JIP3 links lysosome transport to regulation of multiple components of the axonal cytoskeleton.

Lysosome axonal transport is important for the clearance of cargoes sequestered by the endocytic and autophagic pathways. Building on observations that mutations in the JIP3 (MAPK8IP3) gene result in lysosome-filled axonal swellings, we analyzed the impact of JIP3 depletion on the cytoskeleton of human neurons. Dynamic focal lysosome accumulations were accompanied by disruption of the axonal periodic scaffold (spectrin, F-actin and myosin II) throughout each affected axon. Additionally, axonal microtubule organization was locally disrupted at each lysosome-filled swelling. This local axonal microtubule disorganization was accompanied by accumulations of both F-actin and myosin II. These results indicate that transport of axonal lysosomes is functionally interconnected with mechanisms that control the organization and maintenance of the axonal cytoskeleton. They have potential relevance to human neurological disease arising from JIP3 mutations as well as for neurodegenerative diseases associated with the focal accumulations of lysosomes within axonal swellings such as Alzheimer's disease.

Formation of synaptic vesicles.

Synaptic vesicles (SVs) are specialized secretory organelles used for the fast and focal signaling between nerve cells. They are small and homogeneous in size (50 nm), and contain non-peptide neurotransmitters such as glutamate, gamma-aminobutyric acid (GABA) and acetylcholine. The exocytosis of SVs occurs at low rates in resting nerve terminals and is greatly stimulated by depolarization-induced Ca2+ influx. Following exocytosis, SV membranes are rapidly retrieved, refilled locally with neurotransmitters and reused for the assembly of new SVs. Over the past few years, significant progress has been made in characterizing the molecular composition of SVs. From these studies, we know that SVs share a conserved set of membrane proteins with transport vesicles involved in other pathways. Furthermore, these findings have provided us with a new understanding about the evolutionary origin of SVs from recycling vesicles present in all cells.

Dynamin and its partners: a progress report.

Dynamin's role in clathrin-mediated endocytosis is now well established. Here we review new evidence from the past two years for the function of dynamin and related GTPases in other Intracellular trafficking events. We then summarize current information on the domain structure and function of this multidomain GTPase. Finally, we describe dynamin partners and their function in the context of clathrin-mediated endocytosis.

Functional partnership between amphiphysin and dynamin in clathrin-mediated endocytosis.

Amphiphysin, a protein that is highly concentrated in nerve terminals, has been proposed to function as a linker between the clathrin coat and dynamin in the endocytosis of synaptic vesicles. Here, using a cell-free system, we provide direct morphological evidence in support of this hypothesis. Unexpectedly, we also find that amphiphysin-1, like dynamin-1, can transform spherical liposomes into narrow tubules. Moreover, amphiphysin-1 assembles with dynamin-1 into ring-like structures around the tubules and enhances the liposome-fragmenting activity of dynamin-1 in the presence of GTP. These results show that amphiphysin binds lipid bilayers, indicate a potential function for amphiphysin in the changes in bilayer curvature that accompany vesicle budding, and imply a close functional partnership between amphiphysin and dynamin in endocytosis.

The Eps15 C. elegans homologue EHS-1 is implicated in synaptic vesicle recycling.

Eps15 represents the prototype of a family of evolutionarily conserved proteins that are characterized by the presence of the EH domain, a protein-protein interaction module, and that are involved in many aspects of intracellular vesicular sorting. Although biochemical and functional studies have implicated Eps15 in endocytosis, its function in the endocytic machinery remains unclear. Here we show that the Caenorhabditis elegans gene, zk1248.3 (ehs-1), is the orthologue of Eps15 in nematodes, and that its product, EHS-1, localizes to synaptic-rich regions. ehs-1-impaired worms showed temperature-dependent depletion of synaptic vesicles and uncoordinated movement. These phenotypes could be correlated with a presynaptic defect in neurotransmission. Impairment of EHS-1 function in dyn-1(ky51) worms, which express a mutant form of dynamin and display a temperature-sensitive locomotion defect, resulted in a worsening of the dyn-1 phenotype and uncoordination at the permissive temperature. Thus, ehs-1 and dyn-1 interact genetically. Moreover, mammalian Eps15 and dynamin protein were shown to interact in vivo. Taken together, our results indicate that EHS-1 acts in synaptic vesicle recycling and that its function might be linked to that of dynamin.

