P De Camilli

Synaptobrevin: an integral membrane protein of 18,000 daltons present in small synaptic vesicles of rat brain.

A protein with an apparent mol. wt of 18,000 daltons (synaptobrevin) was identified in synaptic vesicles from rat brain. Some of its properties were studied using monoclonal and polyclonal antibodies. Synaptobrevin is an integral membrane protein with an isoelectric point of approximately 6.6. During subcellular fractionation, synaptobrevin followed the distribution of small synaptic vesicles, with the highest enrichment in the purified vesicle fraction. Immunogold electron microscopy of subcellular particles revealed that synaptobrevin is localized in nerve endings where it is concentrated in the membranes of virtually all small synaptic vesicles. No significant labeling was observed on the membranes of peptide‐containing large dense core vesicles. In agreement with these results, synaptobrevin immunoreactivity has a widespread distribution in nerve terminal‐containing regions of the central and peripheral nervous system as shown by light microscopy immunocytochemistry. Outside the nervous system, synaptobrevin immunoreactivity was found in endocrine cells and cell lines (endocrine pancreas, adrenal medulla, PC12 cells, insulinoma cells) but not in other cell types, for example smooth muscle, skeletal muscle and exocrine pancreas. Thus, the distribution of synaptobrevin is similar to that of synaptophysin, a well‐characterized membrane protein of small vesicles in neurons and endocrine cells.

Autoantibodies to Glutamic Acid Decarboxylase in a Patient with Stiff-Man Syndrome, Epilepsy, and Type I Diabetes Mellitus

Stiff-man syndrome is a rare disorder of the central nervous system consisting of progressive, fluctuating muscle rigidity with painful spasms. It is occasionally associated with endocrine disorders, including insulin-dependent diabetes, and with epilepsy. We investigated the possible existence of autoimmunity against the nervous system in a patient with stiff-man syndrome associated with epilepsy and Type I diabetes mellitus. Levels of IgG, which had an oligoclonal pattern, were elevated in the cerebrospinal fluid. The serum and the cerebrospinal fluid produced an identical, intense staining of all gray-matter regions when used to stain brain sections according to an indirect light-microscopical immunocytochemical procedure. The staining patterns were identical to those produced by antibodies to glutamic acid decarboxylase (the enzyme responsible for the synthesis of gamma-aminobutyric acid). A band comigrating with glutamic acid decarboxylase in sodium dodecyl sulfate-polyacrylamide gels appeared to be the only nervous-tissue antigen recognized by cerebrospinal fluid antibodies, and the predominant antigen recognized by serum antibodies. These findings support the idea that an impairment of neuronal pathways that operate through gamma-aminobutyric acid is involved in the pathogenesis of stiff-man syndrome, and they raise the possibility of an autoimmune pathogenesis.

GABA and pancreatic beta-cells: colocalization of glutamic acid decarboxylase (GAD) and GABA with synaptic-like microvesicles suggests their role in GABA storage and secretion.

GABA, a major inhibitory neurotransmitter of the brain, is also present at high concentration in pancreatic islets. Current evidence suggests that within islets GABA is secreted from beta‐cells and regulates the function of mantle cells (alpha‐ and delta‐cells). In the nervous system GABA is stored in, and secreted from, synaptic vesicles. The mechanism of GABA secretion from beta‐cells remains to be elucidated. Recently the existence of synaptic‐like microvesicles has been demonstrated in some peptide‐secreting endocrine cells. The function of these vesicles is so far unknown. The proposed paracrine action of GABA in pancreatic islets makes beta‐cells a useful model system to explore the possibility that synaptic‐like microvesicles, like synaptic vesicles, are involved in the storage and release of non‐peptide neurotransmitters. We report here the presence of synaptic‐like microvesicles in beta‐cells and in beta‐cells. Some beta‐cells in culture were found to extend neurite‐like processes. When these were present, synaptic‐like microvesicles were particularly concentrated in their distal portions. The GABA synthesizing enzyme, glutamic acid decarboxylase (GAD), was found to be localized around synaptic‐like microvesicles. This was similar to the localization of GAD around synaptic vesicles in GABA‐secreting neurons. GABA immunoreactivity was found to be concentrated in regions of beta‐cells which were enriched in synaptic‐like microvesicles. These findings suggest that in beta‐cells synaptic‐like microvesicles are storage organelles for GABA and support the hypothesis that storage of non‐peptide signal molecules destined for secretion might be a general feature of synaptic‐like microvesicles of endocrine cells.

