The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca2+ sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a large cytoplasmic domain that contains two C2 domains, C2A and C2B. Multiple Ca2+ ions bind to the membrane proximal C2A domain. However, it is not known whether the C2B domain also functions as a Ca2+-sensing module. Here, we report that Ca2+ drives conformational changes in the C2B domain of synaptotagmin and triggers the homo- and hetero-oligomerization of multiple isoforms of the protein. These effects of Ca2+ are mediated by a set of conserved acidic Ca2+ ligands within C2B; neutralization of these residues results in constitutive clustering activity. We addressed the function of oligomerization using a dominant negative approach. Two distinct reagents that block synaptotagmin clustering potently inhibited secretion from semi-intact PC12 cells. Together, these data indicate that the Ca2+-driven clustering of the C2B domain of synaptotagmin is an essential step in excitation-secretion coupling. We propose that clustering may regulate the opening or dilation of the exocytotic fusion pore.

The role of myofibroblasts in vocal fold scarring has not been extensively studied partly due to a lack of a robust in vitro model. The objective of this investigation was to develop and characterize a myofibroblast in vitro model that could be utilized to investigate the molecular mechanism of myofibroblast differentiation and function in injured vocal fold tissue. Differentiation of human primary vocal fold fibroblasts (hVFF) to myofibroblasts was stimulated using 5, 10, or 20 ng/ml of recombinant transforming growth factor beta-1 (TGF-β1). Cultures were analyzed using immunofluorescence and western blotting, with an anti-alpha smooth muscle actin (α-SMA) antibody as a myofibroblast marker. Normal rabbit vocal folds were treated with 10 ng/ml of TGF-β1 for 7 days for in vivo corroboration. The effects of interleukin-6 (IL-6) and hepatocyte growth factor (HGF) on myofibroblast differentiation were studied using western blots. hVFF demonstrated positive α-SMA labeling in 10 and 20 ng/ml TGF-β1 stimulated cells indicating that hVFFs were capable of differentiation to myofibroblasts. TGF- β1 induced the largest increase in α-SMA at 10-ng/ml on day 5 of treatment. HGF and IL6 suppressed the expression of TGF-β1 induced α–SMA. Our work characterizes a useful in vitro model of TGF-β1 mediated vocal fibroblast-myofibroblast differentiation. The extent of differentiation appears to be attenuated by HGF suggesting a potential mechanism to support prior work indicating that HGF plays a protective role from scar formation in vocal fold injuries. Paradoxically, IL-6 which has been shown to play a profibrotic role in dermal studies also attenuated the TGF-β1 response.