Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae.

Abstract

The distribution of actin in wild-type cells and in morphogenetic mutants of the budding yeast Saccharomyces cerevisiae was explored by staining cells with fluorochromelabeled phallotoxins after fixing and permeabilizing the cells by several methods . The actin appeared to be localized in a set of cortical spots or patches, as well as in a network of cytoplasmic fibers . Bundles of filaments that may possibly correspond to the fibers visualized by fluorescence were observed with the electron microscope.The putative actin spots were concentrated in small and medium-sized buds and at what were apparently the sites of incipient bud formation on unbudded cells, whereas the putative actin fibers were generally oriented along the long axes of the mother-bud pairs. In several morphogenetic mutants that form multiple, abnormally elongated buds, the actin patches were conspicuously clustered at the tips of most buds, and actin fibers were clearly oriented along the long axes of the buds. There was a strong correlation between the occurrence of active growth at particular bud tips and clustering of actin spots at those same tips. Near the end of the cell cycle in wild-type cells, actin appeared to concentrate (as a cluster of spots or a band) in the neck region connecting the mother cell to its bud .Observations made using indirect immunofluorescence with a monoclonal anti-yeast-tubulin antibody on the morphogenetic mutant cdc4 (which forms multiple, abnormally elongated buds while the nuclear cycle is arrested) revealed the surprising occurrence of multiple bundles of cytoplasmic microtubules emanating from the one duplicated spindle-pole body per cell. It seems that most or all of the buds contain one or more of these bundles of microtubules, which often can be seen to extend to the very tips of the buds.These observations are consistent with the hypotheses that actin, tubulin, or both may be involved in the polarization of growth and localization of cell-wall deposition that occurs during the yeast cell cycle .The cell-division cycle of the yeast Saccharomyces cerevisiae includes the following sequential morphogenetic events (see references 16, 71, and references cited therein): (a) selection of a nonrandom site at which budding will occur ; (b) formation of a ring ofchitin (the "bud scar") in the largely nonchitinous cell wall at that site; (c) localization of new cell-wall growth to the region bounded by the chitin ring, resulting in the appearance and selective growth ofa bud; (d) localization of new cell-wall growth to the tip of the growing bud; (e) cytokinesis and the formation of septal cell wall. In addition, it seems that periods of uniform growth of the bud cell wall precede and follow the period of tip growth; presumably, the relative amounts of tip growth and of uniform growth are adjusted to yield the normal ellipsoidal shape ofthe daughter cell (21). In addition, when the bud is largely grown, the nucleus migrates from a position within the mother cell into the neck connecting mother and bud, and...

The Role of Cdc42p GTPase-activating Proteins in Assembly of the Septin Ring in Yeast

“…The mutant cells produced grossly elongated buds (Figure 5, D and F) in which the nuclear cycle continued ( Figure 5G). This phenotype is very similar to that associated with a complete loss of septin function (Hartwell, 1971;Adams and Pringle, 1984;Longtine et al, 1996), and Figure 1. Suppression of the growth, morphological, and septinorganization defects of a cdc12-6 mutant by multicopy BEM3, RGA1, or RGA2.…”

“…It has long been known that septin mutants produce hyperpolarized, abnormally elongated buds (Hartwell, 1971;Adams and Pringle, 1984). Recent observations have suggested that at least part of the reason for this is that septin mutants have a transient, Swe1p-dependent delay in the G2-M transition, which results in a delay in the switch from polarized to isotropic growth of the bud (Carroll et al, 1998;Barral et al, 1999;Shulewitz et al, 1999;Longtine et al, 2000).…”

“…A pair of hybrid primers (Table 3) was used to amplify HIS3 (for (Adams and Pringle, 1984). b Constructed as described for M-1077 (Longtine et al, 2000).…”

“…The Rho-family GTPase Cdc42p and its activating factor (GEF) Cdc24p are required for the polarized assembly of both the actin cytoskeleton and the septins at the beginning of the cell cycle Johnson, 1999;Pruyne and Bretscher, 2000;Cid et al, 2001;Gladfelter et al, 2001a). However, actin and the septins appear to be independent of each other for their organization at the presumptive bud site (Adams and Pringle, 1984;Ford and Pringle, 1991;Ayscough et al, 1997;Harkins et al, 2001). In the past decade, a major effort has focused on how Cdc42p controls actin organization (Johnson, 1999;Pruyne and Bretscher, 2000), but studies of the role of Cdc42p in septin organization have lagged behind.…”

Stable and Dynamic Axes of Polarity Use Distinct Formin Isoforms in Budding Yeast

“…The myosins move along cables of actin filaments that radiate throughout the cell in arrays oriented toward growth sites (Adams and Pringle, 1984;Kilmartin and Adams, 1984).…”