ANTIGEN LOCALIZATION IN FISSION YEAST

This chapter reviews applications of immunolocalization methodology to the cytology and ultrastructure of fission yeast Schizosaccharomyces pombe and its distant relative Schizasaccharomyces japonicus. Three immunofluorescence methods were developed to localize cytoskeletal proteins in both S. pombe and spindle pole body. The three methods involve fixation in (1) formaldehyde, (2) formaldehyde plus glutaraldehyde, and (3) methanol. Microtubules are best preserved by both aldehydes in combination. Formaldehyde alone appears to give excellent resolution of actin. The state of the cells used depends on the nature of the experiment. In most cases, it is desirable to start with a culture in the midexponential phase of growth, however all three methods work equally well for cells arrested at different phases of the cell cycle or in stationary phase. Fluorescent probes that specifically recognize a particular cellular structure or organelle are a familiar tool to most cell biologists. Some of the probes that work particularly well in S. pombe include 4′,6-diamidino-2-phenylindole (DAPI), calcofluor, and Rhodamine 123. The small size of the fission yeast cell and the limit of resolution of the light microscope both restrict the amount of structural data that can be obtained by optical methods. © 1993, Academic Press Inc.