Date of Award

Summer 2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Pinfen Yang

Second Advisor

Rosemary Stuart

Third Advisor

Stephen Downs, Stephen Munroe, Jane Dorweiler

Abstract

Cilia and flagella are similar organelles found in eukaryotic cells. A microtubule-based axonemal cytoskeleton supports these organelles that project into the extracellular environment to detect various stimuli and propel fluid. These functions enable cells to sense and respond to the changing environment. Because of their importance, the fundamental mechanisms have been conserved throughout evolution. However, it remains largely unknown how axonemes are assembled and how the motility is regulated. This thesis investigated two molecules, RSP2 and RSP23, positioned in the axonemes within a T-shaped complex, the Radial Spoke (RS). Among several axonemal complexes, flagella lacking the evolutionarily conserved RS are paralyzed suggesting that this complex is crucial for motility. It was proposed that the RS transduces mechanical and chemical signals to regulate flagellar beating. These two molecules, RSP2 and RSP23, in Chlamydomonas bind the prototype calcium sensor, calmodulin. They also share a highly conserved DPY-30 domain that resembles the RIIa domain known for dimerization and targeting of cAMP-dependent protein kinase A to specific intracellular locations. Moreover, RSP2 links the head and stalk modules in the RS while RSP23 contains a Nucleoside Diphosphate Kinase (NDK) domain presumably for maintaining the equilibrium of nucleotide species. To reveal the mechanisms mediated by these molecules, mutants defective in these domains were generated. The calmodulin-binding region in RSP2, that is absent in mammalian homologs, is not required for the assembly of RS or the oscillatory beating. However, the motile mutants cannot maintain the typical helical trajectory when cells are exposed to bright light and glass barrier simultaneously. In contrast, mutants lacking the DPY-30 domain in RSP2 are paralyzed, despite the presence of all RSPs. Mutants expressing a fraction of RSP23 with inactive NDK activity generate shorter flagella with reduced amounts of RS. Together these results suggest that DPY-30 domains targets to key locations conserved molecular modules critical for flagellar elongation and motility and the diverged calmodulin-binding regions for steering the biflagellate. Models are proposed to illustrate the various roles of these molecular domains. These discoveries provide new insight on the extraordinary mechanisms in cilia and flagella.

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