Two-dimensional analysis of flagellar proteins from wild-type and paralyzed mutants of Chlamydomonas reinhardtii.

Abstract

(14). Flagella were removed from cells suspended at a concentration of approximately 2 X 107/ml in 10 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes) (pH 7.2), 0.5 mM CaCl2, and 1 mM dithiothreitol by pH shock (3) or by addition of Nonidet P40 (14). Flagella or axonemes were isolated by differential centrifugation, solubilized in sodium dodecyl sulfate (NaDodSO4) and analyzed by two-dimensional gel electrophoresis. Isoelectric focusing was started within 3-5 hr of the time flagella were detached from cell bodies in order to limit artifactual modification of flagellar proteins. The method of analysis described by Ferro-Luzzi Ames and Nikaido (13) was modified as follows: to obtain a linear pH gradient in the range of 3.5-9.5, ampholines were mixed according to the LKB (Bromma, Sweden) application note on the model 2117 Multiphor; the amount of riboflavin added for polymerization of the slab gel used for the isoelectric focusing was 10% of the amount indicated; the polyacrylamide gel slab chosen for the second electrophoresis was prepared by a modification (14) of the method of Neville (16); and, protein samples of approximately 3 ,ug (1 X 105 cpm/,ug) were mixed with one-half volume of undiluted Nonidet P40 and 2 volumes of the sample buffer before being applied to the gels. The proteins used as standards were: spectrin heavy subunit, W-galactosidase, bovine-serum albumin, ovalbumin, chymotrypsinogen, and hemoglobin. Exposure time for autoradiograms was approximately 1 week. RESULTS Two-dimensional separation of flagellar polypeptidesTo detect flagellar components present in low quantities, we labeled cells to high specific activity by including [a5S]sulfate in the medium. We observed that cell growth was not altered by use of the isotope at a specific activity of 500 Ci/mol of sulfate. Consequently, our analyses will detect only sulfurcontaining proteins and the number of components observed will also be a function of the sensitivity of the film and the exposure time used for autoradiography.The two-dimensional method we used (13) included isoelectric focusing in one dimension and gradient gel electrophoresis with NaDodSO4 in the other. In analyzing the maps we obtained, including the gel strip from the first dimension after electrophoresis in the second dimension, we recognized that there were other limits to the types of polypeptides we could resolve. While the majority of components fell into a molecular weight range of 300,000-15,000 with isoelectric points between 7 and 4 and were well resolved (Fig. 1A), molecules with isoelectric points between 8.5 and 7 formed long streaks. We also observed that polypeptides wth molecular weight higher than 300,000 did not migrate to their isoelectric points; when loaded at the anode for the first dimension instead 1600 Abbreviation: NaDodSO4, sodium dodecyl sulfate. * We did the mapping studies after finding that two of these cultures maintained in our collection were incorrectly identified.

Analysis of microtubule sliding patterns inChlamydomonasflagellar axonemes reveals dynein activity on specific doublet microtubules

“…5. The pf14 mutant has paralyzed flagella that fail to assemble radial spokes; the pf17 mutant has paralyzed flagella that retain the radial spoke stalks but lack the radial spoke heads (Piperno et al, 1977). Evidently, in the absence of radial spokes or spoke heads the patterns of dynein-driven microtubule sliding remain unchanged in response to increased Ca 2+ concentration.…”

“…In this study, the principal components summarize the major patterns of variation among axonemes isolated from multiple strains and induced to slide in Table 1. Chlamydomonas strains used in this study Strain Structural/motility defect A54-e18 no axonemal structural defect/wild-type motility (Smith and Lefebvre, 1996) 137c no axonemal structural defect/wild-type motility (Harris, 1989) oda1, pf28 lack outer dynein arms/flagella beat with one-half frequency (Kamiya and Okamoto, 1985;Mitchell and Rosenbaum, 1985) ida1, pf30 lacks inner dynein arm subform I1/ reduced beat frequency, altered waveform (Brokaw and Kamiya, 1987) pf30pf28 lacks outer dynein arms and inner dynein arm I1 (Piperno et al, 1990;Smith and Sale, 1992) pf3 defects in dynein regulatory complex/reduced beat frequency, altered waveform (Brokaw and Kamiya, 1987;Kamiya et al, 1991;Piperno et al, 1992) pf6 central apparatus, lacks 1A projection/flagella paralyzed or twitch (Dutcher et al, 1984) cpc1 central apparatus, lacks 1B projection/reduced beat frequency (Mitchell and Sale, 1999) pf14 lacks radial spokes/paralyzed flagella (Piperno et al, 1977) pf17 lacks radial spoke heads/paralyzed flagella (Huang et al, 1981) Fig conditions of low and high Ca 2+ buffer; these analyses revealed major trends in microtubule sliding patterns and the identities of doublet microtubules with active dynein arms. The results of the PCs analyses for each variable set are shown in Fig.…”

Rotation and twist of the central-pair microtubules in the cilia of Paramecium.

“…These authors postulate that the spoke-central sheath interaction converts the sliding to bending . Mutant cilia missing radial spokes are paralyzed (32,45,53) . The nine spoke heads connected to the nine peripheral doublets face the asymmetric central pair and its associated structures differently, and therefore are not expected to interact with the central structures in the same way.…”

“…The functions in motility of such components as the radial spokes and the central-tubule complex are largely un-THE JOURNAL OF CELL BIOLOGY " VOLUME 87 OCTOBER 1980 [33][34][35][36][37][38][39][40][41][42][43][44][45][46] ©The Rockefeller University Press " 0021-9525/80/10/0033/14 $1 .00 known . Nevertheless, they must be essential because mutational loss of these components results in paralysis (32,45,52) .…”

Analysis of flagellar size control using a mutant of Chlamydomonas reinhardtii with a variable number of flagella.

“…: " j complex, containing well in excess of 100 structural proteins (19 Relationship between cell volume and flagellar length for 50 NO mt+ gametes, (a) before deflagellation and (b) after 60 min of regeneration in the presence of cycloheximide . its ultrastructure is highly ordered, with identical elements repeating numerous times from base to tip, it is in some ways valid to think of the organelle as a polymer, with assembly permitted only at the ends.…”