as leading to an increase in the area of the presynaptic plasma membrane (and possible misalignment with postsynaptic structures). Hence, synaptic vesicle fusion is tightly coupled to a local recycling of synaptic vesicles. For a long time, however, the exact molecular mechanisms coupling fusion and subsequent recycling remained unclear. Recent work now indicates a unique role for the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂), acting together with the vesicular protein synaptotagmin, in coupling these two processes. In this work, we review the evidence for such a mechanism and discuss both the possible advantages and disadvantages for vesicle recycling (and hence signal transduction) in the nervous system. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

Coupling exo- and endocytosis: an essential role for PIP₂ at the synapse

Chemical synapses are specialist points of contact between two neurons, where information transfer takes place. Communication occurs through the release of neurotransmitter substances from small synaptic vesicles in the presynaptic terminal, which fuse with the presynaptic plasma membrane in response to neuronal stimulation. However, as neurons in the central nervous system typically only possess ~200 vesicles, high levels of release would quickly lead to a depletion in the number of vesicles, as well as leading to an increase in the area of the presynaptic plasma membrane (and possible misalignment with postsynaptic structures). Hence, synaptic vesicle fusion is tightly coupled to a local recycling of synaptic vesicles. For a long time, however, the exact molecular mechanisms coupling fusion and subsequent recycling remained unclear. Recent work now indicates a unique role for the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)), acting together with the vesicular protein synaptotagmin, in coupling these two processes. In this work, we review the evidence for such a mechanism and discuss both the possible advantages and disadvantages for vesicle recycling (and hence signal transduction) in the nervous system. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

Compound exocytosis: mechanisms and functional significance

Compound exocytosis occurs in many cell types. It represents a specialized form of secretion in which vesicles undergo fusion with each other as well as with the plasma membrane. In most cases, compound exocytosis occurs sequentially, with deeper-lying vesicles fusing, after a delay, with vesicles that have already fused with the plasma membrane. However, in some cells, vesicles can also apparently fuse with each other intracellularly before any interaction with the plasma membrane. In this review, we discuss the general features of compound exocytosis, and the features that are specific to particular cells. We consider mechanisms that might impose the requirement for vesicles to fuse with the plasma membrane before they become able to fuse with each other, the possibility that there are biochemical differences between vesicle-plasma membrane fusion events and subsequent secondary homotypic vesicle fusion events, and the role that cytoskeletal elements might play in the stabilization of fused vesicles, in order to permit secondary fusion events. Finally, we discuss the likely physiological significance of compound exocytosis in the various cell types in which it exists.     

Translocation of small preformed vesicles is responsible for the insulin activation of glucose transport in adipose cells. Evidence from the in vitro reconstitution assay

Insulin stimulates translocation of the glucose transporter isoform 4 (Glut4) from an intracellular storage compartment to the plasma membrane in fat and skeletal muscle cells. At present, the nature of the Glut4 storage compartment is unclear. According to one model, this compartment represents a population of preformed small vesicles that fuse with the plasma membrane in response to insulin stimulation. Alternatively, Glut4 may be retained in large donor membranes, and insulin stimulates the formation of transport vesicles that deliver Glut4 to the cell surface. Finally, insulin can induce plasma membrane fusion of the preformed vesicles and, also, stimulate the formation of new vesicles. In extracts of fat and skeletal muscle cells, Glut4 is predominantly found in small insulin-sensitive 60-70 S membrane vesicles that may or may not artificially derive from large donor membranes during cell homogenization. Here, we use a cell-free reconstitution assay to demonstrate that small Glut4-containing vesicles are formed from large rapidly sedimenting donor membranes in a cytosol-, ATP-, time-, and temperature-dependent fashion and, therefore, do not represent an artifact of homogenization. Thus, small insulin-responsive vesicles represent the major form of Glut4 storage in the living adipose cell. Fusion of these vesicles with the plasma membrane may be largely responsible for the primary effect of insulin on glucose transport in fat tissue. In addition, our results suggest that insulin may also stimulate the formation of Glut4 vesicles and accelerate Glut4 recycling to the plasma membrane.     

