European Biophysics Journal

, Volume 29, Issue 2, pp 67–89

Tracking chromaffin granules on their way through the actin cortex

  • Martin Oheim
  • Walter Stühmer

DOI: 10.1007/s002490050253

Cite this article as:
Oheim, M. & Stühmer, W. Eur Biophys J (2000) 29: 67. doi:10.1007/s002490050253


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.

Key words Exocytosis Total internal reflection fluorescence Single-particle tracking Evanescent wave microscopy Diffusion 

Copyright information

© Springer-Verlag Berlin Heidelberg 2000

Authors and Affiliations

  • Martin Oheim
    • 1
  • Walter Stühmer
    • 1
  1. 1.Max-Planck Institute for Experimental Medicine, Molecular Biology of Neuronal Signals, Hermann Rein Strasse 3, 37075 Göttingen, GermanyDE