Non-muscle myosin IIA is involved in focal adhesion and actin remodelling controlling glucose-stimulated insulin secretion.
OBJECTIVE
Actin and focal adhesion (FA) remodelling are essential for glucose-stimulated insulin secretion (GSIS). Non-muscle myosin II (NM II) isoforms have been implicated in such remodelling in other cell types, and myosin light chain kinase (MLCK) and Rho-associated coiled-coil-containing kinase (ROCK) are upstream regulators of NM II, which is known to be involved in GSIS. The aim of this work was to elucidate the implication and regulation of NM IIA and IIB in beta cell actin and FA remodelling, granule trafficking and GSIS.


METHODS
Inhibitors of MLCK, ROCK and NM II were used to study NM II activity, and knockdown of NM IIA and IIB to determine isoform specificity, using sorted primary rat beta cells. Insulin was measured by radioimmunoassay. Protein phosphorylation and subcellular distribution were determined by western blot and confocal immunofluorescence. Dynamic changes were monitored by live cell imaging and total internal reflection fluorescence microscopy using MIN6B1 cells.


RESULTS
NM II and MLCK inhibition decreased GSIS, associated with shortening of peripheral actin stress fibres, and reduced numbers of FAs and insulin granules in close proximity to the basal membrane. By contrast, ROCK inhibition increased GSIS and caused disassembly of glucose-induced central actin stress fibres, resulting in large FAs without any effect on FA number. Only glucose-induced NM IIA reorganisation was blunted by MLCK inhibition. NM IIA knockdown decreased GSIS, levels of FA proteins and glucose-induced extracellular signal-regulated kinase 1/2 phosphorylation.


CONCLUSIONS
Our data indicate that MLCK-NM IIA may modulate translocation of secretory granules, resulting in enhanced insulin secretion through actin and FA remodelling, and regulation of FA protein levels.
Department of Genetic Medicine and Development, University Medical Centre, University of Geneva, 1 Michel Servet, 1211 Geneva 4, Switzerland. Caroline.Arous@unige.ch
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D000199:Actins / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002469:Cell Separation; D005434:Flow Cytometry; D022001:Focal Adhesions / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000166:cytology; D051379:Mice; D008856:Microscopy, Fluorescence; D024507:Nonmuscle Myosin Type IIA / Q000378:metabolism*; D010766:Phosphorylation; D020033:Protein Isoforms; D051381:Rats; D015398:Signal Transduction; D054460:rho-Associated Kinases / Q000378:metabolism
D000199:Actins; D007328:Insulin; D020033:Protein Isoforms; D054460:rho-Associated Kinases; D024507:Nonmuscle Myosin Type IIA; D005947:Glucose