Functional assays to identify and characterize regulators of microtubule behavior.
Previous experiments have clearly demonstrated that microtubule dynamic instability is regulated in living cells, but the molecular mechanisms that are responsible for this regulation are not well understood. We describe two rapid, functional assays that can be used to screen cell extracts for regulators of microtubule dynamic instability behavior. In both assays, highly purified tubulin is used to assemble microtubules from Tetrahymena axonemes. In the immunofluorescence assay, microtubules are visualized by fixation and staining with anti-tubulin antibodies. Alternatively, microtubule assembly has been visualized by the addition of rhodamine-labeled tubulin to axonemes, followed by low-light-level fluorescence microscopy. In either case, polymerization is quantified by measuring polymer length, total polymer and the number of microtubules per axoneme. In these assays, addition of brain microtubule-associated proteins (MAPs) results in a 2-fold-3-fold increase in average microtubule length, and addition of vinblastine results in a 50%-75% decrease in average microtubule length. The number of microtubules per axoneme was significantly increased by the addition of MAPs and significantly decreased by the addition of vinblastine. These functional assays can detect molecules that stimulate or suppress net microtubule assembly and provide a useful initial screen to isolate regulators of microtubule dynamic behavior.
McMeekan
J
J
University of Massachusetts, Amherst, USA.
Wadsworth
P
P
D000818:Animals; D001921:Brain; D002417:Cattle; D005455:Fluorescent Antibody Technique; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D012235:Rhodamines; D013347:Subcellular Fractions / Q000737:chemistry; D013552:Swine; D013768:Tetrahymena; D013950:Thymus Gland; D014747:Vinblastine / Q000494:pharmacology
D012235:Rhodamines; D014747:Vinblastine