Sergey V. Popov

Synaptotagmin: A Calcium-Sensitive Inhibitor of Exocytosis?

Sergey V. Popov and Mu-ming Poo Department of Biological Sciences Columbia University New York, New York 16927 The basic steps of excitation-secretion coupling at the presynaptic nerve terminal are well established. Transmit- ters are stored in synaptic vesicles, some of which are docked at specialized sites along the cytoplasmic face of the plasma membrane. An action potential arriving at the nerve terminal opens Ca*+ channels in the plasma mem- brane and results in a local and transient elevation of cyto- solic Ca*+ concentration near the membrane, which in turn triggers the exocytosis of docked synaptic vesicles and the release of transmitters. While rapid advances have been made in the biochemical characterization and clon- ing of various presynaptic proteins (Siidhof and Jahn, 1991; Bennett and Scheller, 1993) our understanding of the functional role of these proteins has lagged. A case in point is an integral membrane protein of synaptic vesicles, synaptotagmin (~65). Recent findings have provided seemingly contradictory evidence for its functional impor- tance in transmitter secretion. This review aims to summa- rize these findings and to search for a model that may resolve the issue. Synaptotagmin as a Docking and Fusion Protein Synaptotagmin is an abundant synaptic vesicle protein in neurons and some endocrine cells (Matthew et al., 1961). It has a single transmembrane domain with the amino ter- minus on the intravesicular side. In vitro biochemical find- ings suggest that synaptotagmin may interact with plasma membrane components. Antibodies to synaptotagmin co- precipitate N-type Ca*+ channels (Leveque et al., 1992). Synaptotagmin also interacts with the plasma membrane proteins syntaxin and neurexin, and syntaxin binds N-type Ca2+ channels (Ushkaryov et al., 1992; Bennett et al., 1992). These findings suggest that synaptotagmin may play a role in the docking of synaptic vesicles at presynap- tic release sites. The cytoplasmic carboxyl terminus of synaptotagmin has two domains (C2 repeats), which are highly conserved among all species from which synaptotagmin has been cloned. Similar domains in protein kinase C and in phos- pholipase A2 are responsible for Ca*dependent binding of these proteins to acidic phospholipids, phosphatidylser- ine in particular. Synaptotagmin also binds through C2 repeats to phosphatidylserine upon elevation of Ca*‘ . Such direct interaction with membrane lipids prompted the suggestion that synaptotagmin may be a Ca2+-dependent fusion protein (Brose et al., 1992). Taken together, these studies suggest an attractive model for the function of synaptotagmin: Synaptotagmin docks synaptic vesicles in the vicinity of Ca2+ channels (by binding either directly or indirectly through syntaxin) and promotes exocytotic fusion upon local elevation of cytosolic Caw. Synaptotagmin thus plays both docking

Calcium-dependent transmitter secretion from fibroblasts: Modulation by synaptotagmin I

Following endocytic uptake of acetylcholine (ACh), CHO fibroblasts exhibit Ca(2+)-dependent spontaneous quantal ACh release and depolarization-evoked ACh release, as detected by a whole-cell voltage-clamped myocyte in contact with the fibroblast. CHO fibroblasts transfected with synaptotagmin I, an integral membrane protein of synaptic vesicles, showed a reduced spontaneous quantal ACh release and an enhanced Ca(2+)-evoked ACh release, as compared with control cells. Biochemical and ultrastructural studies of endocytic activity using horseradish peroxidase as a marker further confirmed the inhibitory action of synaptotagmin I on spontaneous vesicular exocytosis and on elevated exocytosis induced by Ca2+. Through inhibition of exocytosis at the resting intracellular concentration of Ca2+ and removal of the inhibition upon depolarization-induced Ca2+ entry, synaptotagmin I could enhance the efficiency of excitation-secretion coupling.

