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Ultrastructural Imaging of Endocytic Sites in Saccharomyces cerevisiae by Transmission Electron Microscopy and Immunolabeling

Published online by Cambridge University Press:  05 March 2013

Christopher Buser  and
David G. Drubin
Christopher Buser
Affiliation:
Department of Molecular & Cell Biology, University of California, Berkeley, CA 94720, USA
David G. Drubin*
Affiliation:
Department of Molecular & Cell Biology, University of California, Berkeley, CA 94720, USA
*
*Corresponding author. E-mail: drubin@berkeley.edu
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Abstract

Defining the ultrastructure of endocytic sites and localization of endocytic proteins in Saccharomyces cerevisiae by immunoelectron microscopy is central in understanding the mechanisms of membrane deformation and scission during endocytosis. We show that an improved sample preparation protocol based on high-pressure freezing, freeze substitution, and low-temperature embedding allows us to maintain the cellular fine structure and to immunolabel green fluorescent protein–tagged endocytic proteins or actin in the same sections. Using this technique we analyzed the stepwise deformation of endocytic membranes and immunolocalized the endocytic proteins Abp1p, Sla1p, Rvs167p, and actin, and were able to draw a clear ultrastructural distinction between endocytic sites and eisosomes by immunolocalizing Pil1p. In addition to defining the geometry and the fine structure of budding yeast endocytic sites, we observed associated actin filaments forming a cage-like meshwork around the endocytic membrane.

Keywords

yeastendocytosistraffickinghigh-pressure freezingfreeze substitutiontransmission electron microscopyelectron tomographyimmunogold labelingactin filamentsanti-GFP immunolabeling
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 19 , Issue 2 , April 2013 , pp. 381 - 392
Copyright
Copyright © Microscopy Society of America 2013

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