Microvesicle Biogenesis Research Articles

An AP-3-dependent mechanism drives synaptic-like microvesicle biogenesis in pancreatic islet beta-cells.

Pancreatic islet beta-cells contain synaptic-like microvesicles (SLMVs). The origin, trafficking, and role of these SLMVs are poorly understood. In neurons, synaptic vesicle (SV) biogenesis is mediated by two different cytosolic adaptor protein complexes, a ubiquitous AP-2 complex and the neuron-specific AP-3B complex. Mice lacking AP-3B subunits exhibit impaired GABAergic (inhibitory) neurotransmission and reduced neuronal vesicular GABA transporter (VGAT) content. Since beta-cell maturation and exocytotic function seem to parallel that of the inhibitory synapse, we predicted that AP-3B-associated vesicles would be present in beta-cells. Here, we test the hypothesis that AP-3B is expressed in islets and mediates beta-cell SLMV biogenesis. A secondary aim was to test whether the sedimentation properties of INS-1 beta-cell microvesicles are identical to those of bona fide SLMVs isolated from PC12 cells. Our results show that the two neuron-specific AP-3 subunits beta3B and mu3B are expressed in beta-cells, the first time these proteins have been found to be expressed outside the nervous system. We found that beta-cell SLMVs share the same sedimentation properties as PC12 SLMVs and contain SV proteins that sort specifically to AP-3B-associated vesicles in the brain. Brefeldin A, a drug that interferes with AP-3-mediated SV biogenesis, inhibits the delivery of AP-3 cargoes to beta-cell SLMVs. Consistent with a role for AP-3 in the biogenesis of GABAergic SLMV in beta-cells, INS-1 cell VGAT content decreases upon inhibition of AP-3 delta-subunit expression. Our findings suggest that beta-cells and neurons share molecules and mechanisms important for mediating the neuron-specific membrane trafficking pathways that underlie synaptic vesicle formation.

Microvesicle Mediated Genetic Phenotype Modulation.

Abstract 4509 ObjectiveWe have previously reported that lung-derived microvesicles (MVs) can enter target marrow cells, resulting in increased levels of lung-specific mRNAs (Stem Cells 25:2245, 2007). Marrow cells which have been exposed to MVs also show increased production of pulmonary epithelial cells after transplantation into irradiated mice. The present studies have addressed the universality of the mRNA modulation and the underlying mechanisms. Methods/ResultsCo-culture of heart, brain, liver, and lung tissue across from murine marrow, but separated by a 0.4 micron cell-impermeable membrane, show tissue specific elevations of mRNA. MVs were found to contain lung-specific mRNA and 200 microRNAs. Proteomic studies of MVs showed up to 75 individual proteins, some of which are known to be associated with MV biogenesis and trafficking. Studies using rat/mouse hybrid cultures demonstrated that the target cell induced lung-specific mRNA elevations were mediated by transcriptional mechanisms. In these experiments, rat lung was co-cultured across from murine marrow cells and RT-PCR was performed using rat or mouse-specific primers for surfactant B. High levels of rat-specific surfactant B were seen in the co-cultured marrow cells indicating that transcription had been induced in the target cells. These conclusions were supported by additional studies employing the transcription factor inhibitors actinomycin-D and alpha-amantin. RNase treatment of conditioned media prior to marrow cell co-culture suggested that transfer of RNA may be involved in these mRNA elevations. However, our transcriptional studies indicate that we are not observing a simple transfer of MV lung-specific mRNA. One possible mechanism may be transfer of microRNA with epigenetic changes resulting in lung-specific mRNA production. ConclusionIn summary, these observations suggest the existence of unique pathways for information transfer and cell phenotype determination. MV transfer could represent an underlying mechanism for much of the previous reported stem cell plasticity in different tissues. Disclosures:No relevant conflicts of interest to declare.

