Cryo-electron tomography reveals a critical role of RIM1α in synaptic vesicle tethering.

Synaptic vesicles are embedded in a complex filamentous network at the presynaptic terminal. Before fusion, vesicles are linked to the active zone (AZ) by short filaments (tethers). The identity of the molecules that form and regulate tethers remains unknown, but Rab3-interacting molecule (RIM) is a prominent candidate, given its central role in AZ organization. In this paper, we analyzed presynaptic architecture of RIM1α knockout (KO) mice by cryo-electron tomography. In stark contrast to previous work on dehydrated, chemically fixed samples, our data show significant alterations in vesicle distribution and AZ tethering that could provide a structural basis for the functional deficits of RIM1α KO synapses. Proteasome inhibition reversed these structural defects, suggesting a functional recovery confirmed by electrophysiological recordings. Altogether, our results not only point to the ubiquitin-proteasome system as an important regulator of presynaptic architecture and function but also show that the tethering machinery plays a critical role in exocytosis, converging into a structural model of synaptic vesicle priming by RIM1α.

Analyses of the spatiotemporal expression and subcellular localization of liprin-α proteins.

The members of the Liprin-α protein family, Liprin-α1-4, are scaffolding proteins that play important roles in the regulation of synapse assembly and maturation, vesicular trafficking, and cell motility. Recent evidence suggests that despite their high degree of homology, the four isoforms can be differentially regulated and fulfill diverging functions. However, to date their precise regional and subcellular distribution has remained elusive. Here, we examine the spatiotemporal expression patterns of Liprins-α in the rodent by using in situ hybridization, immunoblotting, and immunochemistry of primary cells as well as brain and retina sections. We show that Liprin-α1-4 mRNA and protein are widely expressed throughout the developing and adult central nervous system, with Liprin-α2 and -α3 being the major Liprin-α isoforms in the brain. Our data show that the four Liprin-α proteins differ in their regional distribution, in particular in the hippocampus, the cerebellum, and the olfactory bulb. Liprin-α1 exhibits a unique spatiotemporal expression pattern as its levels decrease during synaptogenesis, and it is the only Liprin-α with substantial non-neuronal expression. Immunocytochemistry of cultured primary neurons with pre- and postsynaptic marker proteins shows all four Liprins-α to be present at synapses and nonsynaptic sites to varying degrees. Together, these results show that neurons in different brain regions express a distinct complement of Liprin-α proteins.

The mouse and human Liprin-alpha family of scaffolding proteins: genomic organization, expression profiling and regulation by alternative splicing.

In the nervous system the Liprin-alpha protein family plays an important role in the regulation of dendrite development, the targeting of photoreceptor axons, and the formation and structure of synapses. To gain a better understanding of Liprin-alpha regulation we have comparatively analyzed the genomic organization of the human and mouse Liprin-alpha genes, characterized the alternative exon use in human and mouse, and studied their expression in adult rodent tissues and brain regions. Our results show that Liprins-alpha1-4 share multiple properties in their genomic structure, exhibit an identical modular organization, and are highly conserved within certain structural domains, indicating strong evolutionary cohesion. We demonstrate that all Liprin-alpha genes are subject to alternative splicing, which is regulated in a developmental manner. Interestingly, regulation via alternative splicing is not conserved between isoforms and across species and represents a post-transcriptional mechanism to independently diversify the properties of the individual isoforms.