Kinesin-Like Proteins in Eukaryotic Flagella and Cilia

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9808654 Bernstein The kinesins are a diverse superfamily of cytoplasmic microtubule (MT)-dependent motor proteins that transduce chemical energy into mechanical forces that are applied during a variety of critical cellular events, including mitosis, meiosis, and vesicular transport. Over eighty different kinesin-like proteins (klps) have been identified, whose known roles fall into two broad categories: those involved in transport of membranous cargo and those required during mitosis. The precise function of most klps, however, is unknown. Recently, klps have been described in flagella and cilia, the MT-based structures that eukaryotic cells use to either swim through an aqueous environment or move the aqueous environment past the cell's surface. The objective of this project is to understand the function of two kinesins in ciliary and flagellar function, using a combination of molecular genetic, biochemical and cytological approaches with the unicellular eukaryotes, Chlamydomonas and Tetrahymena. Klp1 is a Chlamydomonas flagellar klp found in flagellar central pair MTs. It has been suggested that Klp1 is required for flagellar beating. To test this hypothesis, the phenotype of Klp1-deficient flagella will be determined. The deficiency will be achieved by construction of dominant-negative Klp1 mutations in vitro by site-directed alteration of the ATP binding site of Klp1, followed by transformation of the mutant genes back into Chlamydomonas cells. Cells expressing mutant forms of Klp1 will be assayed for fine-structural cytological defects and both in vitro and in vivo for defects in flagellar function. Immunoblot analysis has indicated that Klp1 is also present in the cilia of Tetrahymena. The Tetrahymena Klp1 gene will be cloned using PCR and a klp1 null mutant of Tetrahymena will be constructed. The Tetrahymena klp1 null mutants will be examined for defective ciliary motility and for ultrastructural cytological defects. Two-dimensional gel analysis will be used to det ermine if ciliary polypeptides are missing from Klp1-deficient cilia. To determine if Klp1 is required for proper orientation of central pair MTs, serial thin sections will be examined to compare the position of the central pair MTs relative to the outer doublet MTs in mutant versus wild type cilia. The flagellar kinesin-like protein Fla10 is a member of the kinesin II/Kif3 subfamily of kinesins that is present in Chlamydomonas flagella, and which is required for flagellar membrane-associated motilities and for flagellar growth and maintenance. A new Fla10-related kinesin, Kin5, has been cloned from Tetrahymena by PCR. The complete genomic clone for Kin5 will be obtained and used in transformation and gene replacement protocols to generate a Tetrahymena delta-kin5 deletion mutant. The delta-kin5 mutant will be analyzed for ciliary beating, and video enhanced DIC microscopy will be used to examine mutant cilia for the membrane-associated motility. The results of these experiments will advance our understanding of the structure and function of the cytoskeleton, and specifically the functions of kinesin-like proteins and eukaryotic cilia and flagella. Due to the highly conserved structures and biochemical compositions of cilia and flagella, MTs, and the kinesin-like proteins, the insights into kinesin and ciliary/flagellar function that will be gained by studying these easily manipulated protozoans will be applicable to klps and cilia/flagella of higher eukaryotic cells. In addition, the concepts gained by studying flagellar and ciliary kinesins may be useful in understanding other, more complex and dynamic, MT arrays, including the mitotic spindle. The studies described impact science education and human resource development in that they will be carried out as part of the training of graduate students. By carrying out the studies described, the students will be trained academically and technically to become independent scientists in the field of cell biology who are expected to eventually contribute significantly to intellectual and technical advances in either an academic or industrial setting.