Proteomic Analysis of Microvesicles Derived from Human Colorectal Cancer Cells

Microvesicles (MV) are membrane vesicles secreted from the plasma and endosomal membrane compartment by various cell types such as hematopoietic, epithelial, and tumor cells. Actively growing tumor cells shed MV, and the rate of shedding increases in malignant tumors. Although recent progress in this area has revealed that tumor-derived MV play multiple roles in tumor growth and metastasis via immune escape, tumor invasion, and angiogenesis, the mechanism of vesicle formation and the biological roles of tumor-derived MV are not understood. Here, we report the first global proteomic analysis of highly purified MV from human colorectal cancer cells. Using 1D SDS gel electrophoresis and nano-LC-MS/MS analyses, we identified a total of 547 microvesicular proteins from three independent experiments with high confidence; 416 proteins were identified at least in two trials, including 181 as yet unreported proteins. We identified 49 proteins involved in the biogenesis of MV, including annexins, ADP-ribosylation factors, and Rab proteins. We also identified 28 proteins that may function in tumorigenesis via promotion of migration, invasion, and growth of tumor cells, immune modulation, metastasis, and angiogenesis. Taken together with previously reported results, our observations suggest that tumor-derived MV may act as communicasomes, that is, extracellular organelles that play diverse roles in intercellular communication. This information will help elucidate the biogenesis and functions of tumor-derived MV, and aid in the development of effective vaccines for various cancers, including colorectal cancer.

Cellugyrin and Synaptogyrin Facilitate Targeting of Synaptophysin to a Ubiquitous Synaptic Vesicle-sized Compartment in PC12 Cells

Cellugyrin represents a ubiquitously expressed four-transmembrane domain protein that is closely related to synaptic vesicle protein synaptogyrin and, more remotely, to synaptophysin. We report here that, in PC12 cells, cellugyrin is localized in synaptic-like microvesicles (SLMVs), along with synaptogyrin and synaptophysin. Upon overexpression of synaptophysin in PC12 cells, it is localized in rapidly sedimenting membranes and practically is not delivered to the SLMVs. On the contrary, the efficiency of the SLMV targeting of exogenously expressed cellugyrin and synaptogyrin is high. Moreover, expression of cellugyrin (or synaptogyrin) in PC12 cells dramatically and specifically increases SLMV targeting of endogenous synaptophysin. Finally, we utilized the SLMV purification scheme on a series of non-neuroendocrine cell types including the mouse fibroblast cell line 3T3-L1, the Chinese hamster ovary cell line CHO-K1, and the monkey kidney epithelial cell line COS7 and found that a cellugyrin-positive microvesicular compartment was present in all cell types tested. We suggest that synaptic vesicles have evolved from cellugyrin-positive ubiquitous microvesicles and that neuroendocrine SLMVs represent a step along that pathway of evolution.

Cholesterol binds to synaptophysin and is required for biogenesis of synaptic vesicles.

Here, to study lipid-protein interactions that contribute to the biogenesis of regulated secretory vesicles, we have developed new approaches by which to label proteins in vivo, using photoactivatable cholesterol and glycerophospholipids. We identify synaptophysin as a major specifically cholesterol-binding protein in PC12 cells and brain synaptic vesicles. Limited cholesterol depletion, which has little effect on total endocytic activity, blocks the biogenesis of synaptic-like microvesicles (SLMVs) from the plasma membrane. We propose that specific interactions between cholesterol and SLMV membrane proteins, such as synaptophysin, contribute to both the segregation of SLMV membrane constituents from plasma-membrane constituents, and the induction of synaptic-vesicle curvature.

Biogenesis of Synaptic-like Microvesicles in Perforated PC12 Cells

We have established a system that reconstitutes the biogenesis of synaptic-like microvesicles (SLMVs) in perforated cells of the rat neuroendocrine cell line PC12. The system is based on the biotinylation of synaptophysin, a marker of synaptic vesicles and SLMVs. Biotinylation is performed at 18°C, a temperature at which formation of SLMVs is blocked and biotinylated synaptophysin accumulates in the SLMV donor compartment. The biotinylated PC12 cells are then perforated by scraping and incubated at 37°C in the presence of ATP and cytosolic proteins, conditions required for SLMV biogenesis. After the perforated-cell reaction, the newly formed SLMVs are isolated by differential centrifugation followed by either glycerol gradient centrifugation or a simple single-glycerol-step centrifugation. The latter allows the analysis of many perforated-cell reactions in parallel and, hence, the dissection of the molecular machinery mediating SLMV biogenesis. Using this system, we have found that clathrin, dynamin, phosphatidylinositol transfer protein, and SH3P4 are involved in SLMV biogenesis.