Analyses of the Recycling Receptor, FcRn, in Live Cells Reveal Novel Pathways for Lysosomal Delivery

Lysosomes play a central role in the degradation of proteins and other macromolecules. The mechanisms by which receptors are transferred to lysosomes for constitutive degradation are poorly understood. We have analyzed the processes that lead to the lysosomal delivery of the Fc receptor, FcRn. These studies provide support for a novel pathway for receptor delivery. Specifically, unlike other receptors that enter intraluminal vesicles in late endosomes, FcRn is transferred from the limiting membrane of such endosomes to lysosomes, and is rapidly internalized into the lysosomal lumen. By contrast, LAMP-1 persists on the limiting membrane. Receptor transfer is mediated by tubular extensions from late endosomes to lysosomes, or by interactions of the two participating organelles in kiss-and-linger-like processes, whereas full fusion is rarely observed. The persistence of FcRn on the late endosomal limiting membrane, together with selective transfer to lysosomes, allows this receptor to undergo recycling or degradation. Consequently, late endosomes have functional plasticity, consistent with the presence of the Rab5 GTPase in discrete domains on these compartments.     

The recycling and transcytotic pathways for IgG transport by FcRn are distinct and display an inherent polarity

The Fc receptor FcRn traffics immunoglobulin G (IgG) in both directions across polarized epithelial cells that line mucosal surfaces, contributing to host defense. We show that FcRn traffics IgG from either apical or basolateral membranes into the recycling endosome (RE), after which the actin motor myosin Vb and the GTPase Rab25 regulate a sorting step that specifies transcytosis without affecting recycling. Another regulatory component of the RE, Rab11a, is dispensable for transcytosis, but regulates recycling to the basolateral membrane only. None of these proteins affect FcRn trafficking away from lysosomes. Thus, FcRn transcytotic and recycling sorting steps are distinct. These results are consistent with a single structurally and functionally heterogeneous RE compartment that traffics FcRn to both cell surfaces while discriminating between recycling and transcytosis pathways polarized in their direction of transport.     

Synaptophysin and synapsin I as tools for the study of the exo-endocytotic cycle

Synaptophysin, an integral protein of the synaptic vesicle membrane, and synapsin I, a phosphoprotein associated with the cytoplasmic side of synaptic vesicles, represent useful markers that allow to follow the movements of the vesicle membrane during recycling. The use of antibodies against these proteins to label nerve terminals during experimental treatments which stimulate secretion has provided evidence that during the exo-endocytotic cycle synaptic vesicles transiently fuse with the axolemma, from which they are specifically recovered. When recycling is blocked, exocytosis leads to the permanent incorporation of the synaptic vesicle membrane into the axolemma and to diffusion of the vesicle components in the plane of the membrane.     

Comparison of the intracellular pathways of transferrin recycling and vesicular stomatitis virus membrane glycoprotein exocytosis by ultrastructural double-label cytochemistry

Transferrin is taken up by receptor-mediated endocytosis into intracellular vesicles and tubules, and then recycles rapidly to the plasma membrane (diacytosis). We applied double-label cytochemistry to study whether the recycling structures containing transferrin fuse with the intracellular membranous structures that deliver newly synthesized membrane glycoproteins from the ER to the plasma membrane (exocytosis) or whether they remain independent. KB and Vero cells were infected with the temperature-sensitive transport mutant 0-45 of vesicular stomatitis virus (VSV). Temperature-regulated exocytosis of membrane glycoprotein "G" occurred simultaneously with diacytosis of transferrin. The exocytic "G" protein, as detected by immunoperoxidase electron microscopy, passed through the cisternal Golgi stacks and vacuolar, tubular, vesicular, and pit-like structures of the Golgi system. A transferrin-ferritin conjugate used in ultrastructural double-label experiments was detected in diacytic vesicles and tubules that accumulated in the proximal (trans-reticular) Golgi area of the cell. The ferritin-labeled vesicles/tubules were often close to and intermixed with the VSV-"G" containing membranous structures, but in most cases at early times (15-20 min) the transferrin and VSV-"G" containing vesicular structures remained distinct. At later times (30-45 min), the two labels were occasionally found in the same structures. These results indicate that rapid recycling of endocytosed materials and exocytosis of membrane glycoproteins to the cell surface usually occur in distinct vesicles, possibly along the same general morphologic exit pathway.     

