Cell-wide analysis of secretory granule dynamics in three dimensions in living pancreatic beta-cells

TY - JOUR

T1 - Cell-wide analysis of secretory granule dynamics in three dimensions in living pancreatic beta-cells

T2 - evidence against a role for AMPK-dependent phosphorylation of KLC1 at Ser(517)/Ser(520) in glucose-stimulated insulin granule movement

A1 - McDonald,Angela

A1 - Fogarty,Sarah

A1 - Leclerc,Isabelle

A1 - Hill,Elaine V.

A1 - Hardie,D. Grahame

A1 - Rutter,Guy A.

AU - McDonald,Angela

AU - Fogarty,Sarah

AU - Leclerc,Isabelle

AU - Hill,Elaine V.

AU - Hardie,D. Grahame

AU - Rutter,Guy A.

PY - 2010/2

Y1 - 2010/2

N2 - <p>Glucose-stimulated insulin secretion from pancreatic beta-cells requires the kinesin-1/Kif5B-mediated transport of insulin granules along microtubules. 5'-AMPK (5'-AMP-activated protein kinase) is a heterotrimeric serine/threonine kinase which is activated in beta-cells at low glucose concentrations, but inhibited as glucose levels increase. Active AMPK blocks glucose-stimulated insulin secretion and the recruitment of insulin granules to the cell surface, suggesting motor proteins may be targets for this kinase. While both kinesin-1/Kif5B and KLC1 (kinesin light chain-1) contain consensus AMPK phosphorylation sites (Thr(693) and Ser(520), respectively) only recombinant GST (glutathione transferase)-KLC1 was phosphorylated by purified AMPK in vitro. To test the hypothesis that phosphorylation at this site may modulate kinesin-1-mediated granule movement, we developed an approach to study the dynamics of all the resolvable granules within a cell in three dimensions. This cell-wide approach revealed that the number of longer excursions (&gt;10 mu m) increased significantly in response to elevated glucose concentration (30 versus 3 mM) in control MIN6 beta-cells. However, similar changes were seen in cells overexpressing wild-type KLC1, phosphomimetic (S517D/S520D) or non-phosphorylatable (S517A/S520A) mutants of KLC1 Thus, changes in the phosphorylation state of KLC1 at Ser(517)/Ser(520) seem unlikely to affect motor function.</p>

AB - <p>Glucose-stimulated insulin secretion from pancreatic beta-cells requires the kinesin-1/Kif5B-mediated transport of insulin granules along microtubules. 5'-AMPK (5'-AMP-activated protein kinase) is a heterotrimeric serine/threonine kinase which is activated in beta-cells at low glucose concentrations, but inhibited as glucose levels increase. Active AMPK blocks glucose-stimulated insulin secretion and the recruitment of insulin granules to the cell surface, suggesting motor proteins may be targets for this kinase. While both kinesin-1/Kif5B and KLC1 (kinesin light chain-1) contain consensus AMPK phosphorylation sites (Thr(693) and Ser(520), respectively) only recombinant GST (glutathione transferase)-KLC1 was phosphorylated by purified AMPK in vitro. To test the hypothesis that phosphorylation at this site may modulate kinesin-1-mediated granule movement, we developed an approach to study the dynamics of all the resolvable granules within a cell in three dimensions. This cell-wide approach revealed that the number of longer excursions (&gt;10 mu m) increased significantly in response to elevated glucose concentration (30 versus 3 mM) in control MIN6 beta-cells. However, similar changes were seen in cells overexpressing wild-type KLC1, phosphomimetic (S517D/S520D) or non-phosphorylatable (S517A/S520A) mutants of KLC1 Thus, changes in the phosphorylation state of KLC1 at Ser(517)/Ser(520) seem unlikely to affect motor function.</p>

KW - AMP-activated protein kinase (AMPK)

KW - four-dimensional confocal imaging

KW - insulin secretion

KW - kinesin

KW - protein neuropeptide

KW - ACTIVATED PROTEIN-KINASE

KW - KINESIN LIGHT-CHAIN

KW - HEAVY-CHAIN

KW - CONVENTIONAL KINESIN

KW - GENE-EXPRESSION

KW - EXOCYTOSIS

KW - RELEASE

KW - IDENTIFICATION

KW - MITOCHONDRIA

KW - REGULATOR

U2 - 10.1042/BST0380205

DO - 10.1042/BST0380205

M1 - Article

JO - Biochemical Society Transactions

JF - Biochemical Society Transactions

SN - 0300-5127

VL - 38

SP - 205

EP - 208

ER -