NOVEL FUNCTIONS OF THE SNARE PROTEIN SNAP29IN MEMBRANE TRAFFICKING AND CELL DIVISION

Vesicular trafficking within cells is an important process for tissue development and homeostasis. A key step of vesicular trafficking is the fusion between two membranes, a process in which SNARE (Soluble NSF Attachment Protein Receptors) proteins play a fundamental role. SNAP29 (SyNaptosomal Associated Proteins 29) is a ubiquitous SNARE, regulating membrane fusion in different trafficking compartments and in different contexts in non dividing cells. We isolated a loss of function mutant in usnp, the gene encoding the Drosophila homolog of the human protein SNAP29 (Snap29 hereafter), that, when made homozygous in developing epithelial organs, disrupts epithelial architecture. In vivo, we find that Snap29 interacts with multiple SNARE proteins, localizes to a number of trafficking organelles, and is required for proper Golgi Apparatus morphology. In addition, we show that Snap29 is required for fusion of autophagosomes with lysosomes together with Syx17 and Vamp7, and that lack of Snap29 results in excess secretion, suggesting that Snap29 might act negatively in regulation of vesicle fusion at the plasma membrane. Interestingly, at the onset of mitosis, when trafficking compartments re-shape to allow the formation of the mitotic spindle, Snap29 is found at the outer KT in Drosophila S2 cells and localizes at spindle microtubules and centrosomes in mammalian cells. Depletion of Snap29 in Drosophila and mammalian cells leads to spindle assembly defects, associated to pro-metaphase delay in mammalian cells, and to the formation of daughter cells containing mininuclei. Mechanistically, lack of SNAP29 correlates with absence at KT of ZWINT-1 and ZWILCH, a component of RZZ complex, and with weak KTs-MTs attachments. In addition, we find that SPINDLY, the adaptor for recruitment to KTs of dynein/dynactin and MAD1, a component of the Spindle Assembly Checkpoint machinery, fail to be removed from KTs at the end of metaphase in SNAP29 depleted mammalian cells forced to reassemble the spindle after treatment with microtubules depolymerization drug. Finally, we show that cell division is impaired in Snap29 mutant tissues in vivo, that autophagy defects are not the cause of the altered epithelial tissues architecture in Snap29 mutants and that the trafficking and cell division function of Snap29 are molecularly distinct. All together our findings support a role of Snap29 at key steps of membrane trafficking and in cell division. Our study contribute to shed light on the pathogenesis of CEDNIK, a human congenital syndrome caused by SNAP29 inactivation. In addition to this, we propose that the function of SNAP29 in cell division might be evolutionarly related to that of complexes tethering MTs to vesicular organelles in interphase. We surmise that such function could be potentially relevant to development of aneuploidy in tumor-like masses.