Date of Award

Summer 2008

Degree Type

Thesis - Restricted

Degree Name

Master of Science (MS)

Department

Biology

First Advisor

Stuart, Rosemary

Second Advisor

Munroe, Stephen

Third Advisor

Yang, Pinfen

Abstract

Assembly is a daunting task for the eukaryotic cilia and flagella. More than 600 proteins are present in compartmentalized membrane and the supporting axonemal scaffold. For motile flagella, the burden is especially extraordinary, since many proteins belong to individual structure complexes that play distinct roles in the oscillatory beating. These components synthesized in the cell body have to make their way to the tip to incorporate precisely relative to each other into the stable nanomachine that can sweep viscous fluid at high frequency without disintegrating. Intraflagellar transport (IFT), a transportation mechanism driven by molecular motors, is in place to deliver these components. However, the rest of the assembly processes remain largely unknown. It is assumed that the axonemal components are preassembled first. However, the precursors are not necessarily completely furbished. For example, the precursor for the radial spoke complex, that coordinates dynein motors and possibly transduces signaling of second messengers, is significantly smaller than the complex harvested from the axonemes. One protein absent in the precursor is the dimeric spoke HSP40 that is delivered in a small particle separately and join the rest to strengthen the complex for mechanic transduction. The addition is essential for the function of RS in synchronized flagellar beating. This finding suggests that the transport, assembly and function are coupled. Curiously, a group of low abundance RSPS has not been investigated. The low abundance suggests that they are delivered and assembled differently to build a subpopulation of RS complex for specialized local function. Investigation of these rare proteins will elucidate the assembly and functional mechanism of RS and will shed new light on delivery and assembly mechanism involved in ciliogenesis. This thesis focused on a predicted rare protein, RSP14 and its null mutant. The aims were: Aim 1:- To verify if RSP14 is the second armadillo repeat protein in the axonemal radial spoke complex of Chlamydomonas reinhardtii. Aim 2: To determine the interaction of RSP14 and the other spoke proteins by subfractionating the radial spoke complex. Aim 3: To verify if the transport and assembly of the rare and abundant radial spoke proteins is different by comparing the radial spoke precursor particles. By testing these three aims, this thesis not only confirms that RS indeed contain two ARM molecules and but also makes three major contributions. First, extraction with concentrated KI solution followed by velocity sedimentation defines the content in the sub domains of the spoke complex. The location of each sub domain sheds valuable information about the predicted role of each protein, including the RSP14 ARM. Second, the serial extraction is applicable for dissecting the other axonemal complexes. Last but not the least, the absence of ARM precursor in the flagellar membrane matrix suggests a third way of transporting flagellar protein possibly not mediated by IFT.

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