Abstract
For pancreatic β-cells to secrete insulin in response to elevated blood glucose, insulin granules retained within the subplasmalemmal space must be transported to sites of secretion on the plasma membrane. Using a combination of super-resolution STORM imaging and live cell TIRF microscopy we investigate how the organization and dynamics of the actin and microtubule cytoskeletons in INS-1 β-cells contribute to this process. GFP-labeled insulin granules display 3 different modes of motion (stationary, diffusive-like, and directed). Diffusive-like motion dominates in basal, low glucose conditions. Upon glucose stimulation no gross rearrangement of the actin cytoskeleton is observed but there are increases in the 1) rate of microtubule polymerization; 2) rate of diffusive-like motion; and 3) proportion of granules undergoing microtubule-based directed motion. By pharmacologically perturbing the actin and microtubule cytoskeletons, we determine that microtubule-dependent granule transport occurs within the subplasmalemmal space and that the actin cytoskeleton limits this transport in basal conditions, when insulin secretion needs to be inhibited.
Highlights
Insulin secretion from pancreatic b-cells is critical for proper maintenance of blood glucose levels, with perturbations to this process leading to diabetes [1,2,3]
Evidence for molecular motor function being critical for granule transport is based on perturbation of either kinesin-1 or myosin Va function by expression of dominant negative constructs or knockdown of protein levels with siRNA resulting in diminished insulin release, loss of directed granule movement, and altered granule localization at the plasma membrane [7,9,10,11,12,13,14]
On a log-log axis, the mean square displacement (MSD) slope defines the diffusive exponent, a, where a,0 characterizes the granule as stationary, a,1 as undergoing random motion, and a,2 as directed (Saxton, 1997)
Summary
Insulin secretion from pancreatic b-cells is critical for proper maintenance of blood glucose levels, with perturbations to this process leading to diabetes [1,2,3]. Evidence for molecular motor function being critical for granule transport is based on perturbation of either kinesin-1 or myosin Va function by expression of dominant negative constructs or knockdown of protein levels with siRNA resulting in diminished insulin release, loss of directed granule movement, and altered granule localization at the plasma membrane [7,9,10,11,12,13,14]. Implicit in this model is that the microtubule and actin networks serve as cytoskeletal tracks upon which the motors transport their granule cargos. The actin and microtubule cytoskeletons may not be passive players but actively involved in modulating insulin granule trafficking
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