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F Valtorta

Publications and source records attributed to F Valtorta.

At least 19 recordsLinked to original sources
PubMed1992

Synaptic vesicle-associated Ca2+/calmodulin-dependent protein kinase II is a binding protein for synapsin I.

Synapsin I is a synaptic vesicle-associated phosphoprotein that is involved in the modulation of neurotransmitter release. Ca2+/calmodulin-dependent protein kinase II, which phosphorylates two sites in the carboxy-terminal region of synapsin I, causes synapsin I to dissociate from synaptic vesicles and increases neurotransmitter release. Conversely, the dephosphorylated form of synapsin I, but not the form phosphorylated by Ca2+/calmodulin-dependent protein kinase II, inhibits neurotransmitter release. The amino-terminal region of synapsin I interacts with membrane phospholipids, whereas the C-terminal region binds to a protein component of synaptic vesicles. Here we demonstrate that the binding of the C-terminal region of synapsin I involves the regulatory domain of a synaptic vesicle-associated form of Ca2+/calmodulin-dependent protein kinase II. Our results indicate that this form of the kinase functions both as a binding protein for synapsin I, and as an enzyme that phosphorylates synapsin I and promotes its dissociation from the vesicles.

Amino Acid Sequence↗
PubMed1992

Calcium-dependent transmitter secretion reconstituted in Xenopus oocytes: requirement for synaptophysin.

Calcium-dependent glutamate secretion was reconstituted in Xenopus oocytes by injecting the oocyte with total rat cerebellar messenger RNA (mRNA). Co-injection of total mRNA with antisense oligonucleotides to synaptophysin message decreased the expression of synaptophysin in the oocyte and reduced the calcium-dependent secretion. A similar effect on secretion was observed for oocytes injected with total mRNA together with an antibody to rat synaptophysin. These results indicate that synaptophysin is necessary for transmitter secretion and that the oocyte expression system may be useful for dissecting the molecular events associated with the secretory process.

Animals↗
PubMed1992

Effects of the neuronal phosphoprotein synapsin I on actin polymerization. I. Evidence for a phosphorylation-dependent nucleating effect.

Synapsin I is a synaptic vesicle-specific phosphoprotein which is able to bind and bundle actin filaments in a phosphorylation-dependent fashion. In the present paper we have analyzed the effects of synapsin I on the kinetics of actin polymerization and their modulation by site-specific phosphorylation of synapsin I. We found that dephosphorylated synapsin I accelerates the initial rate of actin polymerization and decreases the rate of filament elongation. The effect was observed at both low and high ionic strength, was specific for synapsin I, and was still present when polymerization was triggered by F-actin seeds. Dephosphorylated synapsin I was also able to induce actin polymerization and bundle formation in the absence of KCl and MgCl2. The effects of synapsin I were strongly decreased after its phosphorylation by Ca2+/calmodulin-dependent protein kinase II. These observations suggest that synapsin I has a phosphorylation-dependent nucleating effect on actin polymerization. The data are compatible with the view that changes in the phosphorylation state of synapsin I play a functional role in regulating the interactions between the nerve terminal cytoskeleton and synaptic vesicles in various stages of the exoendocytotic cycle.

Actins↗
PubMed1992

Effects of the neuronal phosphoprotein synapsin I on actin polymerization. II. Analytical interpretation of kinetic curves.

The general features of the kinetics of actin polymerization are investigated by mathematical models, with the aim of identifying the kinetically relevant parameters in the process and detecting and interpreting the alterations occurring in actin polymerization under various experimental conditions. Polymerization curves, obtained by following the increase in fluorescence of actin derivatized with N-(1-pyrenyl) iodoacetamide, are fitted using analytical equations derived from biochemical models of the actin polymerization process. Particular attention is given to the evaluation of the effects of the neuronal phosphoprotein synapsin I. The models obtained under various ionic conditions reveal that synapsin I interacts with actin in a very complex fashion, sharing some of the properties of classical nucleating proteins but displaying also actions not described previously for other actin-binding proteins. Synapsin I appears to bind G-actin with a very high stoichiometry (1:2-4), and the complex behaves as an F-actin nucleus, producing actin filaments under conditions where spontaneous polymerization is negligible. These actions of synapsin I are markedly affected by site-specific phosphorylation of the protein. An original transformation of the fluorescence data, which estimates the disappearance rate of actin monomer toward the critical concentration, is presented and shown to be of general usefulness for the study of actin-binding proteins.