Identification of a dominant epitope of glutamic acid decarboxylase (GAD-65) recognized by autoantibodies in stiff-man syndrome.

Glutamic acid decarboxylase (GAD) is the enzyme that synthesizes the neurotransmitter gamma-aminobutyric acid (GABA) in neurons and in pancreatic beta cells. It is a major target of autoimmunity in Stiff-Man syndrome (SMS), a rare neurological disease, and in insulin-dependent diabetes mellitus. The two GAD isoforms, GAD-65 and GAD-67, are the products of two different genes. GAD-67 and GAD-65 are very similar to each other in amino acid sequence and differ substantially only at their NH2-terminal region. We have investigated the reactivity of autoantibodies of 30 Stiff-Man syndrome patients to GAD. All patient sera contained antibodies that recognize strongly GAD-65, but also GAD-67, when tested by immunoprecipitation on brain extracts and by immunoprecipitation or immunocytochemistry on cells transfected with either the GAD-65 or the GAD-67 gene. When tested by Western blotting, all patient sera selectively recognized GAD-65. Western blot analysis of deletion mutants of GAD-65 demonstrated that autoantibodies are directed predominantly against two regions of the GAD-65 molecule. All SMS sera strongly recognized a fragment contained between amino acid 475 and the COOH terminus (amino acid 585). Within this region, amino acids 475-484 and 571-585 were required for reactivity. The requirement of these two discontinuous segments implies that the epitope is influenced by conformation. This reactivity is similar to that displayed by the monoclonal antibody GAD 6, suggesting the presence of a single immunodominant epitope (SMS-E1) in this region of GAD-65. In addition, most SMS sera recognized at least one epitope (SMS-E2) in the NH2-terminal domain of GAD-65 (amino acids 1-95). The demonstration in SMS patients of a strikingly homogeneous humoral autoimmune response against GAD and the identification of dominant autoreactive target regions may help to elucidate the molecular mechanisms of GAD processing and presentation involved in GAD autoimmunity. Moreover, the reactivity reported here of GAD autoantibodies in SMS partially differs from the reactivity of GAD autoantibodies in insulin-dependent diabetes mellitus, suggesting a link between the pattern of humoral autoimmunity and the clinical condition.

The synaptic vesicle-associated protein amphiphysin is the 128-kD autoantigen of Stiff-Man syndrome with breast cancer.

Stiff-Man syndrome (SMS) is a rare disease of the central nervous system (CNS) characterized by progressive rigidity of the body musculature with superimposed painful spasms. An autoimmune origin of the disease has been proposed. In a caseload of more than 100 SMS patients, 60% were found positive for autoantibodies directed against the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). Few patients, all women affected by breast cancer, were negative for GAD autoantibodies but positive for autoantibodies directed against a 128-kD synaptic protein. We report here that this antigen is amphiphysin. GAD and amphiphysin are nonintrinsic membrane proteins that are concentrated in nerve terminals, where a pool of both proteins is associated with the cytoplasmic surface of synaptic vesicles. GAD and amphiphysin are the only two known targets of CNS autoimmunity with this distribution. This finding suggests a possible link between autoimmunity directed against cytoplasmic proteins associated with synaptic vesicles and SMS.

Endocytosis proteins and cancer: a potential link?

Recent studies have shown that a variety of proteins participate with clathrin and clathrin adaptors in receptor-mediated endocytosis. The genes encoding some of these proteins are targets of chromosomal rearrangements in human haematopoietic malignancies. In addition, abnormal expression or mutation of some endocytosis proteins has been reported in human cancers. This article discusses these observations and elaborates the potential mechanisms by which the abnormal expression of endocytosis proteins might participate in the biology of cancer.

Dynamic changes of the luminal plasmalemma in stimulated parotid acinar cells. A freeze-fracture study.

