Cysteine string proteins.

Cysteine string protein (CSP) was discovered by use of a synapse-specific, monoclonal antibody to screen a cDNA expression library in Drosophila. A vertebrate CSP homolog was later identified and shown to co-purify with synaptic vesicles. CSP-α is now recognized as a membrane constituent of many regulated secretory organelles.

Fast, synchronous neurotransmitter release: Past, present and future.

This mini-review starts with a summary of the crucial contributions Ricardo Miledi made to our understanding of how the action potential triggers fast, synchronous transmitter release. It then transitions to the discovery of synaptotagmin and its role as the exocytotic Ca sensor at nerve terminals. The final section confronts the array of unique models that have been proposed to explain the membrane fusion step of exocytosis.

Insights into the structure and molecular topography of the fatty acylated domain of synaptotagmin-1.

Abundant attention has focused on synaptotagmin's C2 domains, but less is known about the structure and function of its other regions. Here, we synthesized the N-acetylated, C-end amidated and Cys-palmitated peptide (VLTCCFCICK KCLFKKKNKK K) which includes the fatty acylated cysteine residues in the membrane-affiliated domain of synaptotagmin-1. Fourier-transform infrared spectrometry indicated that this peptide's conformation is influenced by environmental polarity.

A Membrane-Fusion Model That Exploits a β-to-α Transition in the Hydrophobic Domains of Syntaxin 1A and Synaptobrevin 2.

Parallel zippering of the SNARE domains of syntaxin 1A/B, SNAP-25, and VAMP/synaptobrevin 2 is widely regarded as supplying the driving force for exocytotic events at nerve terminals and elsewhere. However, in spite of intensive research, no consensus has been reached concerning the molecular mechanism by which these SNARE proteins catalyze membrane fusion. As an alternative to SNARE-based models, a scenario was developed in which synaptotagmin 1 (or, 2) can serve as a template to guide lipid movements that underlie fast, synchronous exocytosis at nerve terminals.

The Structure of the Synaptic Vesicle-Plasma Membrane Interface Constrains SNARE Models of Rapid, Synchronous Exocytosis at Nerve Terminals.

Contemporary models of neurotransmitter release invoke direct or indirect interactions between the Ca sensor, synaptotagmin and the incompletely zippered soluble, N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) complex. However, recent electron microscopic (EM) investigations have raised pragmatic issues concerning the mechanism by which SNAREs trigger membrane fusion at nerve terminals. The first issue is related to the finding that the area of contact between a "fully primed" synaptic vesicle and the plasma membrane can exceed 600 nm.

Synaptotagmins 1 and 2 as mediators of rapid exocytosis at nerve terminals: the dyad hypothesis.

The dyad model was developed to explain the extremely rapid kinetics of synaptic vesicle exocytosis. In contrast to most hypotheses which invoke interactions among synaptotagmins, SNAREs and other regulatory molecules, the dyad model features a quartet of synaptotagmins arrayed at the synaptic vesicle-plasma membrane interface. Ca(2+)-triggered movements of these synaptotagmins initiate a sequence of events culminating in the fusion of the vesicular and plasma membranes.

Functional neuromuscular junctions formed by embryonic stem cell-derived motor neurons.

A key objective of stem cell biology is to create physiologically relevant cells suitable for modeling disease pathologies in vitro. Much progress towards this goal has been made in the area of motor neuron (MN) disease through the development of methods to direct spinal MN formation from both embryonic and induced pluripotent stem cells. Previous studies have characterized these neurons with respect to their molecular and intrinsic functional properties.

Cysteine string protein beta is prominently associated with nerve terminals and secretory organelles in mouse brain.

Cysteine string proteins (CSPs) are associated with regulated secretory organelles in organisms ranging from fruit flies to man. Mammals have three csp genes (alpha, beta and gamma), and previous work indicated that expression of the csp-beta and -gamma genes was restricted to the testes. For the current investigation, antibodies specific for CSP-beta were developed.

A role for myosin 1e in cortical granule exocytosis in Xenopus oocytes.

Xenopus oocytes undergo dynamic structural changes during maturation and fertilization. Among these, cortical granule exocytosis and compensatory endocytosis provide effective models to study membrane trafficking. This study documents an important role for myosin 1e in cortical granule exocytosis.

Myosin-1c couples assembling actin to membranes to drive compensatory endocytosis.

