Improved Plasmids for Fluorescent Protein Tagging of Microtubules in <i>Saccharomyces cerevisiae</i>

Abstract

The ability to fluorescently label microtubules in live cells has enabled numerous studies of motile and mitotic processes. Such studies are particularly useful in budding yeast owing to the ease with which they can be genetically manipulated and imaged by live cell fluorescence microscopy. Because of problems associated with fusing genes encoding fluorescent proteins (FPs) to the native α-tubulin (TUB1) gene, the FP-Tub1 fusion is generally integrated into the genome such that the endogenous TUB1 locus is left intact. Although such modifications have no apparent consequences on cell viability, it is unknown if these genome-integrated FP-tubulin fusions negatively affect microtubule functions. Thus, a simple, economical and highly sensitive assay of microtubule function is required. Furthermore, the current plasmids available for generation of FP-Tub1 fusions have not kept pace with the development of improved FPs. Here, we have developed a simple and sensitive assay of microtubule function that is sufficient to identify microtubule defects that were not apparent by fluorescence microscopy or cell growth assays. Using results obtained from this assay, we have engineered a new family of 30 FP-Tub1 plasmids that use various improved FPs and numerous selectable markers that upon genome integration have no apparent defect on microtubule function.