Actins↗
PubMed1992

Antibodies to synaptophysin interfere with transmitter secretion at neuromuscular synapses.

The involvement of synaptophysin, a synaptic vesicle-specific protein, in transmitter release at neuromuscular synapses was studied by intracellular application of synaptophysin antibodies into presynaptic neurons. Polyclonal antibodies or their Fab fragments were loaded into spinal neurons by injection into one of the early blastomeres of Xenopus embryos 1 day prior to culturing or, alternatively, directly through a whole-cell recording pipette at the soma of cultured neurons. At synapses made by antibody-loaded neurons in culture, the spontaneous synaptic currents showed marked reduction in frequency without significant change in their mean amplitude. The impulse-evoked synaptic currents showed reduced amplitude and increased failure rate. These results suggest that interference with synaptophysin function by antibody binding inhibits transmitter secretion.

Animals↗
PubMed1992

Synapsin I partially dissociates from synaptic vesicles during exocytosis induced by electrical stimulation.

The distribution of the synaptic vesicle-associated phosphoprotein synapsin I after electrical stimulation of the frog neuromuscular junction was investigated by immunogold labeling and compared with the distribution of the integral synaptic vesicle protein synaptophysin. In resting terminals both proteins were localized exclusively on synaptic vesicles. In stimulated terminals they appeared also in the axolemma and its infoldings, which however exhibited a lower synapsin I/synaptophysin ratio with respect to synaptic vesicles at rest. The value of this ratio was intermediate in synaptic vesicles of stimulated terminals, and an increased synapsin I labeling of the cytomatrix was observed. These results indicate that synapsin I undergoes partial dissociation from and reassociation with synaptic vesicles, following physiological stimulation, and are consistent with the proposed modulatory role of the protein in neurotransmitter release.

Animals↗
PubMed1992

Interaction of free and synaptic vesicle-bound synapsin I with F-actin.

Synapsin I is a neuron-specific phosphoprotein that binds to small synaptic vesicles and F-actin in a phosphorylation-dependent fashion. We have found that dephosphorylated synapsin I induces a dose-dependent increase in the number of actin filaments, which at high ionic strength is abolished by synapsin I phosphorylation. The increase in filament number appears to be due to a nucleating effect of synapsin I and not to a barbed-end capping/severing activity. Synaptic vesicle-bound synapsin I was as effective as free synapsin I in increasing the number of filaments. These data support the view that synapsin I is involved in the regulation of the dynamics of the actin-based network during the exo-endocytotic cycle.

Actin Cytoskeleton↗
PubMed1991

Computer modeling of synapsin I binding to synaptic vesicles and F-actin: implications for regulation of neurotransmitter release.

Synapsin I is a neuron-specific phosphoprotein that binds to small synaptic vesicles and actin filaments in a phosphorylation-dependent fashion. It has been hypothesized that dephosphorylated synapsin I inhibits neurotransmitter release either by forming a cage around synaptic vesicles (cage model) or by anchoring them to the F-actin cytoskeleton of the nerve terminal (crosslinking model). Computer modeling was performed with the aim of testing the impact of phosphorylation on the molecular interactions of synapsin I within the nerve terminal. The results of the simulation experiments demonstrate that in the crosslinking model the phosphorylation of synapsin I causes a severalfold increase in the number of vesicles released from the cytoskeleton and that in the cage model the phosphorylation induces a 2-fold increase in the number of vesicles bearing one or more unsaturated synapsin I binding sites. These data are compatible with the view that the function of synapsin I in the short-term regulation of neurotransmitter release is to induce a phosphorylation-dependent transition of synaptic vesicles from a "reserve pool" to a readily "releasable pool" of vesicles.

Actins↗
PubMed1990

The synapsins and the regulation of synaptic function.