The epsins define a family of proteins that interact with components of the clathrin coat and contain a new protein module.

Epsin (epsin 1) is an interacting partner for the EH domain-containing region of Eps15 and has been implicated in conjunction with Eps15 in clathrin-mediated endocytosis. We report here the characterization of a similar protein (epsin 2), which we have cloned from human and rat brain libraries. Epsin 1 and 2 are most similar in their NH2-terminal region, which represents a module (epsin NH2 terminal homology domain, ENTH domain) found in a variety of other proteins of the data base. The multiple DPW motifs, typical of the central region of epsin 1, are only partially conserved in epsin 2. Both proteins, however, interact through this central region with the clathrin adaptor AP-2. In addition, we show here that both epsin 1 and 2 interact with clathrin. The three NPF motifs of the COOH-terminal region of epsin 1 are conserved in the corresponding region of epsin 2, consistent with the binding of both proteins to Eps15. Epsin 2, like epsin 1, is enriched in brain, is present in a brain-derived clathrin-coated vesicle fraction, is concentrated in the peri-Golgi region and at the cell periphery of transfected cells, and partially colocalizes with clathrin. High overexpression of green fluorescent protein-epsin 2 mislocalizes components of the clathrin coat and inhibits clathrin-mediated endocytosis. The epsins define a new protein family implicated in membrane dynamics at the cell surface.

A role for synaptic vesicles in non-neuronal cells: clues from pancreatic beta cells and from chromaffin cells.

Synaptic vesicles, vesicular carriers highly specialized for the secretion of fast nonpeptide neurotransmitters, until recently were considered neuron‐specific organelles. The identification and characterization of several of the most abundant synaptic vesicle proteins have led to the identification in peptide‐secreting endocrine cells of a class of microvesicles, referred to as synaptic‐like microvesicles (SLMVs), which are similar to synaptic vesicles in membrane composition, biogenesis, and life cycle. Studies of pancreatic β cells and of a chromaffin cell‐derived cell line (PC12 cells) have suggested that SLMVs, like synaptic vesicles, store and secrete neurotransmitter‐like substances that act as paracrine/endocrine signaling molecules. SLMVs of pancreatic β cells store GABA; SLMVs of PC12 cells contain acetylcholine. Both synaptic vesicles and SLMVs are recycling organelles that represent a specialized subcompartment of the receptor‐mediated recycling pathway of all cells. Recently, homologues of membrane proteins of synaptic vesicles and SLMVs have been identified in the endocytic/recycling compartment of non‐neuronal, nonendocrine cells. Tracing the evolution of synaptic vesicles from vesicular carriers present in all cells will help to advance our understanding of neurotransmitter release as well as of molecular mechanisms of vesicular traffic.—Thomas‐Reetz, A. C., De Camilli, P. A role for synaptic vesicles in non‐neuronal cells: clues from pancreatic cells and from chromaffin cells. FASEB J. 8: 209‐216; 1994.

Inositol 1,4,5-Trisphosphate receptor causes formation of ER cisternal stacks in transfected fibroblasts and in cerebellar purkinje cells

The inositol 1,4,5-trisphosphate receptor (IP3R) is expressed at very high levels in cerebellar Purkinje cells. Within these neurons, it has a widespread distribution throughout the endoplasmic reticulum (ER) and is present at particularly high concentrations at sites of membrane appositions within peculiar stacks of ER cisternae. Here we report that stacks of ER cisternae, reminiscent of those observed in Purkinje cells, can be induced by overexpression of full-length IP3R, but not of mutant forms of the protein in COS cells. Within these stacks the IP3R forms a crystalline array at apposed cisternal faces. Additionally, we show that Purkinje cell stacks are not permanent structures. Our findings suggest that massive stack formation in purkinje cells represents an adaptive response of the ER to hypoxic conditions and is due to the presence of the high concentration of IP3R in its membranes.

