Myosin Va transports dense core secretory vesicles in pancreatic MIN6 beta-cells.
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 beta-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 approximately 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 beta-cells.
Varadi
Aniko
A
Henry Wellcome Laboratories for Integrated Cell Signalling, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom.
Tsuboi
Takashi
T
Rutter
Guy A
GA
D000818:Animals; D001692:Biological Transport; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D005089:Exocytosis; D005947:Glucose / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007328:Insulin / Q000302:isolation & purification / Q000378:metabolism; D007515:Islets of Langerhans / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008857:Microscopy, Interference; D009154:Mutation; D018995:Myosin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D024701:Myosin Type V / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010179:Pancreas / Q000166:cytology*; D017434:Protein Structure, Tertiary; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D034622:RNA Interference; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D022142:Secretory Vesicles / Q000378:metabolism*
D015415:Biomarkers; D007328:Insulin; D008565:Membrane Proteins; C513082:Myo5a protein, mouse; D049452:Green Fluorescent Proteins; C077511:MYO5A protein, human; C102889:PTPRN2 protein, human; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D024701:Myosin Type V; D018995:Myosin Heavy Chains; D005947:Glucose