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Ultrastructural Analysis of Nanogold-labeled Endocytic Compartments of Yeast Saccharomyces cerevisiae Using a Cryosectioning Procedure

Janice Griffith and Fulvio Reggiori

Department of Cell Biology and Institute of Biomembranes, University Medical Centre Utrecht, Utrecht, The Netherlands

Correspondence to: Fulvio Reggiori, Department of Cell Biology, University Medical Centre Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail: F.Reggiori{at}umcutrecht.nl

Yeast Saccharomyces cerevisiae has been a valuable model organism for the study of the endosomal system of eukaryotic cells. Morphological analyses, however, have been limited because of the lack of specific protein markers and of procedures that lead to a satisfactory ultrastructural resolution. We have recently developed an immunoelectron microscopy (IEM) protocol adapted from the Tokuyasu method to prepare cryosections from mildly fixed yeast. This novel approach allows excellent cell preservation and a unique resolution of the yeast morphology. Here, we present a protocol that combines this procedure with the specific labeling of the various endosomal compartments with positively charged Nanogold. In particular, we show that this new protocol generates excellent results when applied for the examination of early and late endosomes, and of mutants with an endosomal trafficking defect. Importantly, this method is compatible with immunogold labeling of protein markers, and it is consequently appropriate for localization studies of both resident and cargo proteins. This new IEM protocol will be a valuable tool for the large community of scientists using yeast as a model system to investigate the membrane transport and the biogenesis of the endosomal system. (J Histochem Cytochem 57:801–809, 2009)

Key Words: yeast • endocytosis • endosomes • vacuole • positively charged Nanogold • electron microscopy • Tokuyasu cryosectioning • immunogold labeling

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