In the acinar cells of the rat parotid gland the two membranes participating in exocytosis, i.e., the luminal plasmalemma and the secretory granule membrane, are clearly distinguishable in freeze-fracture because of their different densities in particles. In order to obtain point-specific information about the fusion-fission of these two membranes that occurs during the secretory cycle, glands were studied at various times (5 min to 6 h) after stimulation with isoproterenol. We observed that, in the course of the release of secretion products and shortly afterwards, the enlarged luminal plasmalemma exhibits a mosaic organization consisting of an alternation of membrane patches of high (original plasmalemma) and low (fused granule membrane) particle density. The transition between these two patterns is usually sharp. Later, concomitant with the reformation of acinar canaliculi, the low particle density membrane is found at the cell surface but only bounding vacuolar infoldings, and then it finally disappears. These results suggest that (a) fusion of these membranes does not result in a random intermixing of the molecular components of the participating membranes, which retain their structural identity; and (b) the enlarged luminal plasmalemma reverts to its original size by a progressive, specific removal of the regions of low particle density from the cell surface.

Synapsin I (protein I), a nerve terminal-specific phosphoprotein. I. Its general distribution in synapses of the central and peripheral nervous system demonstrated by immunofluorescence in frozen and plastic sections.

Synapsin I (formerly referred to as protein I) is the collective name for two almost identical phosphoproteins, synapsin Ia and synapsin Ib (protein Ia and protein Ib), present in the nervous system. Synapsin I has previously been shown by immunoperoxidase studies (De Camilli, P., T. Ueda, F. E. Bloom, E. Battenberg, and P. Greengard, 1979, Proc. Natl. Acad. Sci. USA, 76:5977-5981; Bloom, F. E., T. Ueda, E. Battenberg, and P. Greengard, 1979, Proc. Natl. Acad. Sci. USA 76:5982-5986) to be a neuron-specific protein, present in both the central and peripheral nervous systems and concentrated in the synaptic region of nerve cells. In those preliminary studies, the occurrence of synapsin I could be demonstrated in only a portion of synapses. We have now carried out a detailed examination of the distribution of synapsin I immunoreactivity in the central and peripheral nervous systems. In this study we have attempted to maximize the level of resolution of immunohistochemical light microscopy images in order to estimate the proportion of immunoreactive synapses and to establish their precise distribution. Optimal results were obtained by the use of immunofluorescence in semithin sections (approximately 1 micron) prepared from Epon-embedded nonosmicated tissues after the Epon had been removed. Our results confirm the previous observations on the specific localization of synapsin I in nerve cells and synapses. In addition, the results strongly suggest that, with a few possible exceptions involving highly specialized neurons, all synapses contain synapsin I. Finally, immunocytochemical experiments indicate that synapsin I appearance in the various regions of the developing nervous system correlates topographically and temporally with the appearance of synapses. In two accompanying papers (De Camilli, P., S. M. Harris, Jr., W. B. Huttner, and P. Greengard, and Huttner, W. B., W. Schiebler, P. Greengard, and P. De Camilli, 1983, J. Cell Biol. 96:1355-1373 and 1374-1388, respectively), evidence is presented that synapsin I is specifically associated with synaptic vesicles in nerve endings.

Membrane interactions between secretion granules and plasmalemma in three exocrine glands.

