Kinesin I and cytoplasmic dynein orchestrate glucose-stimulated insulin-containing vesicle movements in clonal MIN6 β-cells

https://doi.org/10.1016/j.bbrc.2003.09.208Get rights and content

Abstract

Glucose-stimulated mobilization of large dense-core vesicles (LDCVs) to the plasma membrane is essential for sustained insulin secretion. At present, the cytoskeletal structures and molecular motors involved in vesicle trafficking in β-cells are poorly defined. Here, we describe simultaneous imaging of enhanced green fluorescent protein (EGFP)-tagged LDCVs and microtubules in β-cells. Microtubules exist as a tangled array, along which vesicles describe complex directional movements. Whilst LDCVs frequently changed direction, implying the involvement of both plus- and minus-end directed motors, inactivation of the minus-end motor, cytoplasmic dynein, inhibited only a small fraction of all vesicle movements which were involved in vesicle recovery after glucose-stimulated exocytosis. By contrast, selective silencing of the plus-end motor, kinesin I, with small interfering RNAs substantially inhibited all vesicle movements. We conclude that the majority of LDCV transport in β-cells is mediated by kinesin I, whilst dynein probably contributes to the recovery of vesicles after rapid kiss-and-run exocytosis.

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.

References (50)

  • N. Galjart et al.

    A plus-end raft to control microtubule dynamics and function

    Curr. Opin. Cell Biol.

    (2003)
  • C. Wasmeier et al.

    Molecular cloning of phogrin, a protein–tyrosine phosphatase homologue localized to insulin secretory granule membranes

    J. Biol. Chem.

    (1996)
  • M.J. Donelan et al.

    Ca2+-dependent dephosphorylation of kinesin heavy chain on beta-granules in pancreatic beta-cells. Implications for regulated beta-granule transport and insulin exocytosis

    J. Biol. Chem.

    (2002)
  • T. Lang et al.

    Role of actin cortex in the subplasmalemmal transport of secretory granules in PC-12 cells

    Biophys. J.

    (2000)
  • S. Barg et al.

    Delay between fusion pore opening and peptide release from large dense-core vesicles in neuroendocrine cells

    Neuron

    (2002)
  • D.A. Richards et al.

    Two endocytic recycling routes selectively fill two vesicle pools in frog motor nerve terminals

    Neuron

    (2000)
  • J. Januschke et al.

    Polar transport in the Drosophila oocyte requires Dynein and Kinesin I cooperation

    Curr. Biol.

    (2002)
  • P. Rorsman et al.

    The cell physiology of biphasic insulin secretion

    News Physiol. Sci.

    (2000)
  • C.J. Rhodes

    Introduction: the molecular cell biology of insulin production

    Semin. Cell Dev. Biol.

    (2000)
  • L. AguilarBryan et al.

    Molecular biology of adenosine triphosphate-sensitive potassium channels

    Endocr. Rev.

    (1999)
  • H. Safayhi et al.

    L-type calcium channels in insulin-secreting cells: biochemical characterization and phosphorylation in RINm5F cells

    Mol. Endocrinol.

    (1997)
  • J.C. Henquin

    Triggering and amplifying pathways of regulation of insulin secretion by glucose

    Diabetes

    (2000)
  • G.M. Grodsky et al.

    Further studies on the dynamic aspects of insulin release in vitro with evidence for a two-compartmental storage system

    Acta Diabetol. Lat.

    (1969)
  • M. Komatsu et al.

    Glucose stimulation of insulin release in the absence of extracellular Ca2+ and in the absence of any increase in intracellular Ca2+ in rat pancreatic islets

    Proc. Natl. Acad. Sci. USA

    (1995)
  • A.E. Pouli et al.

    Secretory granule dynamics visualised in vivo with a phogrin-green fluorescent protein chimaera

    Biochem. J.

    (1998)
  • Cited by (0)

    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.

    View full text