Flagellar mutants of Chlamydomonas : Studies of radial spoke-defective strains by dikaryon and revertant analysis

Abstract

The motility mutant of Chlamydomonas reinhardtii pfl4 lacks radial spoke structures in its flagellar axonemes, and 12 proteins present in wild type are missing from a two-dimensional map (isoelectrofocusing/sodium dodecyl sulfate electrophoresis) of its 35S-labeled flagellar proteins, Six of these same proteins are missing in fi, which lacks spokeheads. To determine whether any of the missing roteins represent the mutant gene product two experimenta approaches have been applied. The first makes use of the fact that gametes of either mutant strain when fused with wild-type gametes to form quadriflagellate dikaryons undergo recovery of flagellar function. Recovery at the molecular level was monitored by prelabeling the mutant proteins with 35S and allowing recovery to occur in the absence of protein synthesis. It is to be expected that the mutant gene product would not be restored as a radioactive protein and that recovery would depend on the assembly of the wild-type counterpart that is not labeled. The second technique makes use of revertants induced by UV irradiation. Dikaryon rescue in the case of pfl4 leads to restoration of 11 radioactive components; only protein 3 fails to appear as a radioactive spot. For pfl only two radioactive proteins are restored; proteins 4, 6, 9, and 10 were not radioactive. Analysis of revertants of pfl gave evidence (altered map positions) that protein 4 is the mutant gene product. In the case of pfI4, analysis of 22 revertants has not provided similar positive evidence that protein 3 is the gene product.Previously we have analyzed flagellar proteins in paralyzed mutants of Chlamydomonas reinhardtii using a two-dimensional system of isoelectric focusing followed by electrophoresis in the presence of sodium dodecyl sulfate (1). In the case of the mutant pfl4, a two-dimensional map of flagellar proteins was shown to lack 12 characteristic polypeptides that were regularly present in wild-type flagella. This deficiency could be correlated with the total absence of radial spokes and associated spokeheads in the axonemal structures of pfl4. Another mutant, pfl was shown to lack 6 of these 12 flagellar polypeptides, and analysis of axonemal structures revealed that the radial spokes were present but that spokeheads were absent.Since pfl4 and pfl have been shown to be independent single-site mutations (2), the deficiency of all spoke structures in one case and of spokeheads in the other indicates that the mutant gene products play critical roles in the assembly of as many as 12 different proteins in the case of pfl4 and 6 in the case of pfl. Two complementary approaches have been undertaken to determine if the mutant gene products of pfl4 and/or pfl are structural components of the radial spoke.The first type of experiments makes use of quadriflagellate dikaryon cells which are produced after gametic fusion in the Chlamydomonas mating cycle. It has been observed that in dikaryons of some paralyzed mutants of C. moewusii (3) or C. reinhardtni (4) with the counterpart wild-type stra...

Mutations at twelve independent loci result in absence of outer dynein arms in Chylamydomonas reinhardtii.

“…As in the case of other motility mutants (Luck et al, 1977), zygotes between many, but not all, combinations of different oda mutants recovered a higher level of motility during this period as a result of complementation. In most cases, enhanced motility in heterozygotes could be readily discerned by simply comparing their swimming velocity with the velocity of homozygotes.…”

Central-pair microtubular complex of Chlamydomonas flagella: polypeptide composition as revealed by analysis of mutants.

“…Culture of cells (10), labeling with ['SS]sulfuric acid (4), labeling with ['P]-phosphoric acid (2), preparation of flagella and axonemes (4), and electron microscopy (4) were performed as previously described.…”

Moving to the beat: a review of mammalian sperm motility regulation

“…In green algae, over 200 axonemal and axoneme-associated proteins have been described and it is likely that mammalian axonemes are at least as complex. Defects in axonemal proteins can lead to abnormal assembly of the axoneme and/or aberrant motility (Luck et al 1977;Dutcher 1995;Yagi and Kamiya 1995;Piperno et al 1996;Smith and Lefebvre 1996).…”

“…For example, axonemal defects can result in poor or absent sperm motility and associated infertility (as in primary ciliary dyskinesia; Afzelius 1976;Narayan et al 1994), deafness (if the disorder affects the auditory hair cells, as in Usher's syndrome; Smith et al 1994;Well et al 1995), blindness (if the defect involves the modified ciliated cells (photoreceptor cells) of the retina, as in retinitis pigmentosa; Hunter et al 1988;Humphries et al 1992), chronic respiratory disease (if the defect involves ciliated cells of the respiratory tract, as in primary ciliary dyskinesia; Blouin et al 2000) or some combination of these problems. Large-scale mutagenesis experiments have been performed on axonemal proteins in green algae and, thus, in this organism, many axonemal protein defects have been shown to result in flagellar immotility (Luck et al 1977;Dutcher 1995).…”