Biochemical and Biophysical Research Communications
Kinesin I and cytoplasmic dynein orchestrate glucose-stimulated insulin-containing vesicle movements in clonal MIN6 β-cells☆
Section snippets
Materials and methods
Materials. cDNAs encoding dynactin p50 and p50-EGFP [20]. Human growth hormone-containing plasmid pXGH5 was from Professor R. Burgoyne (University of Liverpool) [21]. Tubulin.EGFP was from Dr. David Stephens (University of Bristol, UK). Cell culture reagents were from Gibco-BRL (Life Science Research, Paisley, UK). The human growth hormone (hGH) ELISA Kit and all molecular biologicals were obtained from Roche Diagnostics (Lewes, UK). Silencer siRNA Construction Kit was purchased from Ambion
Insulin-containing vesicles are moved to / from the cell periphery principally on microtubules
In order to investigate the relative importance of actin- and microtubule-based transport in insulin-containing vesicle mobilization, we visualized microtubules and insulin-containing vesicles simultaneously in live MIN6 cells. Using this approach, microtubule movements as well as changes in microtubule structure could be observed dynamically and directly. Expressed tubulin.EGFP displayed a typical microtubule localization (Fig. 1A, cell II.) whose filamentous structure was clearly distinct
LDCVs move from the cell interior to the plasma membrane principally on microtubules
Our previous studies in β-cells [11], [12] revealed that LDCVs undergo long-range saltatory movements in response to glucose stimulation, indicating that they are likely to represent microtubule-based motility. Simultaneous imaging of LDCVs and microtubules, as achieved here, reveals that LDCVs are transported principally on microtubules from the site of assembly to the plasma membrane in these cells and that actin-based motility has no significant role in these long (several μm) excursions of
Acknowledgements
G.A.R. was supported by Wellcome Trust Program Grant 067081/Z/02/Z, Human Science Frontiers Program grant RGP 0347/2001-M, and by project grants from the Wellcome Trust, Biotechnology and Biological Research Council, UK, and Diabetes UK. We thank Professor Peter Cullen for the use of the UltraView microscope. G.A.R. is a Wellcome Trust Research Leave Fellow.
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Abbreviations: DIC, dynein intermediate chain; DMEM, Dulbecco’s modified Eagle’s medium; EGFP, enhanced green fluorescent protein; FCS, fetal calf serum; hGH, human growth hormone; KHC, kinesin heavy chain; KRH, Krebs–Ringer–Hepes-bicarbonate; LDCV, Large dense-core vesicle; siRNA, small interfering RNA; MT, microtubule; p50, dynamitin; SUK4, anti-kinesin heavy chain antibody; TGN, trans-Golgi network; TIRF, total internal reflection fluorescence.