Flagella regeneration in the bi-flagellate alga Chlamydomonas is being used as a model system in which to define general principles underlying cell organelle formation. When the flagella are detached, transcription of genes for flagellar proteins is rapidly induced followed by translation of mRNAs for flagellar proteins and, within minutes, assembly of a new flagellum begins. We will investigate these processes studying, specifically, the synthesis and assembly of the flagellar radial spoke proteins. The spokes are attached to the A-tubules of the flagellar outer doublet microtubules, have a head and stalk which are composed of 17 polypeptides, and are required for motility. There are paralyzed mutants in several of these polypeptides and the genes for 5 have been cloned. Transformation can be used to rescue the paralyzed mutants back to motility. In vitro mutagenesis and transformation will be used to determine the radial spoke gene sequences responsible for (a) gene up- regulation following flagellar detachment, (b) targeting of radial spoke proteins to the flagellum, and (c) assembly of the proteins onto the outer doublet microtubules. Phosphorylation of radial spoke proteins is necessary for assembly of functional radial spokes. By use of mutants lacking a flagellar protein kinase, as well as in vitro mutagenesis and transformation, the role of radial spoke protein phosphorylation in either the targeting of proteins to the flagellum or their assembly onto the axoneme will be determined. Extracts from wild-type and radial spoke mutant cells will be used to assemble radial spokes onto spokeless axonemes in vitro. Assembly will be determined by electron microscopy, gold-labeled antibody localization, and sedimentation of labeled spoke proteins with the doublet microtubules. These studies will help determine the order of assembly of radial spoke proteins, in a manner similar to that used to analyze T-4 phage morphogenesis. To determine how the flagellar proteins find their way to the end of the cell where flagellar assembly occurs, the site of radial spoke protein synthesis in the cytoplasm will be investigated on the ultrastructural level using gold-labeled antibodies and in situ RNA hybridization. Almost all of the flagellar alpha-tubulin is acetylated and the hypothesis that acetylation plays a role in the attachment of microtubule-associated proteins/structures to the microtubules will be tested by use of acetylated and unacetylated microtubules in an in vitro assembly system with microtubule-associated proteins/structures. Assembly will be analyzed by sedimentation of labeled associated proteins with the microtubules and by electron microscopy.
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