6 research outputs found

    The Tctex1/Tctex2 and LC7/Roadblock families of dynein light chains are required for wild-type assembly, stability, and motor function within the Chlamydomonas flagellum

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    Chlamydomonas is a unicellular green algae that uses its two flagella to swim through its environment. The flagella are composed of a microtubule-based axoneme that provides a scaffold for greater than 450 proteins that collectively regulate motility. The ultimate force-producing components associated with the axoneme are dynein molecular motors. Dyneins are minus-end directed microtubule-based motors that perform an array of functions within the cytoplasm of eukaryotic cells, as well as within cilia and flagella. Cytoplasmic dyneins are involved in such processes as nuclear envelope breakdown, mitosis, maintenance of the golgi apparatus, intraflagellar transport, and the movement of an array of cargoes towards the nucleus, including membrane-bound vesicles, proteins, RNAs, and even viral components. Flagellar dyneins assemble along the length of the axoneme and generate the force necessary for flagellar bending and movement. Cytoplasmic, axonemal outer arm and inner arm I1 dyneins are composed of two or three heavy chains, two or three intermediate chains, and a series of light chains (LCs) that belong to three protein families: LC8, Tctex1/Tctex2, and LC7/Roadblock. Here I demonstrate that members of the Tctex1/Tctex2 family are differentially expressed in a tissue-specific and developmentally-regulated manner, suggesting that within cells, cargo binding specificity may be modulated by LC content. In addition, a novel member of this family associated with axonemal inner arm I1, termed Tctex2b, is required for the stability of this dynein and in its absence results in reduced motor function as measured by three different functional assays, both in vivo and in vitro. Two members of the LC7/Roadblock family associate with two dynein systems in the flagellum: the outer dynein arm and inner arm I1. In addition, one of these proteins is required for wild-type assembly and stability of both dyneins. The second interacts with regulatory components of each motor and identifies the first link between the outer arm motor unit and the trimeric docking complex which is required for the correct placement of the outer arm in the axoneme. Together, these data suggest that dynein LCs are integral components required for assembly, stability, and proper motor function.

    The LC7 Light Chains of Chlamydomonas Flagellar Dyneins Interact with Components Required for Both Motor Assembly and Regulation

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    Members of the LC7/Roadblock family of light chains (LCs) have been found in both cytoplasmic and axonemal dyneins. LC7a was originally identified within Chlamydomonas outer arm dynein and associates with this motor's cargo-binding region. We describe here a novel member of this protein family, termed LC7b that is also present in the Chlamydomonas flagellum. Levels of LC7b are reduced ∼20% in axonemes isolated from strains lacking inner arm I1 and are ∼80% lower in the absence of the outer arms. When both dyneins are missing, LC7b levels are diminished to <10%. In oda9 axonemal extracts that completely lack outer arms, LC7b copurifies with inner arm I1, whereas in ida1 extracts that are devoid of I1 inner arms it associates with outer arm dynein. We also have observed that some LC7a is present in both isolated axonemes and purified 18S dynein from oda1, suggesting that it is also a component of both the outer arm and inner arm I1. Intriguingly, in axonemal extracts from the LC7a null mutant, oda15, which assembles ∼30% of its outer arms, LC7b fails to copurify with either dynein, suggesting that it interacts with LC7a. Furthermore, both the outer arm γ heavy chain and DC2 from the outer arm docking complex completely dissociate after salt extraction from oda15 axonemes. EDC cross-linking of purified dynein revealed that LC7b interacts with LC3, an outer dynein arm thioredoxin; DC2, an outer arm docking complex component; and also with the phosphoprotein IC138 from inner arm I1. These data suggest that LC7a stabilizes both the outer arms and inner arm I1 and that both LC7a and LC7b are involved in multiple intradynein interactions within both dyneins

    Differential Light Chain Assembly Influences Outer Arm Dynein Motor Function

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    Tctex1 and Tctex2 were originally described as potential distorters/sterility factors in the non-Mendelian transmission of t-haplotypes in mice. These proteins have since been identified as subunits of cytoplasmic and/or axonemal dyneins. Within the Chlamydomonas flagellum, Tctex1 is a subunit of inner arm I1. We have now identified a second Tctex1-related protein (here termed LC9) in Chlamydomonas. LC9 copurifies with outer arm dynein in sucrose density gradients and is missing only in those strains completely lacking this motor. Zero-length cross-linking of purified outer arm dynein indicates that LC9 interacts directly with both the IC1 and IC2 intermediate chains. Immunoblot analysis revealed that LC2, LC6, and LC9 are missing in an IC2 mutant strain (oda6-r88) that can assemble outer arms but exhibits significantly reduced flagellar beat frequency. This defect is unlikely to be due to lack of LC6, because an LC6 null mutant (oda13) exhibits only a minor swimming abnormality. Using an LC2 null mutant (oda12-1), we find that although some outer arm dynein components assemble in the absence of LC2, they are nonfunctional. In contrast, dyneins from oda6-r88, which also lack LC2, retain some activity. Furthermore, we observed a synthetic assembly defect in an oda6-r88 oda12-1 double mutant. These data suggest that LC2, LC6, and LC9 have different roles in outer arm assembly and are required for wild-type motor function in the Chlamydomonas flagellum

    Zebrafish Tsc1 reveals functional interactions between the cilium and the TOR pathway

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    The cell surface organelle called the cilium is essential for preventing kidney cyst formation and for establishing left–right asymmetry of the vertebrate body plan. Recent advances suggest that the cilium functions as a sensory organelle in vertebrate cells for multiple signaling pathways such as the hedgehog and the Wnt pathways. Prompted by kidney cyst formation in tuberous sclerosis complex (TSC) patients and rodent models, we investigated the role of the cilium in the TSC–target of rapamycin (TOR) pathway using zebrafish. TSC1 and TSC2 genes are causal for TSC, and their protein products form a complex in the TOR pathway that integrates environmental signals to regulate cell growth, proliferation and survival. Two TSC1 homologs were identified in zebrafish, which we refer to as tsc1a and tsc1b. Morpholino knockdown of tsc1a led to a ciliary phenotype including kidney cyst formation and left–right asymmetry defects. Tsc1a was observed to localize to the Golgi, but morpholinos against it, nonetheless, acted synthetically with ciliary genes in producing kidney cysts. Consistent with a role of the cilium in the same pathway as Tsc genes, the TOR pathway is aberrantly activated in ciliary mutants, resembling the effect of tsc1a knockdown. Moreover, kidney cyst formation in ciliary mutants was blocked by the Tor inhibitor, rapamycin. Surprisingly, we observed elongation of cilia in tsc1a knockdown animals. Together, these data suggest a signaling network between the cilium and the TOR pathway in that ciliary signals can feed into the TOR pathway and that Tsc1a regulates the length of the cilium itself
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