Phospholipase D in endocytosis and endosomal recycling pathways

The discovery that Arf GTPases, mediators of membrane traffic, activate phospholipase D (PLD) raised the possibility that Arfs could facilitate membrane traffic by altering membrane lipid composition. PLD hydrolyzes phosphatidylcholine to generate phosphatidic acid (PA), a lipid that favors membranes with negative curvature and thus can facilitate both membrane fission and fusion. This review examines studies that have reported a role for PLD in endocytosis and membrane recycling from endocytic pathways.     

Powering membrane traffic in endocytosis and recycling

Early in evolution, the diversification of membrane-bound compartments that characterize eukaryotic cells was accompanied by the elaboration of molecular machineries that mediate intercompartmental communication and deliver materials to specific destinations. Molecular motors that move on tracks of actin filaments or microtubules mediate the movement of organelles and transport between compartments. The subjects of this review are the motors that power the transport steps along the endocytic and recycling pathways, their modes of attachment to cargo and their regulation.     

The synaptic vesicle membrane: origin, axonal distribution, protein components, exocytosis and recycling

The paper discusses functional and molecular aspects of the synaptic vesicle membrane during its life cycle. The distribution of the synaptic vesicle membrane compartment in an entire cholinergic neuron is monitored using colloidal gold labelling and a monoclonal antibody against the synaptic vesicle membrane protein SV2. This provides new insights concerning vesicle origin and fate in the various compartments of the neuron. A new synaptic vesicle membrane protein (svp25) of Mr 25,000 with properties similar to synaptophysin as well as a synaptic vesicle binding phosphoprotein of the presynaptic membrane (Mr 92,000) likely to be involved in vesicle exocytosis are described. The membrane compartment recycled on induced transmitter release contains synaptic vesicle but not plasma membrane markers and encloses both newly synthesized transmitter and a sample of extracellular medium.     

Synaptosomes secrete and uptake functionally active microRNAs via exocytosis and endocytosis pathways

In this study, we first characterized synaptosome microRNA (miRNA) profiles using microarray and qRT‐PCR. MicroRNAs were detected in isolated synaptic vesicles, and Ago2 immunoprecipitation studies revealed an association between miRNAs and Ago2. Second, we found that miR‐29a, miR‐99a, and miR‐125a were significantly elevated in synaptosome supernatants after depolarization. MiRNA secretion by the synaptosome was Ca²⁺‐dependent and was inhibited by the exocytosis inhibitor, okadaic acid. Furthermore, application of nerve growth factor increased miRNA secretion without altering the spontaneous release of miRNAs. Conversely, kainic acid decreased miRNA secretion and enhanced the spontaneous release of miRNAs. These results indicate that synaptosomes could secrete miRNAs. Finally, synthesized miRNAs were taken up by synaptosomes, and the endocytosis inhibitor Dynasore blocked this process. After incubation with miR‐125a, additional miR‐125a was bound to Ago2 in the synaptosome, and expression of the miR‐125a target gene (PSD95 mRNA) was decreased; these findings suggest that the ingested miRNAs were assembled in the RNA‐induced silencing complex, resulting in the degradation of target mRNAs. To our knowledge, this is the first study that demonstrates the secretion of miRNAs by synaptosomes under physiological stimulation and demonstrates that secreted miRNAs might be functionally active after being taken up by the synaptic fraction via the endocytic pathway.     

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.     

Enlargeosome traffic: exocytosis triggered by various signals is followed by endocytosis, membrane shedding or both

Enlargeosomes are cytoplasmic organelles discharged by regulated exocytosis, identified by immunofluorescence of their membrane marker, desmoyokin/Ahnak, but never revealed at the ultrastructural level. Among the numerous enlargeosome-positive cells, the richest and most extensively characterized are those of a PC12 clone, PC12-27, defective of classical neurosecretion. By using ultrastructural immunoperoxidase labeling of formaldehyde-fixed, Triton-X-100-permeabilized PC12-27 cells, we have now identified the enlargeosomes as small vesicles scattered in the proximity of, but never docked to, the plasma membrane. Upon stimulation, these vesicles undergo exocytosis [rapid after the Ca(2+) ionophore, ionomycin, much slower after either the phorbol ester, phorbol myristate acetate (PMA), or ATP, working through a P2Y receptor], with appearance in the plasma membrane of typical desmoyokin/Ahnak (d/A)-positive, Omega-shaped and open profiles evolving into flat patches. Postexocytic removal of the exocytized d/A-positive membrane occurs by two processes: generation of endocytic vesicles, predominant after ionomycin and ATP 100-500 microM; and shedding of membrane-bound cytoplasmic bodies, predominant after PMA and 1 mM ATP, containing little or no trace of endoplasmic reticulum, Golgi, endo/lysosomes and also of a plasma membrane marker. Depending on the stimulation, therefore, the cell-surface expansion by enlargeosome exocytosis is not always recycled but can induce release of specific membranes, possibly important in the pericellular environment.     