Expression of synapsin i correlates with maturation of the neuromuscular synapse

Synapsins are a family of neuron-specific phosphoproteins that are localized within the presynaptic terminals in adult brain. Previous work has demonstrated that introduction of exogenous synapsins I(a + b) or IIa into Xenopus spinal neurons promoted maturation of the neuromuscular synapse in a nerve-muscle co-culture system. We have now studied the expression of endogenous Xenopus synapsin I during synaptic maturation in vivo and in culture, using a polyclonal antibody raised against Xenopus synapsin I. Immunoprecipitation experiments indicated that synapsin I was not detectable during the early phase of synaptogenesis in vivo, and exhibited a marked increase during the period of synaptic maturation. In contrast, the expression of synaptophysin, another synaptic vesicle protein, was detected at the start of nervous system formation, and remained at a high level thereafter. Similar expression profiles for the two proteins were also observed in immunocytochemical studies of Xenopus spinal neurons in culture: intense staining of synaptophysin was found on the first day, while synapsin I was not detected until after three days in culture. The expression of synapsin I correlated very well with the appearance of a bell-shaped amplitude distribution of spontaneous synaptic currents, a physiological parameter which reflects functional maturation of the neuromuscular synapse. In one-day-old cultures grown in the absence of laminin, an extracellular matrix protein known to be present at the neuromuscular junction, the amplitude distribution of virtually all synapses was skewed towards smaller values. In contrast, when laminin was used as a culture substrate, many synapses exhibited a bell-shaped amplitude distribution. Laminin treatment also induced synapsin I expression in one-day-old cultures. These results suggest that the expression of endogenous synapsin I may regulate maturation at neuromuscular synapses.

Generation of cell processes in a high frequency electric field

Abstract A new experimental system which allows one to apply force to the plasma membrane of substrate-attached cells is described. The force, which is generated in a special chamber by an alternating current electric field of high frequency, pulls the membrane outward, generating cell processes. The phenomenon of cell outgrowth formation by this membrane-applied force is investigated. The experimental system developed also provides an opportunity to study the mechanical properties of various parts of substrate-attached cells.

Constitutive Secretion of Exogenous Neurotransmitter by Nonneuronal Cells: Implications for Neuronal Secretion

Fibroblasts in cell culture were loaded with exogenous neurotransmitter acetylcholine (ACh). ACh secretion from loaded cells was detected by whole-cell patch clamp recordings from Xenopus myocytes manipulated into contact with ACh-loaded cells. Two different approaches were used for ACh loading. In the first approach, fibroblasts were incubated in the culture medium containing ACh. Recordings from myocytes revealed fast inward currents that resemble miniature endplate currents found at neuromuscular synapses. The currents observed in recordings from myocytes were due to exocytosis of ACh-containing vesicles. Although exogenous ACh penetrated through the plasma membrane of fibroblasts during incubation and was present in the cytoplasm at detectable levels, cytoplasmic ACh did not contribute to the quantal ACh secretion. In the second approach, exogenous ACh was loaded into the cytoplasm of fibroblasts by microinjection. Under these experimental conditions, fibroblasts also exhibited spontaneous quantal ACh secretion. Analysis of the exocytotic events in fibroblasts following two different protocols of ACh loading revealed that the vesicular compartments responsible for uptake of exogenous ACh are associated with the endocytic recycling pathway. Extrapolation of our results to neuronal cells suggest that in cholinergic neurons, in addition to genuine synaptic vesicles, ACh can be secreted by the vesicles participating in endosomal membrane recycling.

Formation of cell protrusions by an electric field : a thermodynamic analysis

This work gives a thermodynamic analysis of outgrowth extraction from the cell body by a pulling force. The results are applied for a case when the pulling force is generated by an external high-frequency electric field. Two equilibrium conditions are analyzed: internal equilibrium of an outgrowth and equilibrium between the outgrowth and the cell body. In both cases the stability of feasible equilibrium states was studied. The work shows that the curvature of an outgrowth equilibrated with a pulling electric force depends on the squared amplitude of the electric field E02, on the outgrowth length l and on the transmembrane pressure differential ΔP, and that at a sufficiently large transmembrane pressure differential the cylindrical form of the outgrowth loses its stability. Long outgrowths are more stable than short ones. The minimal value of critical pressure differential was estimated. The work also shows that outgrowth extraction from the cell body requires that the applied force exceeds a critical value below which no outgrowth is formed. The value of the electric field at which outgrowth formation is feasible was estimated.

Cell Protrusion Formation by External Force

A method of applying a mechanical force to the plasma membrane of cells is described. The force is generated by an alternating current (AC) electrical field (EF) of high frequency. It is applied to the plasma membrane and is directed outwards. This force was sufficient to generate morphologically normal cell protrusions in mouse embryo and 3T3 fibroblasts. Specific inhibitors of actin polymerization were not able to prevent generation of protrusions in an electrical field. Organization of the cytoskeleton inside the processes was examined using a platinum replica method. Bundles of microfilaments morphologically similar to those observed during normal physiological spreading were found in electrical field–generated protrusions. Local application of electrical field to polarized cells demonstrated that different areas of the cell surface differ in their ability to form processes under the action of membrane-applied force.