Microscopy and Microanalysis

Biological Applications

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

Christopher Busera1 and David G. Drubina1 c1

a1 Department of Molecular & Cell Biology, University of California, Berkeley, CA 94720, USA

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.

(Received August 22 2012)

(Accepted November 06 2012)

Key words

  • yeast;
  • endocytosis;
  • trafficking;
  • high-pressure freezing;
  • freeze substitution;
  • transmission electron microscopy;
  • electron tomography;
  • immunogold labeling;
  • actin filaments;
  • anti-GFP immunolabeling

Correspondence

c1 Corresponding author. E-mail: drubin@berkeley.edu