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.

Relative properties and localizations of synaptic vesicle protein isoforms: the case of the synaptophysins

Synaptophysins are abundant synaptic vesicle proteins present in two forms: synaptophysin, also referred to as synaptophysin I (abbreviated syp I), and synaptoporin, also referred to as synaptophysin II (abbreviated syp II). In the present study, the properties and localizations of syp I and syp II were investigated to shed light on their relative functions. Our results reveal that syp II, similar to syp I, is an abundant, N-glycosylated membrane protein that is part of a heteromultimeric complex in synaptic vesicle membranes. Cross-linking studies indicate that syp II is linked to a low-molecular-weight protein in this complex as has been observed before for syp I. Furthermore, after transfection into CHO cells, syp II, similar to syp I, is targeted to the receptor-mediated endocytosis pathway. Immunocytochemistry of rat brain sections reveals that syp II expression is highly heterogeneous, with high concentrations of syp II only in selected neuronal populations, whereas syp I is more homogeneously expressed in most nerve terminals. In general, nerve terminals expressing syp II also express syp I. In addition to high levels of syp II observed in selected neurons, a rostrocaudal gradient of syp II expression was observed in the cerebellar cortex. Immunoelectron microscopy confirmed that syp II is localized to synaptic vesicles. Immunoprecipitations of synaptic vesicles from rat brain with antibodies to syp I demonstrated that syp II is colocalized with syp I on the same vesicles. However, after detergent solubilization, no coimmunoprecipitations of the two proteins were observed, suggesting that they are not complexed with each other although they are on the same vesicles. Together our results demonstrate that syp I and syp II have similar properties and are present on the same synaptic vesicles but do not coassemble. The presence of the two proteins in the same nerve terminal suggests that they have similar but nonidentical functions and that the relative abundance of the two proteins may contribute to the functional heterogeneity of nerve terminals.

Specific interactions of Mss4 with members of the Rab GTPase subfamily.

Mss4 is a mammalian protein that was identified as a suppressor of a yeast secretory mutant harboring a mutation in the GTPase Sec4 and was found to stimulate GDP release from this protein. We have now performed a biochemical characterization of the Mss4 protein and examined the specificity of its association with mammalian GTPases. Mss4 is primarily a soluble protein with a widespread tissue distribution. Recombinant Mss4 binds GTPases present in tissue extracts, and by a gel overlay assay binds specifically Rab Rab10proteins. We further define the Mss4‐GTPase interaction to a subset of Rabs belonging to the same subfamily branch which include Rab1, Rab3, Rab8, Rab10, Sec4 and Ypt1 but not Rab2, Rab4, Rab5, Rab6, Rab9 and Rab11. Accordingly, Mss4 co‐precipitates from a brain extract with Rab3a but not Rab5. Mss4 only stimulates GDP release from, and the association of GTP gamma S with, this Rab subset. Recombinant Mss4 and Rab3a form a stable complex in solution that is dissociated with either GDP or GTP gamma S. Injection of Mss4 into the squid giant nerve terminal enhances neurotransmitter release. These results suggest that Mss4 behaves as a guanylnucleotide exchange factor (GEF) for a subset of Rabs to influence distinct vesicular transport steps along the secretory pathway.

Extracellular calcium and the organization of tight junctions in pancreatic acinar cells.

Abstract In pancreatic lobules incubated in Ca2+-free Krebs-Ringer bicarbonate solution +0.5 mM EGTA tight junctions are first disarrayed and then break up into fasciae occludentes and small fibrillar fragments, which move laterally in the plane of the plasmalemma and often wind up around the gap junctions. The interruption of the continuity of tight junctions results in the disappearance of the difference in intramembranous particle density between the lateral and luminal regions of the plasmalemma. These results are consistent with the interpretation of tight junctions as dynamic structures, probably resulting from a specific polymerization of intramembranous particles and confirm that tight junctions might have a role in establishing and maintaining the regional differences of the plasmalemma.