The synaptic vesicle: cycle of exocytosis and endocytosis

Synaptic vesicles are clustered at the presynaptic terminal where they fuse and recycle in response to stimulation. Vesicles appear to be sorted into pools, but we do not yet understand how physiologically defined pools relate to morphological pools. The advent of dynamic imaging approaches has led to an appreciation of the regulation of vesicle mobility. Newly endocytosed vesicles are highly mobile but appear to become transiently trapped as they re-enter the recycling pool. Recent experiments indicate that endocytosis might have a constant rate, but limited capacity. How endocytosis is linked to exocytosis remains unclear, although calcium emerges as an important player.     

Function of intersectin in endocytosis and exocytosis

Intersectin is an evolutionarily conserved multifunctional adaptor protein with multifunctional domains. These domains interact with components of the endocytic and exocytic pathways, such as the clathrin mediating synaptic vesicle recycling, the protein related to endocytosis via caveolae, the with-no-lysine kinases related to the regulation of renal outer medullar potassium, and the Cdc42 mediating exocytic pathway. Recently, the understanding of intersectin function in the pathogenesis of endocrine tumor and many neurodegenerative diseases such as Down syndrome, Alzheimer disease has been deepened. This article reviewed the structure and roles in endocytosis/exocytosis and diseases of intersectin.     

Hrs and hbp: possible regulators of endocytosis and exocytosis

The molecular mechanisms of endocytosis and exocytosis are not yet fully understood. Hrs and Hbp, two tightly associated proteins in eukaryotic cells, have been implicated in these cellular processes. Hrs is homologous to Vps27p, an endosomal protein required for vacuolar and endocytic trafficking in yeast. Hrs is localized to early endosomes and is required for the normal morphology of early endosomes in mammalian cells. Hrs also associates with proteins implicated in endocytosis and exocytosis such as SNAP-25 and Eps15. Hrs treatment inhibits neurotransmitter release in permeabilized neuronal cells and its overexpression inhibits internalization of transferrin. Overexpression of dominant-negative Hbp mutants inhibits ligand-induced downregulation of growth factor/receptor complexes and immunoglobulin E receptor-triggered degranulation of secretory granules in mast cells. These observations suggest an important role for the Hrs/Hbp protein complex in vesicular trafficking during endocytosis and exocytosis.     

Voltage-dependent calcium channels at the plasma membrane, but not vesicular channels, couple exocytosis to endocytosis

Although calcium influx triggers endocytosis at many synapses and non-neuronal secretory cells, the identity of the calcium channel is unclear. The plasma membrane voltage-dependent calcium channel (VDCC) is a candidate, and it was recently proposed that exocytosis transiently inserts vesicular calcium channels at the plasma membrane, thus triggering endocytosis and coupling it to exocytosis, a mechanism suggested to be conserved from sea urchin to human. Here, we report that the vesicular membrane, when inserted into the plasma membrane upon exocytosis, does not generate a calcium current or calcium increase at a mammalian nerve terminal. Instead, VDCCs at the plasma membrane, including the P/Q-type, provide the calcium influx to trigger rapid and slow endocytosis and, thus, couple endocytosis to exocytosis. These findings call for reconsideration of the vesicular calcium channel hypothesis. They are likely to apply to many synapses and non-neuronal cells in which VDCCs control exocytosis, and exocytosis is coupled to endocytosis.     

Effects of leupeptin on endocytosis and membrane recycling in rat visceral yolk-sac endoderm

Summary The effect of exposure to leupeptin (25 g/ml for 24 h) on the endocytotic activity and the membrane flow of apical cell membranes was studied in endodermal cells of cultured rat visceral yolk sacs by applying a doublelabelling method using concanavalin-A ferritin (Con-A Fer) and horseradish peroxidase (HRP). Control and leupeptintreated yolk sacs were labelled with Con-A Fer at 4°C and then incubated with HRP for 5, 15 or 60 min at 37°C. In controls, HRP reaction product was detected after 5 min in many of the apical vacuoles as well as a few lysosomes; after 15 min, reaction product was observed in all apical vacuoles and in lysosomes of various sizes. These HRP-positive structures usually contained a variable amount of membrane-bound Fer. After 60 min, all apical vacuoles and almost all lysosomes exhibited HRP reactions, but only some of these structures contained Fer particles. At this time, many apical canaliculi (which are involved in membrane recycling) exhibited positive HRP reactions and sometimes also contained Fer particles. In leupeptin-treated cells, HRP reaction product and variable amounts of membrane-bound Fer particles were found in apical vacuoles after 5 min; after 15 min, both labels were also observed in some small lysosomes, and after 60 min, they were found in all apical vacuoles as well as some small and middle-sized lysosomes. Significantly fewer labelled apical vacuoles, lysosomes and apical canaliculi were present after leupeptin treatment than in controls at corresponding times. At all times examined, the giant lysosomes found in leupeptintreated cells did not exhibit any labelling. These findings indicate that, after leupeptin treatment, both endocytotic activity and membrane recycling decrease, and that fusions of the apical vacuolar system with giant lysosomes are retarded or inhibited.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Effects of leupeptin on endocytosis and membrane recycling in rat visceral yolk-sac endoderm

