complex required for transport; E(spl)mb-HLH, enhancer of split mb, helix-loop-helix; FE, follicular epithelium; histone H3, His3; hop-Stat92E, hopscotch-signal transducer and activator of transcription protein at 92E; GFP, green fluorescent protein; MENE, mutant eye no eclosion; MVB, multivesicular body; N, Notch; NECD, N extracellular domain; NPF, asparagine-proline-phenylalanine; os, outstretched; ref(2)P, refractory to sigma P; Snap29, synaptosomal-associated protein 29 kDa; SNARE, soluble NSF attachment protein receptor; Socs36E, suppressor of cytokine signaling at 36E; Syb, Synaptobrevin; Syx, syntaxin; Vamp, vesicle-associated membrane protein; V-ATPase, vacuolar H C -ATPase; Vps25, vacuolar protein sorting 25; WT, wild type.How autophagic degradation is linked to endosomal trafficking routes is little known. Here we screened a collection of uncharacterized Drosophila mutants affecting membrane transport to identify new genes that also have a role in autophagy. We isolated a loss of function mutant in Snap29 (Synaptosomal-associated protein 29 kDa), the gene encoding the Drosophila homolog of the human protein SNAP29 and have characterized its function in vivo. Snap29 contains 2 soluble NSF attachment protein receptor (SNARE) domains and a asparagine-proline-phenylalanine (NPF motif) at its N terminus and rescue experiments indicate that both SNARE domains are required for function, whereas the NPF motif is in part dispensable. We find that Snap29 interacts with SNARE proteins, localizes to multiple trafficking organelles, and is required for protein trafficking and for proper Golgi apparatus morphology. Developing tissue lacking Snap29 displays distinctive epithelial architecture defects and accumulates large amounts of autophagosomes, highlighting a major role of Snap29 in autophagy and secretion. Mutants for autophagy genes do not display epithelial architecture or secretion defects, suggesting that the these alterations of the Snap29 mutant are unlikely to be caused by the impairment of autophagy. In contrast, we find evidence of elevated levels of hop-Stat92E (hopscotch-signal transducer and activator of transcription protein at 92E) ligand, receptor, and associated signaling, which might underlie the epithelial defects. In summary, our findings support a role of Snap29 at key steps of membrane trafficking, and predict that signaling defects may contribute to the pathogenesis of cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma (CEDNIK), a human congenital syndrome due to loss of Snap29.

Despite extensive study, regulation of membrane trafficking is incompletely understood. In particular, the specific role of SNARE (Soluble NSF Attachment REceptor) proteins for distinct trafficking steps and their mechanism of action, beyond the core function in membrane fusion, are still elusive. Snap29 is a SNARE protein related to Snap25 that gathered a lot of attention in recent years. Here, we review the study of Snap29 and its emerging involvement in autophagy, a self eating process that is key to cell adaptation to changing environments, and in other trafficking pathways. We also discuss Snap29 role in synaptic transmission and in cell division, which might extend the repertoire of SNARE-mediated functions. Finally, we present evidence connecting Snap29 to human disease, highlighting the importance of Snap29 function in tissue development and homeostasis.

The kinetochore is an essential structure that mediates accurate chromosome segregation in mitosis and meiosis. While many of the kinetochore components have been identified, the mechanisms of kinetochore assembly remain elusive. Here, we identify a novel role for Snap29, an unconventional SNARE, in promoting kinetochore assembly during mitosis in Drosophila and human cells. Snap29 localizes to the outer kinetochore and prevents chromosome mis‐segregation and the formation of cells with fragmented nuclei. Snap29 promotes accurate chromosome segregation by mediating the recruitment of Knl1 at the kinetochore and ensuring stable microtubule attachments. Correct Knl1 localization to kinetochore requires human or Drosophila Snap29, and is prevented by a Snap29 point mutant that blocks Snap29 release from SNARE fusion complexes. Such mutant causes ectopic Knl1 recruitment to trafficking compartments. We propose that part of the outer kinetochore is functionally similar to membrane fusion interfaces.

Homozygous mutations in SNAP29, encoding a SNARE protein mainly involved in membrane fusion, cause CEDNIK (Cerebral Dysgenesis, Neuropathy, Ichthyosis and Keratoderma), a rare congenital neurocutaneous syndrome associated with short life expectancy, whose pathogenesis is unclear. Here, we report the analysis of the first genetic model of CEDNIK in zebrafish. Strikingly, homozygous snap29 mutant larvae display CEDNIK-like features, such as microcephaly and skin defects. Consistent with Snap29 role in membrane fusion during autophagy, we observe accumulation of the autophagy markers p62 and LC3, and formation of aberrant multilamellar organelles and mitochondria. Importantly, we find high levels of apoptotic cell death during early development that might play a yet uncharacterized role in CEDNIK pathogenesis. Mutant larvae also display mouth opening problems, feeding impairment and swimming difficulties. These alterations correlate with defective trigeminal nerve formation and excess axonal branching. Since the paralog Snap25 is known to promote axonal branching, Snap29 might act in opposition with, or modulate Snap25 activity during neurodevelopment. Our vertebrate genetic model of CEDNIK extends the description in vivo of the multisystem defects due to loss of Snap29 and could provide the base to test compounds that might ameliorate traits of the disease.

No abstract