Three types of membrane interactions were studied in three exocrine systems (the acinar cells of the rat parotid, rat lacrimal gland, and guinea pig pancrease) by freeze- fracture and thin-section electron microscopy: exocytosis, induced in vivo by specific pharmacological stimulations; the mutual apposition of secretory granule membranes in the intact cell; membrane appositions induced in vitro by centrifugation of the isolated granules. In all three glandular cells, the distribution of intramembrane particles (IMP) on the fracture faces of the luminal plasmagranule membrane particles (IMP) on the fracture faces of the lumenal plasmalemma appeared random before stimulation. However, after injection of secretagogues, IMP were rapidly clearly from the areas of granule- plasmalemma apposition in the parotid cells and, especially, in lacrimocytes. In the latter, the cleared areas appeared as large bulges toward the lumen, whereas in the parotid they were less pronounced. Exocytotic openings were usually large and the fracture faces of their rims were covered with IMP. In contrast, in stimulated pancreatic acinar cells, the IMP distribution remained apparently random after stimulation. Exocytoses were established through the formation of narrown necks, and no images which might correspond to early stages of membrane fusion were revealed. Within the cytoplasm of parotid and lacrimal cells (but not in the pancreas), both at rest and after stimulation, secretion granules were often closely apposed by means of flat, circular areas, also devoid of IMP. In thin sections, the images corresponding to IMP-free areas were close granule-granule and granule-plasmalemma appositions, sometimes with focal merging of the membrane outer layers to yield pentalaminar structures. Isolated secretion granules were forced together in vitro by centrifugation. Under these conditions, increasing the centrifugal force from 1,600 to 50,000 g for 10 min resulted in a progressive, statistically significant increase of the frequency of IMP-free flat appositions between parotid granules. In contrast, no such areas were seen between freeze-fractured pancreatic granules, although some focal pentalaminar appositions appeared in section after centrifugation at 50 and 100,000 g for 10 min. On the basis of the observation that, in secretory cells, IMP clearing always develops in deformed membrane areas (bulges, depressions, flat areas), it is suggested that it might result from the forced mechanical apposition of the interacting membranes. This might be a preliminary process not sufficient to initiate fusion. In the pancreas, IMP clearing could occur over surface areas too small to be detected. In stimulated parotid and lacrimal glands they were exceptional. These structures were either attached at the sites of continuity between granule and plasma membranes, or free in the acinar lumen, with a preferential location within exocytotic pockets or in their proximity. Experiments designed to investigate the nature of these blisters and vesicles revealed that they probably arise artifactually during glutaraldehyde fixation. In fact, (a) they were large and numerous in poorly fixed samples but were never observed in thin sections of specimens fixed in one step with glutaraldehyde and OsO(4); and (b) no increase in concentration of phospholipids was observed in the parotid saliva and pancreatic juice after stimulation of protein discharge, as was to be expected if release of membrane material were occurring after exocytosis.

The synaptic vesicle cycle: a single vesicle budding step involving clathrin and dynamin.

Strong evidence implicates clathrin-coated vesicles and endosome-like vacuoles in the reformation of synaptic vesicles after exocytosis, and it is generally assumed that these vacuoles represent a traffic station downstream from clathrin-coated vesicles. To gain insight into the mechanisms of synaptic vesicle budding from endosome-like intermediates, lysed nerve terminals and nerve terminal membrane subfractions were examined by EM after incubations with GTP gamma S. Numerous clathrin-coated budding intermediates that were positive for AP2 and AP180 immunoreactivity and often collared by a dynamin ring were seen. These were present not only on the plasma membrane (Takei, K., P.S. McPherson, S.L.Schmid, and P. De Camilli. 1995. Nature (Lond.). 374:186-190), but also on internal vacuoles. The lumen of these vacuoles retained extracellular tracers and was therefore functionally segregated from the extracellular medium, although narrow connections between their membranes and the plasmalemma were sometimes visible by serial sectioning. Similar observations were made in intact cultured hippocampal neurons exposed to high K+ stimulation. Coated vesicle buds were generally in the same size range of synaptic vesicles and positive for the synaptic vesicle protein synaptotagmin. Based on these results, we suggest that endosome-like intermediates of nerve terminals originate by bulk uptake of the plasma membrane and that clathrin- and dynamin-mediated budding takes place in parallel from the plasmalemma and from these internal membranes. We propose a synaptic vesicle recycling model that involves a single vesicle budding step mediated by clathrin and dynamin.

Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation.

