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First published online on May 3, 2007.
 Copyright © 2007 by The Physiological Society
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Received February 26, 2007
Revised March 22, 2007
Accepted after revision May 1, 2007

Macroendocytosis and Endosome Processing in Snake Motor Boutons

Haibing Teng1, Michael Y. Lin1, and Robert S. Wilkinson1*

1 Washington University School of Medicine

* To whom correspondence should be addressed. E-mail: wilk{at}wustl.edu.

We have examined the processing of endosomes formed by macroendocytosis (ME), or bulk membrane retrieval, in active motor terminal boutons at the snake nerve-muscle synapse. Endocytic probes were imaged at light (FM1-43) and electron (HRP) levels over stimulus frequencies representing low, intermediate, and high levels of use. Endosomes formed rapidly (1-2 s) at all frequencies, concomitant with clathrin-mediated vesicular endocytosis (CME). Endosomes dissipated rapidly into vesicles (~10 s). The dissipation rate was not influenced by activity. Many endosomes split into clusters of 2-20 smaller endosomes of varying size. Vesicles budded from these smaller endosomes, from large endosomes that had not undergone fission into smaller ones, and from precursor membrane infoldings that had not yet internalized. In snake, exocytosed vesicular membrane is not competent for reuse until after a delay (>3 min). We found that time required for endosome processing is not responsible for this delay. Endosome processing might, however, limit availability of some vesicles for release at very high levels of use. Generally, endosome processing paralleled that of vesicles internalized directly from the plasma membrane via CME, regardless of stimulus frequency. There was no evidence for differential recruitment of ME versus CME depending upon level of use


Key words: Endocytosis • Fluorescence • Nerve terminal • 3D rendering • Confocal and elctronic microscopy • Vesicle recycling




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