MOLECULAR ANALYSIS OF FLAGELLAR DYNEIN FUNCTION

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Dyneins are microtuble-based molecular motors involved in a wide variety of cellular functions: e.g. sperm motility and intracellular vesicle transport. Dyneins from the Chlamydomonas flagellum are especially useful as a model system as they are amenable to both biochemical and molecular genetic analysis. The outer dynein arm consists of 3 heavy chains (greater than or equal to 500 kDa), 2 intermediate chains of 69 and 78 kDa (IC69 and IC78) and 10 light chains. The heavy chains contain the motor activity of the complex. The ICs are related to each other and to the ICs of cytoplasmic dynein. Previous studies have raised the possibility that these IC components are involved in the regulation of arm activity and in the binding of the dynein motor to the appropriate cargo (which in the case of a flagellar dynein is another microtubule). Thr proposed research plan has three related specific aims. In the first, the regions of IC78 that are involved in binding to microtubules will be defined by site-directed mutagenesis and in vitro binding assays. Subsequently, the functional role of these domains in the targeting of outer arm dynein will be assessed in vivo by transformation of altered genes into Chlamydomonas strains that are true nulls for this dynein component. Detailed understanding of how microtubule-binding is achieved also requires identification of the regions of tubulin involved. The second specific aim is to determine the IC78 binding sites on tubulin using in vitro assays to examine the binding of mutant tubulins synthesized in vitro or of tubulin segments expressed using phage display technology. This analysis ultimately will be extended using the reagents generated here to identify the tubulin regions involved in binding to other microtubule-associated proteins including the motor domains of dynein and kinesin. Finally, there is molecular genetic and biochemical evidence that dynein ICs may be involved in the regulation of outer arm activity and that one IC may contain a nucleotide binding site. Thus, the third specific aim of this project is to identify this domain within IC78 in vitro using molecular biological and/or protein chemistry approaches. The functional significance of this domain then will be determined in vivo by transforation of altered genes into the IC78-nullo background. In summary, these studies will provide insight into the functional roles of dynein ICs in targeting of the dynein complex to its cargo, in the regulation of motor activity and also will provide insight into the regions of tubulin involved in mediating both structural and mechanochemical interactions.