v-SNAREs control exocytosis of vesicles from priming to fusion

SNARE proteins (soluble NSF-attachment protein receptors) are thought to be central components of the exocytotic mechanism in neurosecretory cells, but their precise function remained unclear. Here, we show that each of the vesicle-associated SNARE proteins (v-SNARE) of a chromaffin granule, synaptobrevin II or cellubrevin, is sufficient to support Ca(2+)-dependent exocytosis and to establish a pool of primed, readily releasable vesicles. In the absence of both proteins, secretion is abolished, without affecting biogenesis or docking of granules indicating that v-SNAREs are absolutely required for granule exocytosis. We find that synaptobrevin II and cellubrevin differentially control the pool of readily releasable vesicles and show that the v-SNARE's amino terminus regulates the vesicle's primed state. We demonstrate that dynamics of fusion pore dilation are regulated by v-SNAREs, indicating their action throughout exocytosis from priming to fusion of vesicles.     

C3G/Rapgef1 Is Required in Multipolar Neurons for the Transition to a Bipolar Morphology during Cortical Development

The establishment of a polarized morphology is essential for the development and function of neurons. During the development of the mammalian neocortex, neurons arise in the ventricular zone (VZ) from radial glia cells (RGCs) and leave the VZ to generate the cortical plate (CP). During their migration, newborn neurons first assume a multipolar morphology in the subventricular zone (SVZ) and lower intermediate zone (IZ). Subsequently, they undergo a multi-to-bipolar (MTB) transition to become bipolar in the upper IZ by developing a leading process and a trailing axon. The small GTPases Rap1A and Rap1B act as master regulators of neural cell polarity in the developing mouse neocortex. They are required for maintaining the polarity of RGCs and directing the MTB transition of multipolar neurons. Here we show that the Rap1 guanine nucleotide exchange factor (GEF) C3G (encoded by the Rapgef1 gene) is a crucial regulator of the MTB transition in vivo by conditionally inactivating the Rapgef1 gene in the developing mouse cortex at different time points during neuronal development. Inactivation of C3G results in defects in neuronal migration, axon formation and cortical lamination. Live cell imaging shows that C3G is required in cortical neurons for both the specification of an axon and the initiation of radial migration by forming a leading process.     

Effect of Latrotoxin-Like Protein on Spontaneous Postsynaptic Activity in Hippocampal Cell Cultures

-Latrotoxin, an active component of black widow spider venom, is known to enhance spontaneous neurotransmitter release. In cultured rat hippocampal neurons, we studied the effects of latrotoxin-like protein (protein purified from the bovine brain and exhibiting some functional properties similar to those of -latrotoxin) on spontaneous GABA-ergic inhibitory currents (IPSC). Latrotoxin-like protein was found to dramatically increase the frequency of spontaneous IPSC recorded in cell cultures of dissociated hippocampal neurons in the presence of tetrodotoxin. Possible mechanisms of the action of latrotoxin-like protein on transmitter release are discussed.     

Disturbance of synaptic vesicle recycling resulting from deletion of a mammalian actin-binding protein, mAbp1

Physiological and ultrastructural studies of synapses between hippocampal neurons of animals with knock-out of a mammalian actin-binding protein, mAbp1, demonstrated that recycling of synaptic vesicles undergoes, in this case, significant modifications. Thus, mAbp1 is rather important from this aspect, which can be related to the noticeable role of actin in clathrin-mediated endocytosis of synaptic vesicles. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 390–391, July–October, 2007.     

Some characteristics of fusogenic activity of cytoplasmic latrotoxin-like brain protein

We studied fusion of negatively charged artificial phospholipid vesicles (liposomes) in the presence of two electrophoretic fractions (molecular mass of about 90 and 50 kdalton) of latrotoxin-like (L) protein. It was shown that both fractions are capable of causing liposome fusion in acidic media. Treatment of native preparations of L protein with NEM depressed their fusogenic activity. Some common characteristics of L protein and well-known fusogenic proteins allow us to account for the possibility of participation of L protein in fusion of the membranes in the cell.     

Membrane fusion in model systems

The mechanism of biological membrane fusion was studied with the use of a fluorescent dye, R18. It was shown that ATP-induced membrane fusion is supporessed by calcium/calmodulin-dependent protein kinase inhibitors. L protein changed the character of fusion of different types of membranes.     

SH3P7/mAbp1 deficiency leads to tissue and behavioral abnormalities and impaired vesicle transport

The intracellular adaptor protein SH3P7 is the mammalian ortholog of yeast actin-binding protein 1 and thus alternatively named as mAbp1 (or HIP55). Structural properties, biochemical analysis of its interaction partners and siRNA studies implicated mAbp1 as an accessory protein in clathrin-mediated endocytosis (CME). Here, we describe the generation and characterization of mice deficient for SH3P7/mAbp1 owing to targeted gene disruption in embryonic stem cells. Mutant animals are viable and fertile without obvious deficits during the first weeks of life. Abnormal structure and function of organs including the spleen, heart, and lung is observed at about 3 months of age in both heterozygous and homozygous mouse mutants. A moderate reduction of both receptor-mediated and synaptic endocytosis is observed in embryonic fibroblasts and in synapses of hippocampal neurons, respectively. Recycling of synaptic vesicles in hippocampal boutons is severely impaired and delayed four-fold. The presynaptic defect of SH3P7/mAbp1 mouse mutants is associated with their constricted physical capabilities and disturbed neuromotoric behaviour. Our data reveal a nonredundant role of SH3P7/mAbp1 in CME and places its function downstream of vesicle fission.     

Key role of clathrin-mediated endocytosis in synaptic vesicle recycling

Using novel fluorescent markers, virus-induced modulation of amphiphysin 1 expression, and electron microscopy, we demonstrated that clathrin-mediated endocytosis is the main mechanism of synaptic vesicle retrieval; a hypothesis on the role of a fast “kiss-and-run” mechanism has not been supported. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 388–389, July–October, 2007.     

ATPase Activity of Latrotoxin-Like Protein

We investigated the ability of latrotoxin-like protein for ATP hydrolysis. It was shown that latrotoxin-like protein possesses ATPase activity, which increases with shift of pH toward alkaline values. A comparison of the enzymatic activity of latrotoxin-like protein at different pH values showed that the kinetic parameters of ATP hydrolysis depend on pH of the incubation medium. It is suggested that the characteristics of latrotoxin-like protein are similar to those of proteins from the AAA-ATPase family, which mediate fusion of cell membranes.