Temperature-sensitive random insulin granule diffusion is a prerequisite for recruiting granules for release

Glucose-evoked insulin secretion exhibits a biphasic time course and is associated with accelerated intracellular granule movement. We combined live confocal imaging of EGFP-labelled insulin granules with capacitance meas-urements of exocytosis in clonal INS-1 cells to explore the relation between distinct random and directed modes of insulin granule movement, as well as exocytotic capacity. Reducing the temperature from 34 C to 24 C caused a dramatic 81 % drop in the frequency of directed events, but reduced directed velocities by a mere 25%. The much stronger temperature sensitivity of the frequency of direc-ted events (estimated energy of activation ~ 135kJ/mol) than that of the granule velocities (~22kJ/mol) suggests that cooling-induced suppression of insulin granule move-ment is attributable to factors other than reduced motor protein adenosine 5’-triphosphatase activity. Indeed, cool-ing suppresses random granule diffusion by ~50%. In the single cell, the number of directed events depends on the extent of granule diffusion. Finally, single-cell exocytosis exhibits a biphasic pattern corresponding to that observed in vivo, and only the component reflecting 2nd phase insulin secretion is affected by cooling. We conclude that random diffusive movement is a prerequisite for directed insulin granule transport and for the recruitment of insulin granules released during 2nd phase insulin secretion. Key words: diffusion, granule movement, insulin, micro-tubule, temperature sensitivity Received 17 May 2004, revised and accepted for publica