Synapsin I and II are a family of synaptic vesicle-associated phosphoproteins involved in the short-term regulation of neurotransmitter release. In this review, we discuss a working model for the molecular mechanisms by which the synapsins act. We propose that synapsin I links synaptic vesicles to actin filaments in the presynaptic nerve terminal and that these interactions are modulated by the reversible phosphorylation of synapsin I through various signal transduction pathways. The high degree of homology between the synapsins suggests that some of the functional properties of synapsin I are also shared by synapsin II.

Animals↗
PubMed1990

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.

Animals↗
PubMed1990

The synapsins.

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Animals↗
PubMed1989

Synapsin I, a neuron-specific phosphoprotein interacting with small synaptic vesicles and F-actin.

Synapsin I is a neuron-specific phosphoprotein which is a substrate for cAMP- and Ca2+/calmodulin-dependent protein kinases. It is specifically localized to the cytoplasmic side of small synaptic vesicles. The interaction of synapsin I with the synaptic vesicle membrane is complex in nature, since it is modulated by phosphorylation and involves binding of different domains of the molecule to phospholipid and protein components of synaptic vesicles. Synapsin I is also able to interact with actin filaments in a phosphorylation-dependent manner. Because of these properties, it has been hypothesized that synapsin I acts as a dynamic link between synaptic vesicles an the actin meshwork of the nerve terminal, thereby modulating the release of neurotransmitter.

Actins↗
PubMed1989

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.

Animals↗
PubMed1989

Characterization of synapsin I fragments produced by cysteine-specific cleavage: a study of their interactions with F-actin.

Synapsin I is a neuron-specific phosphoprotein that is concentrated in the presynaptic nerve terminal in association with the cytoplasmic surface of synaptic vesicles. It has been demonstrated to bundle F-actin in a phosphorylation-dependent manner in vitro, a property consistent with its proposed role in linking synaptic vesicles to the cytoskeleton and its involvement in the regulation of neurotransmitter release. Synapsin I is composed of two distinct domains, a COOH terminal, collagenase-sensitive, hydrophilic, and strongly basic tail region, and an NH2 terminal, collagenase-resistant head region relatively rich in hydrophobic amino acids. To elucidate the structural basis for the interactions between synapsin I and F-actin and how it relates to other characteristics of synapsin I, we have performed a structure-function analysis of fragments of synapsin I produced by cysteine-specific cleavage with 2-nitro-5-thiocyanobenzoic acid. The fragments were identified and aligned with the parent molecule using the deduced primary structure of synapsin I and the known phosphorylation sites as markers. We have purified these fragments and examined their interactions with F-actin. Two distinct fragments, a 29-kD NH2-terminal fragment and a 15-kD middle fragment, were shown to contain F-actin binding sites. A 51/54-kD middle/tail fragment retained the F-actin binding and bundling activity of synapsin I, but the isolated tail fragment did not retain either activity. In contrast to phosphorylation of sites two and three in intact synapsin I, which abolishes F-actin bundling activity, phosphorylation of these sites in the middle/tail fragment failed to abolish this activity. In conclusion, three domains of synapsin I appear to be involved in F-actin binding and bundling.

Actins↗
PubMed1988

Localization of synapsin I at the frog neuromuscular junction.

We report here the results of immunocytochemical and biochemical studies on the localization of synapsin I, a nerve terminal--specific phosphoprotein, at the frog neuromuscular junction. Our results show that in this in situ synapse synapsin I is concentrated in the presynaptic compartment, where it appears to be associated with the synaptic vesicle membrane. Double immunoprecipitated synapsin I from homogenates of frog cutaneous pectoris muscles could be phosphorylated by the catalytic subunit of cyclic adenosine 5'-monophosphate-dependent protein kinase after gel electrophoresis and blotting onto nitrocellulose and could be subsequently identified by an immunoperoxidase technique. Experiments carried out in frog brain preparations indicate that frog synapsin I, like the mammalian protein, can be phosphorylated at different sites by exogenously added catalytic subunit of cyclic adenosine 5'-monophosphate-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase II prepared from mammalian sources. The phosphorylation sites of frog synapsin I, as judged by phosphopeptide mapping, are somewhat different from those of mammalian synapsin I. The study of synapsin I and of the regulation of its state of phosphorylation at the neuromuscular junction may provide important information on its role in synaptic function, since at the present time this is one of the few systems in which a correlation among biochemical, immunocytochemical and electrophysiological results is possible.

Animals↗