IMAGE ANALYSIS OF INNER DYNEIN ARM MUTANTS IN CHLAMYDOMONAS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The motility of eukaryotic cilia and flagella depends on the presence of multiple dynein motors that convert the chemical energy derived from ATP binding and hydrolysis into mechanical forces that drive microtubule sliding within the axoneme. One unresolved issue is the structural arrangement of inner arm dynein subunits within the axoneme and their relationship to the regulatory machinery that coordinates their activity. The current model for the regulation of flagellar motility is that mechanical interactions between the central pair microtubules and radial spokes are converted into a biochemical signaling pathway that ultimately alters the phosphorylation state of the dynein arms within the 96 nm axoneme repeat. We have focused on the I1 inner arm dynein, which is an important target of the radial spoke-central pair complex. The I1 dynein is composed of two distinct heavy chains (1-alpha and 1-beta), three intermediate chains (IC140, IC138, IC97), and several light chains, and its activity is altered by changes in the phosphorylation state of IC138. Thus far we have characterized mutations in both DHC subunits and two IC subunits that alter the assembly of the I1 dynein. Analysis of wild-type and I1 mutant axonemes using chemical fixation and conventional thin section microscopy in combination with computer image averaging have provided a 2-D image of the I1 dynein as a tri-lobed structure located proximal to the first radial spoke within each 96 nm axoneme repeat. Transformations with dynein heavy chain constructs encoding only the N-terminal stem domain result in partial rescue of the mutant phenotypes and reassembly of I1 dynein complexes lacking either the 1-alpha or 1-beta motor domains. Image analysis of the rescued strains demonstrated that each motor domain could be correlated with one lobe of the I1 structure. These studies further suggested the IC/LC complex would be located within the third lobe of the I1 structure, in close proximity to the axonemal kinases and phosphatases associated with the radial spokes. Recent studies of a new mutant, 6F5, which assembles an I1 dynein lacking the IC138 phosphoprotein, has revealed defects within the third lobe of the I1 structure, consistent with previous predictions