Synaptotagmin 11 interacts with components of the RNA-induced silencing complex RISC in clonal pancreatic ß-cells.

Synaptotagmins are two C2 domain-containing transmembrane proteins. The function of calcium-sensitive members in the regulation of post-Golgi traffic has been well established whereas little is known about the calcium-insensitive isoforms constituting half of the protein family. Novel binding partners of synaptotagmin 11 were identified in ß-cells. A number of them had been assigned previously to ER/Golgi derived-vesicles or linked to RNA synthesis, translation and processing. Whereas the C2A domain interacted with the Q-SNARE Vti1a, the C2B domain of syt11 interacted with the SND1, Ago2 and FMRP, components of the RNA-induced silencing complex (RISC). Binding to SND was direct via its N-terminal tandem repeats. Our data indicate that syt11 may provide a link between gene regulation by microRNAs and membrane traffic.

The calcium-sensing protein synaptotagmin 7 is expressed on different endosomal compartments in endocrine, neuroendocrine cells or neurons but not on large dense core vesicles.

Synaptotagmin (syt) isoforms function as calcium sensor in post-Golgi transport although the precise transport step and compartment(s) concerned are still not fully resolved. As syt7 has been proposed to operate in lysosomal exocytosis and in exocytosis of large dense core vesicles (LDCVs), we have addressed the distribution of endogenous syt7 in insulin-secreting cells. These cells express different syt7 isoforms comparable to neurons. According to subcellular fractionation and quantitative confocal immunocytochemistry, syt7 is not found on LDCVs or on synaptic-like microvesicles but colocalizes with Rab7 on endosomes and to structures near to or at the plasma membrane. Similarly, endogenous syt7 was absent from LDCVs in pheochromocytoma PC12 cells. In contrast, syt7 localised to lysosomes in both, PC12 cells and hippocampal neurons. In conclusion, endogenous syt7 shows a wider distribution than previously reported but does not qualify as vesicular calcium sensor in SLMV or LDCV exocytosis according to its localisation.

Distinct roles of the C2A and the C2B domain of the vesicular Ca2+ sensor synaptotagmin 9 in endocrine beta-cells.

Synaptotagmins form a family of calcium-sensor proteins implicated in exocytosis, and these vesicular transmembrane proteins are endowed with two cytosolic calcium-binding C2 domains, C2A and C2B. Whereas the isoforms syt1 and syt2 have been studied in detail, less is known about syt9, the calcium sensor involved in endocrine secretion such as insulin release from large dense core vesicles in pancreatic beta-cells. Using cell-based assays to closely mimic physiological conditions, we observed SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor)-independent translocation of syt9C2AB to the plasma membrane at calcium levels corresponding to endocrine exocytosis, followed by internalization to endosomes. The use of point mutants and truncations revealed that initial translocation required only the C2A domain, whereas the C2B domain ensured partial pre-binding of syt9C2AB to the membrane and post-stimulatory localization to endosomes. In contrast with the known properties of neuronal and neuroendocrine syt1 or syt2, the C2B domain of syt9 did not undergo calcium-dependent membrane binding despite a high degree of structural homology as observed through molecular modelling. The present study demonstrates distinct intracellular properties of syt9 with different roles for each C2 domain in endocrine cells.

Synaptotagmin 8 is expressed both as a calcium-insensitive soluble and membrane protein in neurons, neuroendocrine and endocrine cells.

Synaptotagmins (syt) form a large family of transmembrane proteins and some of its isoforms are known to regulate calcium-induced membrane fusion during vesicular traffic. In view of the reported implication of the isoform syt8 in exocytosis we investigated the expression, localisation and calcium-sensitivity of syt8 in secretory cells. An immunopurified antipeptide antibody was generated which is directed against a C-terminal sequence and devoid of crossreactivity towards syt1 to 12. Subcellular fractionation and immunocytochemistry revealed two forms of synaptotagmin 8 (50 and 40 kDa). Whereas the 40-kDa was present in the cytosol in brain, in PC12 and in clonal beta-cells, the 50-kDa form was localised in very typical clusters and partially colocalised with the SNARE protein Vti1a. Moreover, in primary hippocampal neurons syt8 was only found within the soma. Amplification of syt8 by RT-PCR indicated that the observed protein variants were not generated by alternative splicing of the 6th exon and are most likely linked to variations in the N-terminal region. In contrast to the established calcium sensor syt2, endogenous cytosolic syt8 and transiently expressed syt8-C2AB-eGFP did not translocate upon a raise in cytosolic calcium in living cells. Syt8 is therefore not a calcium sensor in exocytotic membrane fusion in endocrine cells.