Sustained insulin release requires Brownian-like granule oscillations and microtubule-dependent granule transport
Field:	Other
Authors:	Renström, Erik Barg, Sebastian Galvanovskis, Juris Ivarsson, Rosita Jing, Xing-Jun
Address of presenting author:	Sölvegatan 19 S-223 62 Lund Sweden
E-mail:	erik.renstrom@mphy.lu.se
Phone:	+46 46 222 06 33
Fax:	+46 46 222 77 63
Text of abstract:	Intracellular pre-fusion granular movements in insulin-releasing INS-1 cells were investigated by confocal LysotrackerRed microfluorimetry in combination with monitoration of single-cell exocytosis by capacitance measurements. Two distinct types of intracellular granular motion was observed: 1) rapid translocations, or saltatory jumps. 2) In addition, the granules revealed an intense oscillatory behaviour reminiscent of Brownian movements. The intensity of these oscillations was quantified by calculating abstract temperature (TA) and the diffusion coefficient (D). Granular movements were observed already at non-stimulatory conditions (0 glucose), and stimulation with 10 mM glucose had no obvious effect. After treatment with microtubule inhibitors, saltatory jumps did not occur, whereas granular oscillations remained unperturbed. Disruption of actin filaments by cytochalasin D did not affect either type of granular movement. Both types of granular movements were virually abolished under two conditions: a) lowering the temperature from 32oC to 20oC, or b) intracellular dialysis using the standard whole-cell configuration of the patch-clamp technique. Single-cell measurements of exocytosis by capacitance recordings revealed that depolarisation-elicited Ca2+-dependent exocytosis was rapidly exhausted at 20oC, when replacing the cytosol with the electrode-solution using the standard whole-cell configuration and after vindesine treatment. By contrast, when using the perforated-patch configuration (intact cells), the granular movements remained unperturbed and exocytosis could be reproducibly stimulated for up to one hour, even after cytochalasin D treatment. These data suggest that: 1) intracellular mobilization of insulin granules for release involves both Brownian-like granular oscillations and microtubule-dependent saltatory jumps, and that the granular oscillations are critically dependent on the composition of the cytosol, 2) that microtubule-dependent transport mechanisms control the number of release-ready granules in the B-cell, and that 3) the B-cell actin network serves as a barrier controlling the supply of insulin granules to the release sites. Furthermore, it can be speculated granule oscillations are required for the coupling granules to microtubules and, after transport to the plasma membrane, for the formation of exocytotic fusion protein complexes.
Keywords:	exocytosis, insulin, pre-exocytotic granule motion, microtubules, actin

Created 2000-03-15