Synapsin I (protein I) is a neuron-specific phosphoprotein, which is a substrate for cAMP-dependent and Ca/calmodulin-dependent protein kinases. In two accompanying studies (De Camilli, P., R. Cameron, and P. Greengard, and De Camilli, P., S. M. Harris, Jr., W. B. Huttner, and P. Greengard, 1983, J. Cell Biol. 96:1337-1354 and 1355-1373) we have shown, by immunocytochemical techniques at the light microscopic and electron microscopic levels, that synapsin I is present in the majority of, and possibly in all, nerve terminals, where it is primarily associated with synaptic vesicles. In the present study we have prepared a highly purified synaptic vesicle fraction from rat brain by a procedure that involves permeation chromatography on controlled-pore glass as a final purification step. Using immunological methods, synapsin I concentrations were determined in various subcellular fractions obtained in the course of vesicle purification. Synapsin I was found to copurify with synaptic vesicles and to represent approximately 6% of the total protein in the highly purified synaptic vesicle fraction. The copurification of synapsin I with synaptic vesicles was dependent on the use of low ionic strength media throughout the purification. Synapsin I was released into the soluble phase by increased ionic strength at neutral pH, but not by nonionic detergents. The highly purified synaptic vesicle fraction contained a calcium-dependent protein kinase that phosphorylated endogenous synapsin I in its collagenase-sensitive tail region. The phosphorylation of this region appeared to facilitate the dissociation of synapsin I from synaptic vesicles under the experimental conditions used.

Colocalization of synaptophysin with transferrin receptors: implications for synaptic vesicle biogenesis.

We have reported previously that the synaptic vesicle (SV) protein synaptophysin, when expressed in fibroblastic CHO cells, accumulates in a population of recycling microvesicles. Based on preliminary immunofluorescence observations, we had suggested that synaptophysin is targeted to the preexisting population of microvesicles that recycle transferrin (Johnston, P. A., P. L. Cameron, H. Stukenbrok, R. Jahn, P. De Camilli, and T. C. Südhof. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:2863-2872). In contrast to our results, another group reported that expression of synaptophysin in cells which normally do not express SV proteins results in the generation of a novel population of microvesicles (Leube, R. E., B. Wiedenmann, and W. W. Franke. 1989. Cell. 59:433-446). We report here a series of morphological and biochemical studies conclusively demonstrating that synaptophysin and transferrin receptors are indeed colocalized on the same vesicles in transfected CHO cells. These observations prompted us to investigate whether an overlap between the distribution of the two proteins also occurs in endocrine cell lines that endogenously express synaptophysin and other SV proteins. We have found that endocrine cell lines contain two pools of membranes positive for synaptophysin and other SV proteins. One of the two pools also contains transferrin receptors and migrates faster during velocity centrifugation. The other pool is devoid of transferrin receptors and corresponds to vesicles with the same sedimentation characteristics as SVs. These findings suggest that in transfected CHO cells and in endocrine cell lines, synaptophysin follows the same endocytic pathway as transferrin receptors but that in endocrine cells, at some point along this pathway, synaptophysin is sorted away from the recycling receptors into a specialized vesicle population. Finally, using immunofluorescent analyses, we found an overlap between the distribution of synaptophysin and transferrin receptors in the dendrites of hippocampal neurons in primary cultures before synapse formation. Axons were enriched in synaptophysin immunoreactivity but did not contain detectable levels of transferrin receptor immunoreactivity. These results suggest that SVs may have evolved from, as well as coexist with, a constitutively recycling vesicular organelle found in all cells.

Protein p38: an integral membrane protein specific for small vesicles of neurons and neuroendocrine cells.

An intrinsic membrane protein of brain synaptic vesicles with Mr 38,000 (p38, synaptophysin) has recently been partially characterized (Jahn, R., W. Schiebler, C. Ouimet, and P. Greengard, 1985, Proc. Natl. Acad. Sci. USA, 83:4137-4141; Wiedenmann, B., and W. W. Franke, 1985, Cell, 41:1017-1028). We have now studied the presence of p38 in a variety of tissues by light and electron microscopy immunocytochemistry and by immunochemistry. Our results indicate that, within the nervous system, p38, like the neuron-specific phosphoprotein synapsin I, is present in virtually all nerve terminals and is selectively associated with small synaptic vesicles (SSVs). No p38 was detectable on large dense-core vesicles (LDCVs). p38 and synapsin I were found to be present in similar concentrations throughout the brain. Outside the nervous system, p38 was found in a variety of neuroendocrine cells, but not in any other cell type. In neuroendocrine cells p38 was localized on a pleiomorphic population of small, smooth-surfaced vesicles, which were interspersed among secretory granules and concentrated in the Golgi area, but not on the secretory granules themselves. Immunoblot analysis of endocrine tissues and cell lines revealed a band with a mobility slightly different from that of neuronal p38. This difference was attributable to a difference in glycosylation. The finding that p38, like synapsin I, is a component of SSVs of virtually all neurons, but not of LDCVs, supports the idea that SSVs and LDCVs are organelles of two distinct pathways for regulated neuronal secretion. In addition, our results indicate the presence in a variety of neuroendocrine cells of an endomembrane system, which is related to SSVs of neurons but is distinct from secretory granules.

