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  • 1
    Electronic Resource
    Electronic Resource
    Multiple stimulation-dependent processes regulate the size of the releasable pool of vesicles (1999)
    Springer
    European biophysics journal 28 (1999), S. 91-101 
    Details
    ISSN: 1432-1017
    Keywords: Key words Total internal reflection ; Evanescent wave microscopy ; Optical sectioning ; Ca2+-triggered exocytosis ; Vesicle fusion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract In neuroendocrine cells and neurones, changes in the size of a limited pool of readily releasable vesicles contribute to the plasticity of secretion. We have studied the dynamic alterations in the size of a near-membrane pool of vesicles in living neuroendocrine cells. Using evanescent wave microscopy we monitored the behaviour of individual secretory vesicles at the plasma membrane. Vesicles undergo sequential transitions between several states of differing fluorescence intensity and mobility. The transitions are reversible, except for the fusion step, and even in nonstimulated conditions the vesicles change states in a dynamic equilibrium. Stimulation selectively speeds up the three forward transitions leading towards exocytosis. Vesicles lose mobility in all three dimensions upon approach of the plasma membrane. Their movement is directed and targeted to the docking fusion sites. Sites of vesicle docking and exocytosis are distributed non-uniformly over the studied “footprint” region of the cell. While some areas are the sites of repeated vesicle docking and fusion, others are completely devoid of spots. Vesicular mobility at the membrane is confined, as if the vesicle were imprisoned in a cage or tethered to a binding site.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002490050188
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  • 2
    Electronic Resource
    Electronic Resource
    The last few milliseconds in the life of a secretory granule (1998)
    Springer
    European biophysics journal 27 (1998), S. 83-98 
    Details
    ISSN: 1432-1017
    Keywords: Key words TIRF ; Evanescent wave microscopy ; Optical sectioning ; Ca2+-triggered exocytosis ; Visible pool ; Chromaffin cell ; Vesicle fusion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002490050114
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  • 3
    Electronic Resource
    Electronic Resource
    Tracking chromaffin granules on their way through the actin cortex (2000)
    Springer
    European biophysics journal 29 (2000), S. 67-89 
    Details
    ISSN: 1432-1017
    Keywords: Key words Exocytosis ; Total internal reflection fluorescence ; Single-particle tracking ; Evanescent wave microscopy ; Diffusion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract Quantitative time-lapse evanescent-wave imaging of individual fluorescently labelled chromaffin granules was used for kinetic analysis of granule trafficking through a ∼300-nm (1/e2) optical section beneath the plasma membrane. The mean squared displacement (MSD) was used to estimate the three-dimensional diffusion coefficient (D (3)). We calculated the granules' speed, frame-to-frame displacement and direction and their autocorrelation to identify different stages of approach to the membrane. D (3) was about 10,000 times lower than expected for free diffusion. Granules located ∼60 nm beneath the plasma membrane moved on random tracks (D (3)≈10−10 cm2 s−1) with several reversals in direction before they approached their docking site at angles larger than 45∘. Docking was observed as a loss of vesicle mobility by two orders of magnitude within 〈100 ms. For longer observation times the MSD saturated, as if the granules' movement was confined to a volume only slightly larger than the granule. Rarely, the local random motion was superimposed with a directed movement in a plane beneath the membrane. Stimulation of exocytosis selectively depleted the immobile, near-membrane granule population and caused a recruitment of distant granules to sites at the plasma membrane. Their absolute mobility levels were not significantly altered. Application of latrunculin or jasplakinolide to change F-actin polymerisation caused a change in D (3) of the mobile granule population as well as a reduction of the rate of release, suggesting that granule mobility is constrained by the filamentous actin meshwork and that stimulation-dependent changes in actin viscosity propel granules through the actin cortex.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002490050253
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