Localized topological changes of the plasma membrane upon exocytosis visualized by polarized TIRFM

Author:

Anantharam Arun1,Onoa Bibiana22,Edwards Robert H.22,Holz Ronald W.1,Axelrod Daniel11

Affiliation:

1. Department of Pharmacology, Department of Physics, and Department of Biophysics, University of Michigan, Ann Arbor, MI 48109

2. Department of Neurology and Department of Physiology, School of Medicine, University of California, San Francisco, San Francisco, CA 94143

Abstract

Total internal reflection fluorescence microscopy (TIRFM) images the plasma membrane–cytosol interface and has allowed insights into the behavior of individual secretory granules before and during exocytosis. Much less is known about the dynamics of the other partner in exocytosis, the plasma membrane. In this study, we report the implementation of a TIRFM-based polarization technique to detect rapid submicrometer changes in plasma membrane topology as a result of exocytosis. A theoretical analysis of the technique is presented together with image simulations of predicted topologies of the postfusion granule membrane–plasma membrane complex. Experiments on diI-stained bovine adrenal chromaffin cells using polarized TIRFM demonstrate rapid and varied submicrometer changes in plasma membrane topology at sites of exocytosis that occur immediately upon fusion. We provide direct evidence for a persistent curvature in the exocytotic region that is altered by inhibition of dynamin guanosine triphosphatase activity and is temporally distinct from endocytosis measured by VMAT2-pHluorin.

Publisher

Rockefeller University Press

Subject

Cell Biology

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