Microvesicles of the neurohypophysis are biochemically related to small synaptic vesicles of presynaptic nerve terminals.

Nerve endings of the posterior pituitary are densely populated by dense-core neurosecretory granules which are the storage sites for peptide neurohormones. In addition, they contain numerous clear microvesicles which are the same size as small synaptic vesicles of typical presynaptic nerve terminals. Several of the major proteins of small synaptic vesicles of presynaptic nerve terminals are present at high concentration in the posterior pituitary. We have now investigated the subcellular localization of such proteins. By immunogold electron microscopy carried out on bovine neurohypophysis we have found that three of these proteins, synapsin I, Protein III, and synaptophysin (protein p38) were concentrated on microvesicles but were not detectable in the membranes of neurosecretory granules. In addition, we have studied the distribution of the same proteins and of the synaptic vesicle protein p65 in subcellular fractions of bovine posterior pituitaries obtained by sucrose density centrifugation. We have found that the intrinsic membrane proteins synaptophysin and p65 had an identical distribution and were restricted to low density fractions of the gradient which contained numerous clear microvesicles with a size range the same as that of small synaptic vesicles. The peripheral membrane proteins synapsin I and Protein III exhibited a broader distribution extending into the denser part of the gradient. However, the amount of these proteins clearly declined in the fractions preceding the peak of neurosecretory granules. Our results suggest that microvesicles of the neurohypophysis are biochemically related to small synaptic vesicles of all other nerve terminals and argue against the hypothesis that such vesicles represent an endocytic byproduct of exocytosis of neurosecretory granules.

A signal located within amino acids 1-27 of GAD65 is required for its targeting to the Golgi complex region.

The mechanisms involved in the targeting of proteins to different cytosolic compartments are still largely unknown. In this study we have investigated the targeting signal of the 65-kD isoform of glutamic acid decarboxylase (GAD65), a major autoantigen in two autoimmune diseases: Stiff-Man syndrome and insulin-dependent diabetes mellitus. GAD65 is expressed in neurons and in pancreatic beta-cells, where it is concentrated in the Golgi complex region and in proximity to GABA-containing vesicles. GAD65, but not the similar isoform GAD67 which has a more diffuse cytosolic distribution, is palmitoylated within its first 100 amino acids (a.a.). We have previously demonstrated that the domain corresponding to a.a. 1-83 of GAD65 is required for the targeting of GAD65 to the Golgi complex region. Here we show that this domain is sufficient to target an unrelated protein, beta-galactosidase, to the same region. Site-directed mutagenesis of all the putative acceptor sites for thiopalmitoylation within this domain did not abolish targeting of GAD65 to the Golgi complex region. The replacement of a.a. 1-29 of GAD67 with the corresponding a.a. 1-27 of GAD65 was sufficient to target the otherwise soluble GAD67 to the Golgi complex region. Conversely, the replacement of a.a. 1-27 of GAD65 with a.a. 1-29 of GAD67 resulted in a GAD65 protein that had a diffuse cytosolic distribution and was primarily hydrophilic, suggesting that targeting to the Golgi complex region is required for palmitoylation of GAD65. We propose that the domain corresponding to a.a. 1-27 of GAD65, contains a signal required for the targeting of GAD65 to the Golgi complex region.

Heterogeneous distribution of the cAMP receptor protein RII in the nervous system: evidence for its intracellular accumulation on microtubules, microtubule-organizing centers, and in the area of the Golgi complex.