Rab22a regulates the recycling of membrane proteins internalized independently of clathrin

Plasma membrane proteins that are internalized independently of clathrin, such as major histocompatibility complex class I (MHCI), are internalized in vesicles that fuse with the early endosomes containing clathrin-derived cargo. From there, MHCI is either transported to the late endosome for degradation or is recycled back to the plasma membrane via tubular structures that lack clathrin-dependent recycling cargo, e.g., transferrin. Here, we show that the small GTPase Rab22a is associated with these tubular recycling intermediates containing MHCI. Expression of a dominant negative mutant of Rab22a or small interfering RNA-mediated depletion of Rab22a inhibited both formation of the recycling tubules and MHCI recycling. By contrast, cells expressing the constitutively active mutant of Rab22a exhibited prominent recycling tubules and accumulated vesicles at the periphery, but MHCI recycling was still blocked. These results suggest that Rab22a activation is required for tubule formation and Rab22a inactivation for final fusion of recycling membranes with the surface. The trafficking of transferrin was only modestly affected by these treatments. Dominant negative mutant of Rab11a also inhibited recycling of MHCI but not the formation of recycling tubules, suggesting that Rab22a and Rab11a might coordinate different steps of MHCI recycling.     

Endocytosis of VAMP is facilitated by a synaptic vesicle targeting signal

After synaptic vesicles fuse with the plasma membrane and release their contents, vesicle membrane proteins recycle by endocytosis and are targeted to newly formed synaptic vesicles. The membrane traffic of an epitope-tagged form of VAMP-2 (VAMP-TAg) was observed in transfected cells to identify sequence requirements for recycling of a synaptic vesicle membrane protein. In the neuroendocrine PC12 cell line VAMP-TAg is found not only in synaptic vesicles, but also in endosomes and on the plasma membrane. Endocytosis of VAMP-TAg is a rapid and saturable process. At high expression levels VAMP-TAg accumulates at the cell surface. Rapid endocytosis of VAMP-TAg also occurs in transfected CHO cells and is therefore independent of other synaptic proteins. The majority of the measured endocytosis is not directly into synaptic vesicles since mutations in VAMP-TAg that enhance synaptic vesicle targeting did not affect endocytosis. Nonetheless, mutations that inhibited synaptic vesicle targeting, in particular replacement of methionine-46 by alanine, inhibited endocytosis by 85% in PC12 cells and by 35% in CHO cells. These results demonstrate that the synaptic vesicle targeting signal is also used for endocytosis and can be recognized in cells lacking synaptic vesicles.     

An emerging view of presynaptic structure from electron microscopic studies

In response to calcium influx, some of the synaptic vesicles in presynaptic terminals fuse rapidly with the presynaptic membrane, allowing fast synaptic transmission. The regulated recycling of synaptic vesicles at the terminals is required for a sustained release of neurotransmitters. Localization of 'ready to be released' vesicles in close vicinities to voltage-gated calcium channels enables the rapid release of neurotransmitters. Thus, recycling vesicles must translocate from the sites of endocytosis to these release sites. However, the sub-cellular organization that supports this local vesicular traffic remains poorly understood. We will review the results of various electron microscopy studies, which have begun to unveil the structure of presynaptic terminals.     

Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes

Classically, the polymeric immunoglobulin receptor and its ligand, IgA, are thought to be sorted from basolateral early endosomes into transcytotic vesicles that directly fuse with the apical plasma membrane. In contrast, we have found that in MDCK cells IgA is delivered from basolateral endosomes to apical endosomes and only then to the apical cell surface. When internalized from the basolateral surface of MDCK cells IgA is found to accumulate under the apical plasma membrane in a compartment that is accessible to two apically added membrane markers: anti-secretory component Fab fragments, and avidin internalized from the biotinylated apical pole of the cell. This accumulation occurs in the presence of apical trypsin, which prevents internalization of the ligand from the apical cell surface. Using a modification of the diaminobenzidine density-shift assay, we estimate that approximately 80% of basolaterally internalized IgA resides in the apical endosomal compartment. In addition, approximately 50% of basolaterally internalized transferrin, a basolateral recycling protein, has access to this apical endosomal compartment and is efficiently recycled back to the basolateral surface. Microtubules are required for the organization of the apical endosomal compartment and it is dispersed in nocodazole-treated cells. Moreover, this compartment is largely inaccessible to fluid-phase markers added to either pole of the cell, and therefore seems analogous to the recycling endosome described in nonpolarized cells. We propose a model in which transcytosis is not a specialized pathway that uses unique transcytotic vesicles, but rather combines portions of pathways used by non- transcytosing molecules.     