Compensatory endocytosis follows regulated exocytosis in cells ranging from eggs to neurons, but the means by which it is accomplished are unclear. In Xenopus eggs, compensatory endocytosis is driven by dynamic coats of assembling actin that surround and compress exocytosing cortical granules (CGs). We have identified Xenopus laevis myosin-1c (XlMyo1c) as a myosin that is upregulated by polyadenylation during meiotic maturation, the developmental interval that prepares eggs for fertilization and regulated CG exocytosis.

Lithium enhances secretion from large dense-core vesicles in nerve growth factor-differentiated PC12 cells.

Considerable attention has been focused on the therapeutic role of lithium (Li) in bipolar disorders. Although no consensus has emerged, Li presumably influences the behavior of neurons that regulate mood and behavior. Using PC12 cells to study cellular and molecular actions of Li, we previously reported that Li modulates the expression of proteins associated with large dense-core vesicles (LDCVs; organelles typically containing monoamines, neuropeptides and other cargo proteins).

Interaction between constitutively expressed heat shock protein, Hsc 70, and cysteine string protein is important for cortical granule exocytosis in Xenopus oocytes.

In many species, binding of sperm to the egg initiates cortical granule exocytosis, an event that contributes to a sustained block of polyspermy. Interestingly, cortical granule exocytosis can be elicited in immature Xenopus oocytes by the protein kinase C activator, phorbol-12-myristate-13-acetate. In this study, we investigated the role of cysteine string protein (csp) in phorbol-12-myristate-13-acetate-evoked cortical granule exocytosis.

Protein synthesis is required for the transition to Ca(2+)-dependent regulated secretion in progesterone-matured Xenopus oocytes.

Calcium (Ca) ionophores trigger cortical granule exocytosis in progesterone-matured Xenopus oocytes (eggs), but not in immature oocytes. Prior work suggested that this secretory transition involved a Ca-dependent isoform of protein kinase C (PKC). To address this possibility, we treated eggs with several different inhibitors of Ca-dependent PKCs.

Convergent effects of lithium and valproate on the expression of proteins associated with large dense core vesicles in NGF-differentiated PC12 cells.

Lithium and valproate are chemically unrelated compounds that are used to treat manic-depressive illness. Previously, we reported that lithium ions upregulate genes encoding proteins primarily associated with large dense core vesicles (LDCV) in nerve growth factor (NGF)-differentiated PC12 cells, but not in undifferentiated PC12 cells. Moreover, lithium did not alter the expression of proteins associated with small-clear, synaptic-like vesicles (SSV) in these cells.

Dietary lithium induces regional increases of mRNA encoding cysteine string protein in rat brain.

Lithium salts are used to treat manic-depressive disorders; however, the mechanism by which lithium produces its therapeutic benefit remains obscure. The action of lithium may involve alterations of proteins important for regulating synaptic function. In this context, we observed recently that lithium at therapeutically relevant concentrations enhanced expression of cysteine string protein (csp) at the level of both mRNA and protein, in cell culture and in rat brain.

Lithium ions modulate the expression of VMAT2 in rat brain.

Recent work has indicated that lithium (at 1 mM, a concentration that is efficacious in the treatment of manic-depressive disorders) modulates the level of vesicular monoamine transporter 1 (VMAT1) mRNA in PC12 cells as a function of the differentiation status of these cells. To ascertain whether VMAT expression in neurons is sensitive to lithium, in vivo, rats were fed a lithium-supplemented diet for 21 days (which raised serum lithium to 0.98+/-0.

Nerve growth factor signals via preexisting TrkA receptor oligomers.

Nerve growth factor (NGF) promotes neuronal survival and differentiation by activating TrkA receptors. Similar to other receptor tyrosine kinases, ligand-induced dimerization is thought to be required for TrkA receptor activation. To study this process, we expressed TrkA receptors in Xenopus laevis oocytes and analyzed their response to NGF by using a combination of functional, biochemical, and structural approaches.

Activation of protein kinase Ceta triggers cortical granule exocytosis in Xenopus oocytes.

Previous work has shown that phorbol esters or diacylglycerol trigger cortical granule exocytosis in Xenopus oocytes. We sought to identify the isoform(s) of protein kinase C (PKC) that mediate(s) this regulated secretory event. Because this process is initiated by lipid activators of PKC but is independent of calcium ions, we focused on the family of novel (calcium-independent) PKCs.