Journal
MICROSCOPY AND MICROANALYSIS
Volume 19, Issue 2, Pages 381-392Publisher
CAMBRIDGE UNIV PRESS
DOI: 10.1017/S1431927612014304
Keywords
yeast; endocytosis; trafficking; high-pressure freezing; freeze substitution; transmission electron microscopy; electron tomography; immunogold labeling; actin filaments; anti-GFP immunolabeling
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Funding
- Swiss National Science Foundation (SNF)
- National Institutes of Health [R01 GM50399]
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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 immuno-localized 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.
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