The cellular and subcellular distribution of the regulatory subunit RII of cAMP-dependent protein kinase was studied by light and electron microscopy immunocytochemistry in tissue sections from rat brain and in primary cultures of brain cells. RII immunoreactivity was present in most neurons, although at variable concentration. In addition, RII was also detectable in other cell types including glia, neuroepithelial cells, and cells of mesenchymal origin. In the cell cytoplasm, RII immunoreactivity was concentrated at certain sites. An accumulation of RII immunoreactivity was found in all RII-positive cells at the Golgi area, precisely at a region directly adjacent to one of the two major faces of the Golgi complex. RII was also highly concentrated in some microtubule-rich cell processes such as cilia and neuronal dendrites, but was below detectability in most axons. In neurons, its concentration in dendrites is consistent with the previously demonstrated high affinity interaction between RII and the dendritic microtubule-associated protein 2. In addition, RII was accumulated at basal bodies of cilia and at centrosomes, i.e., sites known to act as microtubule organizers. RII-labeled centrosomes, however, were visible only in cells where the Golgi complex had a pericentrosomal organization, and not in cells where the Golgi complex was perinuclear such as in neurons and glia in situ. We hypothesize that centrosomal RII is bound to the pericentriolar microtubule-organizing material and that this material remains associated with the trans region of the Golgi complex when the latter is no longer associated with the centrosome. Our results suggest a key but not obligatory role of cAMP in the Golgi-centrosomal area, the headquarters of cell polarity, mobility and intracellular traffic, and in the function of a subpopulation of microtubules.

Exo-endocytotic recycling of synaptic vesicles in developing processes of cultured hippocampal neurons.

In mature neurons synaptic vesicles (SVs) undergo cycles of exo-endocytosis at synapses. It is currently unknown whether SV exocytosis and recycling occurs also in developing axons prior to synapse formation. To address this question, we have developed an immunocytochemical assay to reveal SV exo-endocytosis in hippocampal neurons developing in culture. In this assay antibodies directed against the lumenal domain of synaptotagmin I (Syt I), an intrinsic membrane protein of SVs, are used to reveal exposure of SV membranes at the cell surface. Addition of antibodies to the culture medium of living neurons for 1 hr at 37 degrees C resulted in their rapid and specific internalization by all neuronal processes and, particularly, by axons. Double immunofluorescence and electron microscopy immunocytochemistry indicated that the antibodies were retained within SVs in cell processes and underwent cycles of exo-endocytosis in parallel with SV membranes. In contrast, another endocytotic marker, wheat germ agglutinin, was rapidly cleared from the processes and transported to the cell body. Antibody-labeled SVs were still present in axons several days after antibody loading and became clustered at presynaptic sites in parallel with synaptogenesis. These results demonstrate that SVs undergo multiple cycles of exo-endocytosis in developing neuronal processes irrespective of the presence of synaptic contacts.

Redistribution of synaptophysin and synapsin I during alpha-latrotoxin-induced release of neurotransmitter at the neuromuscular junction.

The distribution of two synaptic vesicle-specific phosphoproteins, synaptophysin and synapsin I, during intense quantal secretion was studied by applying an immunogold labeling technique to ultrathin frozen sections. In nerve-muscle preparations treated for 1 h with a low dose of alpha-latrotoxin in the absence of extracellular Ca2+ (a condition under which nerve terminals are depleted of both quanta of neurotransmitter and synaptic vesicles), the immunolabeling for both proteins was distributed along the axolemma. These findings indicate that, in the presence of a block of endocytosis, exocytosis leads to the permanent incorporation of the synaptic vesicle membrane into the axolemma and suggest that, under this condition, at least some of the synapsin I molecules remain associated with the vesicle membrane after fusion. When the same dose of alpha-latrotoxin was applied in the presence of extracellular Ca2+, the immunoreactivity patterns resembled those obtained in resting preparations: immunogold particles were selectively associated with the membrane of synaptic vesicles, whereas the axolemma was virtually unlabeled. Under this condition an active recycling of both quanta of neurotransmitter and vesicles operates. These findings indicate that the retrieval of components of the synaptic vesicle membrane is an efficient process that does not involve extensive intermixing between molecular components of the vesicle and plasma membrane, and show that synaptic vesicles that are rapidly recycling still have the bulk of synapsin I associated with their membrane.