Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane.

Abstract

Abstract. We characterized the yeast actin cytoskeleton at the ultrastructural level using immunoelectron microscopy. Anti-actin antibodies primarily labeled dense, patchiike cortical structures and cytoplasmic cables. This localization recapitulates results obtained with immunofluorescence light microscopy, but at much higher resolution. Immuno-EM double-labeling experiments were conducted with antibodies to actin together with antibodies to the actin binding proteins Abplp and cofilin. As expected from immunofluorescence experiments, Abplp, cofilin, and actin colocalized in immuno-EM to the dense patchlike structures but not to the cables. In this way, we can unambiguously identify the patches as the cortical actin cytoskeleton. The cortical actin patches were observed to be associated with the cell surface via an invagination of plasma membrane. This novel cortical cytoskeleton-plasma membrane interface appears to consist of a fingerlike invagination of plasma membrane around which actin filaments and actin binding proteins are organized. We propose a possible role for this unique cortical structure in wall growth and osmotic regulation.

Actin and Septin Ultrastructures at the Budding Yeast Cell Cortex

“…We did not detect any actin cable-like structures in our preparations, nor are cables apparent in intact spheroplasts, which lack obvious cell polarity. Our results extend upon a previous model in which actin filaments wind around finger-like invaginations of the plasma membrane (Mulholland et al, 1994). We show that actin patches are conical or mound-like structures rising out of the plasma membrane and suggest that they are composed of short filaments arranged into an apparently cross-linked scaffold that surrounds a membrane core.…”

“…However, actin filaments do not show up well in the crowded yeast cytoplasm, except for rare actin cables in Saccharomyces cerevisiae and pathogenic yeasts (Adams and Pringle, 1984;Kopecka et al, 2001;Yamaguchi et al, 2002), cytoplasmic "filasomes" distinct from cortical patches in fission yeast (Kanbe et al, 1989;Takagi et al, 2003), and aberrant filamentous actin structures in mutant budding yeast defective in endocytosis (Sekiya-Kawasaki et al, 2003). Immunolabeled thin sections have in some cases revealed finger-like invaginations of the plasma membrane apparently entwined in an actin helix (Mulholland et al, 1994). However, these structures are two-dimensional and thus provide limited information about geometry and spatial relations within cortical actin patches.…”

“…An ultrastructural analysis of the cortical actin cytoskeleton would provide a powerful basis for understanding the function of actin in endocytosis by allowing highresolution visual comparisons of actin and membrane structures in wild-type and mutant yeast strains. Attempts have been made to visualize yeast actin filaments by thinsection electron microscopy (Adams and Pringle, 1984;Mulholland et al, 1994). However, actin filaments do not show up well in the crowded yeast cytoplasm, except for rare actin cables in Saccharomyces cerevisiae and pathogenic yeasts (Adams and Pringle, 1984;Kopecka et al, 2001;Yamaguchi et al, 2002), cytoplasmic "filasomes" distinct from cortical patches in fission yeast (Kanbe et al, 1989;Takagi et al, 2003), and aberrant filamentous actin structures in mutant budding yeast defective in endocytosis (Sekiya-Kawasaki et al, 2003).…”

BED1, a gene encoding a galactosyltransferase homologue, is required for polarized growth and efficient bud emergence in Saccharomyces cerevisiae.

“…At cytokinesis, the actin cytoskeleton is reorganized and actin patches are relocalized to the mother-daughter neck where the cell wall is modified for cell separation. The mechanisms by which actin mediates polarized secretion are not well understood, but it has been shown that cortical actin patches are associated with the cell surface through an invagination of the plasma membrane (Mulholland et al, 1994) and it has been suggested that components of the secretory pathway (endoplasmic reticulum [ER] and Golgi) could be transported into the bud to direct localized growth presumably via an actin-dependent mechanism (Preuss et al, 1992).A variety of proteins have been shown to be required for either bud emergence or for bud site selection (for recent reviews see Bretscher et al, 1994;Chant, 1994; Welch and Drubin, 1994). CDC42, encoding a small GTP-binding protein, and several genes encoding its regulators are involved in bud emergence: cells mutated in these genes arrest as large unbudded cells with a disorganized actin cytoskeleton and delocalized chitin.…”

Interdependence of Filamentous Actin and Microtubules for Asymmetric Cell Division

“…Immunoelectron microscopy indicated that at least some of the actin patches represent coils of actin filaments surrounding membrane invaginations (Mulholland et al, 1994). These structures might anchor the cytoskeleton to the plasma membrane.…”