Alison E M Adams

Immunofluorescence methods for yeast

Publisher Summary This chapter provides protocols for the application of immunofluorescence procedures to yeast. It should perhaps be stressed that immunofluorescence and other light microscopic techniques play a role that is separate from but equal to the role of electron microscopy. Although in some situations the greater resolving power of the electron microscope is clearly essential to obtain the needed structural information, in other situations the necessary information can be obtained more easily, more reliably, or both, by light microscopy. The potential advantages of light microscopic approaches derive from various facts: (1) they can be applied to lightly processed or (in some cases) living cells, (2) Much larger numbers of cells can be examined than by electron microscopy (note especially the great labor involved in visualizing the structure of whole cells by serial-section methods), and (3) Some structures (for example, the cytoplasmic microtubules) have simply been easier to see by light microscopy than by electron microscopy.

Fluorescence microscopy methods for yeast.

Publisher Summary This chapter reviews and provides detailed protocols for the application of immunofluorescence and other fluorescence-microscopic procedures to yeast. These procedures play a role that is separate from but equal to the role of electron microscopy. Although in some situations the greater resolving power of the electron microscope is clearly essential to obtain the needed structural information, in other situations the necessary information can be obtained more easily, more reliably, or both, by light (including fluorescence) microscopy. The potential advantages of light-microscopic approaches derive from the facts (1) that they can be applied to lightly processed or living cells, (2) that much larger numbers of cells can be examined than by electron microscopy (note especially the great labor involved in visualizing the structure of whole cells by serial-section methods), and (3) that some structures have simply been easier to see by light microscopy than by electron microscopy. The methods are also effective with other yeasts such as Schizosaccharomyces pombe and Candida albicans .

Cdc53p acts in concert with cdc4p and cdc34p to control the G1-to-S- phase transition and identifies a conserved family of proteins

Regulation of cell cycle progression occurs in part through the targeted degradation of both activating and inhibitory subunits of the cyclin-dependent kinases. During G1, CDC4, encoding a WD-40 repeat protein, and CDC34, encoding a ubiquitin-conjugating enzyme, are involved in the destruction of these regulators. Here we describe evidence indicating that CDC53 also is involved in this process. Mutations in CDC53 cause a phenotype indistinguishable from those of cdc4 and cdc34 mutations, numerous genetic interactions are seen between these genes, and the encoded proteins are found physically associated in vivo. Cdc53p defines a large family of proteins found in yeasts, nematodes, and humans whose molecular functions are uncharacterized. These results suggest a role for this family of proteins in regulating cell cycle proliferation through protein degradation.

Staining of actin with fluorochrome-conjugated phalloidin

Publisher Summary This chapter describes the staining of actin with fluorochrome-conjugated phalloidin. Phalloidin interacts specifically with yeast and other actins; the interaction appears to be specific for polymerized rather than unpolymerized actin. The coupling of fluorochromes to phalloidin thus provides a quick and convenient means of visualizing the actin cytoskeleton in various types of cells; in some cases, staining of living as well as fixed cells has been achieved. In yeast, phallotoxin staining can be accomplished to date only with fixed cells; this staining reveals patterns of localization very similar to those seen by immunofluorescence. Good results with both the fluorescein and tetramethylrhodamine derivatives of phalloidin. It is suggested that in microscopy methods, staining of bigger cells is generally more informative and facilitates photomicroscopy; thus, diploids are generally more satisfactory than haploids, and tetraploids are better yet. The importance of rapid fixation should also be stressed; like many other aspects of cell structure, the actin network rearranges rapidly when the cells are subjected to stresses such as the loss of an energy source during washes with glucose-free buffer.

Isoform-specific complementation of the yeast sac6 null mutation by human fimbrin.

The actin cytoskeleton is a fundamental component of eukaryotic cells, with both structural and motile roles. Actin and many of the actin-binding proteins found in different cell types are highly conserved, showing considerable similarity in both primary structure and biochemical properties. To make detailed comparisons between homologous proteins, it is necessary to know whether the various proteins are functionally, as well as structurally, conserved. Fimbrin is an example of a cytoskeletal component that, as shown by sequence determinations and biochemical characterizations, is conserved between organisms as diverse as Saccharomyces cerevisiae and humans. In this study, we examined whether the human homolog can substitute for the yeast protein in vivo. We report here that two isoforms of human fimbrin, also referred to as T- and L-plastin, can both substitute in vivo for yeast fimbrin, also known as Sac6p, whereas a third isoform, I-fimbrin (or I-plastin), cannot. We demonstrate that the human T- and L-fimbrins, in addition to complementing the temperature-sensitive growth defect of the sac6 null mutant, restore both normal cytoskeletal organization and cell shape to the mutant cells. In addition, we show that human T- and L-fimbrins can complement a sporulation defect caused by the sac6 null mutation. These findings indicate that there is a high degree of functional conservation in the cytoskeleton, even between organisms as diverse as S. cerevisiae and humans.

1 The Yeast Cytoskeleton

INTRODUCTION An internal cytoskeleton consisting of a network of protein polymer filaments is a common feature of all eukaryotic cells. The cytoskeleton serves to organize the cytoplasm, to provide the means for generating force within the cell, and to determine and maintain the shape of the cell and its structural integrity. Three different types of cytoskeletal filaments are found. One type is based on long flexible polymers of actin called microfilaments (5–7 nm in diameter), another is based on more rigid polymers of tubulin called microtubules (hollow tubes 25 nm in diameter), and the third type is based on polymers of any of a number of related fibrous coiled-coil proteins called intermediate filaments (~10 nm in diameter). The microfilaments and microtubules of Saccharomyces cerevisiae closely resemble those of animal cells, and the structure and functions of these cytoskeletal elements have been extensively studied in this yeast (Botstein 1986; Huffaker et al. 1987; Drubin 1989; Stearns 1990; Welch et al. 1994; Cid et al. 1995). Potential nuclear lamins and 10-nm filament structures have also been reported in Saccharomyces, but relatively little is known about the structure and functions of intermediate filaments in yeast (Haarer and Pringle 1987; Kim et al. 1991; Chant 1996). The cytoskeleton is a complex architecture of polymers and associated proteins that plays a part in many aspects of cellular physiology. Understanding the elaborate system of interactions upon which cytoskeletal function is based requires tools that can be used on the intact organism. Indeed, much of what...