The last few milliseconds in the life of a secretory granule (original) (raw)

Abstract

We have monitored single vesicles (granules) in bovine adrenal chromaffin cells using an optical sectioning technique, total internal reflection fluorescence microscopy (TIRFM). With TIR, fluorescence excitation is limited to an optical slice near a glass/water interface. In cells located at the interface, granules loaded with fluorescent dye can be visualized near to or docked at the plasma membrane. Here we give evidence that (1) TIRFM resolves single vesicles and (2) the fluorescence signal originates from vesicles of roughly 350 nm diameter, presumably large dense core vesicles (LDCVs). (3) Diffusional spread of released vesicle contents can be resolved and serves as a convenient criterion for a fusion event. (4) We give details on vesicle properties in resting cells, such as lateral mobility of chromaffin granules, number density, and frequency of spontaneous fusion or withdrawal into the cytoplasm. (5) Upon stimulation with high extracellular potassium, TIRFM reports depletion of the `visible pool' of vesicles closest to the plasma membrane within hundreds of milliseconds, consistent with previous concepts of a release-ready pool. We conclude that TIRFM constitutes an independent assay for pool depletion. TIRFM will allow us to study aspects of secretion that have previously been inaccessible in living cells, in particular the spatial relations and dynamics of vesicles prior to and during exocytosis and re-supply of the near-membrane pool of vesicles.

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Authors and Affiliations

  1. Max-Planck-Institute for Experimental Medicine, Department of Molecular Biology of Neuronal Signals, Hermann-Rein Strasse 3, D-37075 Göttingen, Germany (e-mail: oheim@mail.mpiem.gwdg.de) (e-mail: loerke@mail.mpiem.gwdg.de) (e-mail: stuhmer@mail.mpiem.gwdg.de), , , , , , DE
    M. Oheim, Dinah Loerke & Walter Stühmer
  2. University of Edinburgh Medical School, Department of Physiology, Membrane Biology Group, Teviot Place, EH8 9AG, Scotland, UK (e-mail: rchow@holyrood.ed.ac.uk), , , , , , GB
    Robert H. Chow

Authors

  1. M. Oheim
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  2. Dinah Loerke
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  3. Walter Stühmer
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  4. Robert H. Chow
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Received: 26 June 1997 / Accepted: 26 September 1997

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Oheim, M., Loerke, D., Stühmer, W. et al. The last few milliseconds in the life of a secretory granule .Eur Biophys J 27, 83–98 (1998). https://doi.org/10.1007/s002490050114

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