Extragenic suppressors of paralyzed flagellar mutations in Chlamydomonas reinhardtii identify loci that alter the inner dynein arms.

Abstract

Abstract. We have analyzed extragenic suppressors of paralyzed flagella mutations in Chlamydomonas reinhardtii in an effort to identify new dynein mutations. A temperature-sensitive allele of the Pb16 locus was mutagenized and then screened for revertants that could swim at the restrictive temperature (Dutcher et al. 1984. J. Cell Biol. 98:229-236). In backcrosses of one of the revertant strains to wild-type, we recovered both the original pf16 mutation and a second, unlinked suppressor mutation with its own flagellar phenotype. This mutation has been identified by both recombination and complementation tests as a new allele of the previously uncharacterized PF9 locus on linkage group XII/XnI. SDS-PAGE analysis of isolated flagellar axonemes and dynein extracts has demonstrated that the pfl) strains are missing four polypeptides that form the I1 inner arm dynein subunit. The primary effect of the loss of the I1 subunit is a decrease in the forward swimming velocity due to a change in the flagellar waveform. Both the flagellar beat frequency and the axonemal ATPase activity are nearly wild-type. Examination of axonemes by thin section electron microscopy and image averaging methods reveals that a specific domain of the inner arm complex is missing in the pfl) mutant strains (see accompanying paper by Mastronarde et al.). When combined with other flageUar defects, the loss of the I1 subunit has synergistic effects on both flagellar assembly and flagellar motility. These synthetic phenotypes provide a screen for new suppressor mutations in other loci. Using this approach, we have identified the first interactive suppressors of a dynein arm mutation and an unusual bypass suppressor mutation.T HE dynein ATPases are large and complex multisubunit enzymes that provide the force for a variety ofmicrotubule based movements inside cells (reviewed in Gibbons, 1988;Porter and Johnson, 1989). In cilia and flagella, at least nine different dynein heavy chains have been described; these and other polypeptides are arranged into at least four different dynein arm subunits (Piperno et al., 1990). The presence of these multiple dynein isoforms within the axoneme provides the cell with several different potential mechanisms for affecting the flagellar waveform, but how the activity of the different dyneins is coordinated is not well understood. Both the inner and outer dynein arms can drive the sliding movements between adjacent outer doublet microtubules that serve as the basis for ciliary movement. However, study of flagellar mutations in Chlamydomonas has revealed that the different dynein arms have different functions in the generation of flagellar motility. For instance, mutant strains that lack outer arm polypeptides swim with a reduced flagellar beat frequency, whereas mutant strains that lack subsets of inner arm polypeptides swim with an altered flagellar waveform (Brokaw and Kamiya, 1987).The flagellar dyneins also play an important but poorly understood role in modifying the flagellar waveform in response to signals f...

Casein Kinase I Is Anchored on Axonemal Doublet Microtubules and Regulates Flagellar Dynein Phosphorylation and Activity

“…Reconstitution and functional assays demonstrate that the radial spokes are required for wild-type dynein activity, and the velocity of dyneindriven microtubule sliding is mediated by posttranslational modification of the inner dynein arms (20). These results are consistent with other evidence indicating that the central pair/ radial spoke apparatus along with the dynein regulatory complex controls dynein activity (14,17,(21)(22)(23)(24)(25)(26). The challenge has been to define the biochemical signaling pathway and to determine how this biochemistry can be altered by mechanical interaction between the central pair structures, radial spoke structures, and outer doublet microtubule components to control dynein activity.…”

The radial spokes and central apparatus: Mechano‐chemical transducers that regulate flagellar motility

“…In contrast, for double mutants lacking I1 and the central apparatus, microtubule sliding velocities are restored to nearly wild-type velocity in the absence of any kinase inhibitors [Smith, 2002b]. And, mutations that affect I1 assembly can suppress paralysis in mutants lacking the C1 microtubule of the central apparatus [Porter et al, 1992]. These observations reveal a distinct role for the I1 dynein subtype and provide a functional link between the central apparatus, radial spokes, and inner dynein arm I1.…”

Asymmetry of the central apparatus defines the location of active microtubule sliding in Chlamydomonas flagella

“…3). Recent evidence has revealed that inner dynein arm subform I1 may play a role in controlling flagellar bending (23,31,(35)(36)(37)(38)(39). Therefore, we analyzed central apparatus orientation in axonemes isolated from the I1 defective strain ida1.…”