The effect of exposure to leupeptin (25 micrograms/ml for 24 h) on the endocytotic activity and the membrane flow of apical cell membranes was studied in endodermal cells of cultured rat visceral yolk sacs by applying a double-labelling method using concanavalin-A ferritin (Con-A Fer) and horseradish peroxidase (HRP). Control and leupeptin-treated yolk sacs were labelled with Con-A Fer at 4 degrees C and then incubated with HRP for 5, 15 or 60 min at 37 degrees C. In controls, HRP reaction product was detected after 5 min in many of the apical vacuoles as well as a few lysosomes; after 15 min, reaction product was observed in all apical vacuoles and in lysosomes of various sizes. These HRP-positive structures usually contained a variable amount of membrane-bound Fer. After 60 min, all apical vacuoles and almost all lysosomes exhibited HRP reactions, but only some of these structures contained Fer particles. At this time, many apical canaliculi (which are involved in membrane recycling) exhibited positive HRP reactions and sometimes also contained Fer particles. In leupeptin-treated cells, HRP reaction product and variable amounts of membrane-bound Fer particles were found in apical vacuoles after 5 min; after 15 min, both labels were also observed in some small lysosomes, and after 60 min, they were found in all apical vacuoles as well as some small and middle-sized lysosomes. Significantly fewer labelled apical vacuoles, lysosomes and apical canaliculi were present after leupeptin treatment than in controls at corresponding times. At all times examined, the giant lysosomes found in leupeptin-treated cells did not exhibit any labeling.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mammary gland secretion: hormonal coordination of endocytosis and exocytosis

The mammary epithelium coordinates the uptake of milk precursors and the transport of milk components in order to produce milk of relatively constant composition at a particular stage of lactation, as long as the mammary gland is healthy. The mammary epithelial cell controls the uptake of blood-borne molecules at its basal side and the release of products into milk at its apical side, through mechanisms of internalization (endocytosis) and mechanisms of release (exocytosis). These events are strictly dependent on the physiological stage of the mammary gland. This review addresses the mechanisms responsible for these processes and points out new questions that remain to be answered concerning possible interconnections between them, for an optimal milk secretion.     

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.     

Multiple forms of glycosyltransferases in Golgi complex membrane fractions

The distribution of multiple forms of galactosyltransferase (EC 2.4.1.22) and sialyltransferase (EC 2.4.99.1) from the microsomes and Golgi complex membrane fractions of rat liver was investigated. Three fractions of Golgi membranes, namely GF1, GF2, and GF3, differing in their morphology and marker enzyme activity, were obtained. A simultaneous increase of glycosyltransferases under study was observed in fractions GF3 less than GF2 less than GF1. Using isoelectrofocusing, the presence of at least 6-8 forms of galactosyl- and sialyltransferases in the microsomes and Golgi fraction was revealed. The distribution patterns of multiple forms along the pH gradient for each membrane fraction were found to be identical. However, the ratios of highly active and low active forms were specific for each fraction. The similarity of multiple form spectra for galactosyl-and sialyltransferase suggest their tight functional interaction and a possible "en block" packing of membrane glycosyltransferases.     

Recovery of human neutrophils from complement attack: removal of the membrane attack complex by endocytosis and exocytosis

Nucleated cells can resist lysis by and recover from complement attack even after formation of the potentially cytolytic membrane attack complex on the cell surface. We have found that human neutrophils resist complement lysis by the physical removal of membrane attack complexes by both endocytic and exocytic process. The latter mechanism predominates, vesiculation being detectable within 60 sec of initiating the complement cascade. Sixty-five percent of the formed complexes are removed on plasma membrane vesicles, although only 2% of the cell surface is lost. Ultrastructural examination revealed that these vesicles were covered with ring-like "classical" complement lesions. Analysis of these vesicles by gel electrophoresis indicated that C9 was present exclusively in the form of a sodium dodecyl sulfate-resistant, high m.w. complex. In contrast, the 35% of C9 that remained associated with the cells was found to be inaccessible to a C9-specific monoclonal antibody, and was partly degraded, suggesting internalization of the membrane attack complex and proteolysis of some C9 molecules. The molar ratio of C9 to C8 was 12 to 1 on shed vesicles and on recovered cells.