Digestion in the peritrich ciliateOphrydium versatile

Summary Digestion in the peritrich ciliateOphrydium versatile O.F.M. involves a complex sequence of intracytotic and exocytotic membrane fusion and recycling events. Food particulates are concentrated in the lower cytopharynx which forms a fusiform-shaped food vacuole. Upon release from the cytopharynx, this food vacuole begins to condense, concentrating the food particulates. Excess membrane is removed intracytotically. These released membranes pieces form discoidal vesicles which are recycled to the base of the cytopharynx, thus providing additional membrane for subsequent food vacuole formation. In the condensed food vacuole, digestion proceeds; hydrolytic enzymes are delivered to the food vacuole via rough endoplasmic reticulum and/or by the cup-shaped coated vesicles (CSCV). As these vesicles fuse with the food vacuole, the food vacuole enlarges, digestion proceeds and an electron-dense membrane coat appears along the luminal surface of the food vacuole. Prior to defecation, the food vacuole undergoes a final condensation; irregularly-shaped, electron dense, single-membrane bound vesicles are cut-off intracytotically from the old food vacuole. These vesicles undergo condensation and invagination to form the cup-shaped coated vesicles (CSCV) which fuse with younger food vacuoles.     

Role of microtubules in fusion of post-Golgi vesicles to the plasma membrane

Biosynthetic cargo is transported away from the Golgi in vesicles via microtubules. In the cell periphery the vesicles are believed to engage actin and then dock to fusion sites at the plasma membrane. Using dual-color total internal reflection fluorescence microscopy, we observed that microtubules extended within 100 nm of the plasma membrane and post-Golgi vesicles remained on microtubules up to the plasma membrane, even as fusion to the plasma membrane initiated. Disruption of microtubules eliminated the tubular shapes of the vesicles and altered the fusion events: vesicles required multiple fusions to deliver all of their membrane cargo to the plasma membrane. In contrast, the effects of disrupting actin on fusion behavior were subtle. We conclude that microtubules, rather than actin filaments, are the cytoskeletal elements on which post-Golgi vesicles are transported until they fuse to the plasma membrane.     

Albumin turnover: FcRn-mediated recycling saves as much albumin from degradation as the liver produces

It is now understood that the nonclassical major histocompatibility complex-I molecule FcRn binds albumin and retrieves it from an intracellular degradative fate. Whether FcRn in the liver modulates albumin turnover through effects on biosynthesis and production is not known. Thus we quantified the appearance of biosynthetically labeled albumin in plasma after an intravenous bolus injection of [(3)H]leucine in FcRn-deficient mice. The production rates for both albumin (FcRn substrate) and transferrin (nonsubstrate) are increased by approximately 20% in FcRn-deficient mice compared with normal mice, likely compensating for the lowered plasma oncotic pressure caused by hypoalbuminemia in FcRn-deficient mice. Determining the magnitude of FcRn-mediated effects on albumin turnover, we then measured the steady-state plasma concentrations of biosynthetically labeled albumin and transferrin during [(3)H]leucine infusion. The concentration of albumin was approximately 40% lower in FcRn-deficient mice compared with normal mice. Furthermore, the approximately 40% lower plasma albumin concentration in FcRn-deficient mice along with the approximately 20% increase in albumin production indicate, by the mass-balance equation, that albumin degradation in FcRn-deficient mice is twice that of normal mice. These studies of biosynthetically labeled, and thus native, albumin support our previous finding that FcRn protects albumin from degradation. Permitting quantification of the magnitude of FcRn-mediated recycling, they further indicate that FcRn has extraordinary capacity: the amount of albumin saved from degradation by FcRn-mediated recycling is the same as that produced by the liver.     

Exocytosed protons feedback to suppress the Ca2+ current in mammalian cone photoreceptors.

A proton pump acidifies synaptic vesicles and provides the electrochemical gradient for transmitter uptake. Although external protons can modulate membrane voltage- and ligand-gated conductances, the fate of the protons released when vesicles fuse with the plasma membrane is unclear. In the dark, the glutamate-laden vesicles of cone photoreceptors fuse continuously with the plasma membrane. I now show that vesicular protons feed back to block the nearby calcium channels that mediate release. This local proton-mediated feedback is a novel mechanism through which neurons may regulate the release of transmitter.