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Published online 2 April 2001. doi:10.1083/jcb.153.1.177
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© The Rockefeller University Press, 0021-9525/2001//177 $5.00
The Journal of Cell Biology, Volume 153, Number 1, , 2001 177-190

Original Article

Restriction of Secretory Granule Motion near the Plasma Membrane of Chromaffin Cells



Laura M. Johnsa, Edwin S. Levitand, Eric A. Sheldenb, Ronald W. Holza, and Daniel Axelrodc

a Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan 48109
b Department of Cell and Developmental Biology, The University of Michigan, Ann Arbor, Michigan 48109
c Department of Physics and Biophysics Research Division, The University of Michigan, Ann Arbor, Michigan 48109
d Department of Pharmacology, The University of Pittsburgh, Pittsburgh, Pennsylvania 15261
Department of Physics and Biophysics Research Division, The University of Michigan, 930 N. University, Ann Arbor, Michigan 48109.(734) 764-3323(734) 764-5280

daxelrod{at}umich.edu

We used total internal reflection fluorescence microscopy to study quantitatively the motion and distribution of secretory granules near the plasma membrane (PM) of living bovine chromaffin cells. Within the ~300-nm region measurably illuminated by the evanescent field resulting from total internal reflection, granules are preferentially concentrated close to the PM. Granule motion normal to the substrate (the z direction) is much slower than would be expected from free Brownian motion, is strongly restricted over tens of nanometer distances, and tends to reverse directions within 0.5 s. The z-direction diffusion coefficients of granules decrease continuously by two orders of magnitude within less than a granule diameter of the PM as granules approach the PM. These analyses suggest that a system of tethers or a heterogeneous matrix severely limits granule motion in the immediate vicinity of the PM. Transient expression of the light chains of tetanus toxin and botulinum toxin A did not disrupt the restricted motion of granules near the PM, indicating that SNARE proteins SNAP-25 and VAMP are not necessary for the decreased mobility. However, the lack of functional SNAREs on the plasma or granule membranes in such cells reduces the time that some granules spend immediately adjacent to the PM.

Key Words: evanescent field • exocytosis • diffusion • fluorescence microscopy • green fluorescent protein

© 2001 The Rockefeller University Press

Abbreviations used in this paper: ANP, atrial natriuretic peptide; BoNT/A, botulinum neurotoxin type; DMPP, 1,1-dimethyl-4-phenylpiperazinium; GFP, green fluorescent protein; GH, growth hormone; PSS, physiological salt solution; TeNT, tetanus toxin; TIRFM, total internal reflection fluorescence microscopy.


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