Rabbit Anti-Human Synaptotagmin (a.a. 276-325) Polyclonal antibody

Membrane contact sites (MCS) have increasingly received attention because of their general role in a number of important cellular processes. SYNAPTOTAGMIN 1 (SYT1) is a tethering factor connecting the endoplasmic reticulum (ER) and the plasma membrane (PM) in plant cells. Confocal microscopy using fluorescent protein fusion is an indispensable tool for studying protein localisation and functions. However, several studies have reported that fluorescent protein dimerisation affects the subcellular localisation of proteins tagged by the fluorescent protein. Here, we investigate the effects of fluorescent protein dimerisation by comparing the subcellular localisation of SYT1 fused with a synthetic GFP (SYT1-sGFP) and SYT1 fused with a monomeric GFP (SYT1-mGFP). SYT1-mGFP was confined to specific domains in the ER, whereas SYT1-sGFP spread along the ER when transiently overexpressed. SYT1-localised regions were suggested to correspond to ER-PM contact sites because of its immobility. Similar results were obtained in the transgenic Arabidopsis, even though SYT1-sGFP and SYT1-mGFP were expressed at comparable levels. It is suggested that SYT1-mGFP more accurately reproduced SYT1 localisation in intact cells because the proportion of persistent area in the ER was more similar between the wild type and the plant expressing SYT1-mGFP than between the wild type and the plant expressing SYT1-sGFP. Taken together, these results suggest that the fusion of sGFP makes SYT1-sGFP form excessive ER-PM contact sites in the ER.

It is well-established that syntaxin 1A (Sx1A), SNAP-25 and synaptotagmin (Syt1) either alone or in combination, modify the kinetic properties of voltage-gated Ca2+ channels (VGCCs). The interaction interface resides mainly at the cytosolic II-III domain of the alpha1 subunit of the channels, while Sx1A interacts with the channel also via two highly conserved cysteine residues at the transmembrane domain. In the present study, we characterized Ca2+-independent coupling of the human neuronal P/Q-type calcium channel (Ca(v)2.1) with Sx1A, SNAP-25, Syt1 and synaptobrevin (VAMP) in BAPTA-injected Xenopus oocytes. The co-expression of Ca(v)2.1 with Sx1A, SNAP-25 and Syt1, produced a multiprotein complex with distinctive kinetic properties analogous to the excitosome complexes generated by Ca(v)1.2, Ca(v)2.2 and Ca(v)2.3. The distinct kinetic properties of Ca(v)2.1 acquired by close association with Syt1 and t-SNAREs, suggests that the vesicle is tethered to the neuronal channel and to the exocytotic machinery independently of intracellular Ca2+. To explore the relevance of these interactions to secretion we exploited a BotC1-and a BotA-sensitive secretion system developed for Xenopus oocytes not buffered by BAPTA, in which depolarization-evoked secretion is monitored by a change in membrane capacitance. The reconstituted release mediated by Ca(v)2.1 is consistent with the model in which the VGCC plays a signaling role in triggering release, acting from within the exocytotic complex. The relevance of these results to secretion posits the role of possible rearrangements within the excitosome subsequent to Ca2+ entry, setting the stage for the fusion of channel-tethered-vesicles upon the arrival of an action potential.