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Purification of basal body and basal body complexes from Sourcebook

Purification of basal body and basal body complexes from Sourcebook. fragmented into numerous short segments, presumably by disruption of tubulinCtubulin bonds at indiscriminate points along the length of the microtubule, eventually leading to total disassembly. In contrast, depolymerization via dynamic instability is restricted to loss of tubulin subunits from your ends of microtubules (Mitchison and Kirschner, 1984 ). The microtubule-severing activity was shown to be regulated by the cell cycle; activity was low in interphase extracts and stimulated in extracts prepared from M phase oocytes (Vale, 1991 ) or interphase extracts activated by p34cdc2 kinase (Verde extracts, katanin requires the hydrolysis of ATP to disassemble microtubules. Neither p56, which has low severing activity compared with katanin, nor EF-1 requires ATP for severing activity (examined by Shiina may provide a useful system for the study of microtubule severing. deflagellation has focused mainly on analysis of the variable flagella number MIR96-IN-1 mutant mutation is usually a point mutation in the centrin gene resulting in gross abnormalities in all centrin-containing structures including complete loss of the contractile stellate fibers within the transition zone (Jarvik and Suhan, 1991 ; Taillon cells fail to deflagellate under certain experimental conditions, we (Lohret and Quarmby, unpublished observations) as well as others (Jarvik and Suhan, 1991 ) have found that cells deflagellate normally. We conclude that centrin is not necessary for the flagellar excision process. In the absence of a centrin-induced microtubule severing, Jarvik and Suhan (1991) speculated that a transition zone-localized microtubule-severing activity, comparable to that reported by Vale (1991) , may be responsible for outer doublet severing. In this study, we investigate the mechanism responsible for outer doublet severing during deflagellation. We find that micromolar free calcium induces axonemal severing in preparations of purified flagellar-basal body complexes (FBBCs) demonstrating that both the calcium sensor and microtubule-severing activity isolate with this cytoskeletal complex of axonemes plus basal body. The severing of axonemal doublet microtubules may proceed by a mechanism analogous to that of single microtubules (Vale, 1991 ; McNally and Vale, 1993 ; McNally axonemes. Like the severing of in vitro polymerized microtubules, the activity required ATP hydrolysis. This is a critical obtaining because it is the first demonstration of a microtubule-severing protein breaking the complex doublet microtubules of an axoneme and raises the exciting possibility that MIR96-IN-1 this severing of outer doublet microtubules during deflagellation may involve the specific action of a katanin-like severing activity. In support of this model, we show that affinity-purified antibodies raised against the 60-kDa subunit of human katanin recognize a single predominant protein at 55 kDa on Western BMP13 blots of both whole-cell and purified FBBCs. In addition, the antibody produced an intense staining of the basal body/flagellar transition region using indirect immunofluorescence in both whole cells and purified FBBCs. Importantly, the human p60 antibody significantly blocked Ca2+-stimulated axonemal severing in preparations of FBBCs. Taken together, these data provide evidence that an endogenous katanin may be involved in outer doublet severing during deflagellation. MATERIALS AND METHODS Chlamydomonas Strains and Culture Conditions wild-type strains 137c, cc620, and cc621 were obtained from Dr. E. Harris (Genetics Center, Botany Department, Duke University or college, Durham, NC). Cells MIR96-IN-1 were produced on 1.5% agar TAP plates (Harris, 1989 ) at 21C under constant illumination for 4C5 d. Cells were transferred from Tris acetate phosphate (TAP) plates into 4 ml of M-N media (M media of Sagar and Granick, 1953 [Harris, 1989 ] excluding NH4N03) and MIR96-IN-1 MIR96-IN-1 incubated for 3C5 h under constant light and agitation. Cells were collected by brief centrifugation, washed inside a Ca2+-free deflagellation buffer ([DB] 10 mM PIPES, pH 7.0, 5 mM EGTA, 0.5 mM MgCl2), and resuspended in DB to an approximate final density of 1 1 107 cells/ml. Detergent Permeabilization and Deflagellation Assay Cells (1 107 cells/ml) were permeabilized by addition of 10 quantities of DB comprising 0.05% Nonidet P-40 (Sigma Chemical, St. Louis, MO). For a typical experiment, 5 ml of 0.05% NP-40 in DB was added to 500 l cells. Deflagellation was induced.