Synaptic vesicles fuse at active zone (AZ) membranes where Ca2+ channels are clustered and that are typically decorated by electron-dense projections. Recently, mutants of the Drosophila melanogaster ERC/CAST family protein Bruchpilot (BRP) were shown to lack dense projections (T-bars) and to suffer from Ca2+ channel–clustering defects. In this study, we used high resolution light microscopy, electron microscopy, and intravital imaging to analyze the function of BRP in AZ assembly. Consistent with truncated BRP variants forming shortened T-bars, we identify BRP as a direct T-bar component at the AZ center with its N terminus closer to the AZ membrane than its C terminus. In contrast, Drosophila Liprin-α, another AZ-organizing protein, precedes BRP during the assembly of newly forming AZs by several hours and surrounds the AZ center in few discrete punctae. BRP seems responsible for effectively clustering Ca2+ channels beneath the T-bar density late in a protracted AZ formation process, potentially through a direct molecular interaction with intracellular Ca2+ channel domains.

Aggregation of a-synuclein (aS) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of aS is largely unknown. We demonstrate with in vitro vesicle fusion experiments that aS has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, aS binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age-dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous aS. In contrast, siRNA-mediated downregulation of aS results in elongated mitochondria in cell culture. aS can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, aS prevents fusion of differently labelled mitochondrial populations. Thus, aS inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of aS is rescued by coexpression of PINK1, parkin or DJ-1 but not the PD-associated mutations PINK1 G309D and parkin D1-79 or by DJ-1 C106A.

The relation of a-synuclein (aS) aggregation to Parkinson's disease (PD) has long been recognized, but the mechanism of toxicity, the pathogenic species and its molecular properties are yet to be identified. To obtain insight into the function different aggregated aS species have in neurotoxicity in vivo, we generated aS variants by a structurebased rational design. Biophysical analysis revealed that the aS mutants have a reduced fibrillization propensity, but form increased amounts of soluble oligomers. To assess their biological response in vivo, we studied the effects of the biophysically defined pre-fibrillar aS mutants after expression in tissue culture cells, in mammalian neurons and in PD model organisms, such as Caenorhabditis elegans and Drosophila melanogaster. The results show a striking correlation between aS aggregates with impaired b-structure, neuronal toxicity and behavioural defects, and they establish a tight link between the biophysical properties of multimeric aS species and their in vivo function.