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Vol. 16, Issue 6, 2670-2680, June 2005
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-Cells
* Henry Wellcome Laboratories for Integrated Cell Signalling and Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom;
Genomics Research Institute, Centre for Research in Biomedicine, University of the West of England, Bristol BS16 1QY, United Kingdom
Submitted November 16, 2004;
Revised February 7, 2005;
Accepted March 14, 2005
Monitoring Editor: Benjamin Glick
The role of unconventional myosins in neuroendocrine cells is not fully understood, with involvement suggested in the movement of both secretory vesicles and mitochondria. Here, we demonstrate colocalization of myosin Va (MyoVa) with insulin in pancreatic 
-cells and show that MyoVa copurifies with insulin in density gradients and with the vesicle marker phogrin-enhanced green fluorescent protein upon fluorescence-activated sorting of vesicles. By contrast, MyoVa immunoreactivity was poorly colocalized with mitochondrial or other markers. Demonstrating an important role for MyoVa in the recruitment of secretory vesicles to the cell surface, a reduction of MyoVa protein levels achieved by RNA interference caused a significant decrease in glucose- or depolarization-stimulated insulin secretion. Similarly, expression of the dominant-negative–acting globular tail domain of MyoVa decreased by 
50% the number of vesicles docked at the plasma membrane and by 87% the number of depolarization-stimulated exocytotic events detected by total internal reflection fluorescence microscopy. We conclude that MyoVa-driven movements of vesicles along the cortical actin network are essential for the terminal stages of regulated exocytosis in -cells.
Abbreviations used: [Ca2+]i, intracellular free calcium ion concentration; ER, endoplasmic reticulum; EGFP, enhanced green fluorescent protein; FACS, fluorescence activated cell sorting; KRH, KrebsRinger-HEPES; LDCV, large dense core vesicle (secretory granule); MGTD, myosin Va globular tail domain; MT, microtubule; MyoVa, myosin Va; NPY, neuropeptide Y; siRNA, small interfering RNA; TIRF, total internal reflection fluorescence.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).
Address correspondence to: Aniko Varadi (Aniko.Varadi{at}uwe.ac.uk) or Guy A. Rutter (g.a.rutter{at}bris.ac.uk).
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