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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