Staining of actin with fluorochrome-conjugated phalloidin

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.