Abstract
Dynein light chain 1 (LC1/DNAL1) is one of the most highly conserved components of ciliary axonemal outer arm dyneins, and it associates with both a heavy chain motor unit and tubulin located within the A-tubule of the axonemal outer doublet microtubules. In a variety of model systems, lack of LC1 or expression of mutant forms leads to profound defects in ciliary motility, including the failure of the hydrodynamic coupling needed for ciliary metachronal synchrony, random stalling during the power/recovery stroke transition, an aberrant response to imposed viscous load, and in some cases partial failure of motor assembly. These phenotypes have led to the proposal that LC1 acts as part of a mechanical switch to control motor function in response to alterations in axonemal curvature. Here we have used NMR chemical shift mapping to define the regions perturbed by a series of mutations in the C-terminal domain that yield a range of phenotypic effects on motility. In addition, we have identified the subdomain of LC1 involved in binding microtubules and characterized the consequences of an Asn → Ser alteration within the terminal leucine-rich repeat that in humans causes primary ciliary dyskinesia. Together, these data define a series of functional subdomains within LC1 and allow us to propose a structural model for the organization of the dynein heavy chain-LC1-microtubule ternary complex that is required for the coordinated activity of dynein motors in cilia.
Highlights
Ciliary dyneins monitor and respond to the mechanical state or curvature of the microtubular axoneme
Perturbations Caused by Dominant Negative Mutations in light chain 1 (LC1) C-terminal Domain—Previous single residue mutagenesis experiments (Ref. 12 and see Table 1) suggested that the C-terminal domain might represent a key functional subdomain of LC1, intriguingly there are significant sequence differences observed within this region when assessed across a broad phylogenetic spectrum (Fig. 1, a– c)
As long range perturbations could disrupt other segments of the molecule, we set out to determine the extent to which the introduced mutations affected the structure of LC1 and define the region responsible for the observed phenotypic effects
Summary
Ciliary dyneins monitor and respond to the mechanical state or curvature of the microtubular axoneme. We found that expression of mutant forms of LC1 in which these Arg residues or the Met/Asp exhibiting high dynamics were altered yielded dominant negative phenotypes in a wild-type background [12] These Chlamydomonas cells swam slowly as they were unable to coordinate their flagella movement due to random stalling during the beat cycle and especially at the power/recovery stroke transition. To gain further insight into the mechanisms involved, we have used targeted mutagenesis to delineate the segment of this molecule involved in associating the dynein motor unit with the outer doublet A-tubule and have characterized the structural consequences of the PCD-causing mutation within the terminal LRR Together, these data allow functional subdomains within LC1 and the overall geometry to be defined. The results implicate the LC1 C-terminal domain in controlling motor function through direct interactions within the HC AAA domains
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