Synaptotagmin I, synaptobrevin II, and syntaxin I are coexpressed in rat and gerbil pinealocytes.

Recent studies have shown that mammalian pinealocytes contain a compartment of synaptic-like microvesicles that may serve secretory functions; however, knowledge of the molecular composition of these microvesicles is still incomplete. Therefore, we have analyzed rat and gerbil pineal glands for the presence of synaptotagmin I, synaptobrevin I and II, syntaxin I, and synaptoporin (synaptophysin II) by immunoblot analyses and immunostaining of serial semithin sections. These proteins, which are components of the synaptic vesicle membrane or presynaptic plasmalemma, are thought to be essential for synaptic vesicle trafficking and exocytosis. Antibodies against synaptotagmin I, synaptobrevin II, and syntaxin I label pinealocytes (identified with an antiserum directed against synaptophysin I) in pineal glands of both species, the coexpression of the latter proteins being demonstrable at the single cell level. In contrast, pinealocytes are not or only weakly stained by the synaptoporin antibody. Immunoreactivity for synaptobrevin I is restricted to intrapineal nerve terminals, thus indicating a differential expression of synaptic vesicle protein isoforms within endocrine tissues. Immunogold staining has been performed in the gerbil pineal and reveals that synaptobrevin II and synaptotagmin I can be localized to the synaptic-like microvesicles that are concentrated in pinealocyte process terminals. Syntaxin immunoreactivity is associated with clear microvesicles and with the plasma membrane. Our findings corroborate the hypothesis that the synaptic-like microvesicles of pinealocytes can be considered as the endocrine equivalent of neuronal synaptic vesicles. Since pinealocytes of several mammalian species contain abundant clear microvesicles, the pinealocyte may serve as a paradigm for studies aimed at elucidating the biogenesis and functions of synaptic-like microvesicles in neuroendocrine cells.

Selective storage of acetylcholine, but not catecholamines, in neuroendocrine synaptic-like microvesicles of early endosomal origin

We have defined, in the neuroendocrine cell line PC12, the catecholamine- and acetylcholine-storing organelles in the context of the biogenesis of secretory granules and synaptic-like microvesicles (SLMVs). SLMVs were found to originate directly from early endosomes. Both early endosomes and SLMVs exhibited uptake and storage of biosynthetic acetylcholine. Surprisingly, however, despite the presence of a reserpine-sensitive vesicular amine transporter in early endosomes, SLMVs lacked detectable uptake and storage of catecholamines. This was confined to two populations of mature secretory granules, referred to as small and large mature secretory granules, which both derived from immature secretory granules. Our results show that PC12 cells lack small dense core vesicles, i.e., the catecholamine-storing, but secretory protein-lacking, vesicles found in sympathetic neurons and imply that the biogenesis of these vesicles requires the expression of a distinct type of vesicular amine transporter and/or a change in endosomal protein sorting.

Newly synthesized synaptophysin is transported to synaptic-like microvesicles via constitutive secretory vesicles and the plasma membrane.

The biogenesis of synaptic-like microvesicles (SLMVs) in neuroendocrine cells was investigated by studying the traffic of newly synthesized synaptophysin to SLMVs in PC12 cells. Synaptophysin was found to be sulfated, which facilitated the determination of its exit route from the trans-Golgi network (TGN). Virtually all [35S]sulfate-labeled synaptophysin was found to leave the TGN in vesicles which were indistinguishable from constitutive secretory vesicles but distinct from immature secretory granules and SLMVs. [35S]sulfate-labeled synaptophysin was rapidly transported from the TGN to the cell surface, with a t1/2 of approximately 10 min in resting cells. After arrival at the cell surface, [35S]sulfate-labeled synaptophysin cycled for at least 1 h between the plasma membrane and an intracellular compartment likely to be the early endosome. Up to approximately 40% of the [35S]sulfate-labeled synaptophysin eventually (after 3 h and later) reached SLMVs, which could be distinguished from the other post-TGN compartments by their lower buoyant density in a sucrose gradient and their selective inclusion upon permeation chromatography using a controlled-pore glass column. Our results suggest that newly synthesized membrane proteins of SLMVs in neuroendocrine cells, and possibly of small synaptic vesicles in neurons, reach these organelles via the TGN----plasma membrane----early endosome.