Graduate School of Biomedical Sciences
Intracellular Signaling Peptides and Proteins; Protein Transport; Dynein ATPase; Chlamydomonas; Flagella; Protozoan Proteins; Molecular Motor Proteins; Academic Dissertations
Life Sciences | Medicine and Health Sciences
Intraflagellar transport (IFT), the bi-directional movement of particles along the length of flagella, is required for flagellar assembly. The IFT particles are moved by kinesin II from the base to the tip of the flagellum, where flagellar assembly occurs. The IFT particles are then moved in the retrograde direction by cytoplasmic dynein 1b/2 to the base of the flagellum. The IFT particles of Chlamydomonas are composed of ~16 proteins, organized into complexes A and B. Alhough IFT is believed to transport cargoes into flagella, few cargoes have been identified and little is known about how the cargos are transported. To study the mechanism of IFT and how IFT is involved in flagellar assembly, this thesis focuses on two questions. 1) In addition to a heavy chain, DHC1b, and a light chain, LC8, what other proteins are responsible for the retrograde movement of IFT particles? 2) What is the specific function of an individual IFT-particle protein? To address these two questions, I screened for Chlamydomonas mutants either defective in retrograde IFT by immunofluorescence microscopy, or defective in IFT-particle proteins and D1bLIC, a dynein light intermediate chain possibly involved in retrograde IFT, by Southern blotting. I identified several mutants defective in retrograde IFT and one of them is defective in the D1bLIC gene. I also identified several mutants defective in several IFT-particle protein genes. I then focused on the mutant defective in D1bLIC and the one defective in IFT46, which was briefly reported as an IFT complex B protein. My results show that as a subunit of the retrograde IFT motor, D1bLIC is required for the stability of DHC1b and is involved in the attachment of IFT particles to the retrograde motor. The P-loop in D1bLIC is not necessary for the function of D1bLIC in retrograde IFT. My results also show that as a complex B protein, IFT46 is necessary for complex B stability and is required for the transport of outer dynein arms into flagella. IFT46 is phosphorylated in vivo and the phosphorylation is not critical for IFT46’s function in flagellar assembly.
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Chapter V was originally published in The Journal of Cell Biology, 176:653-665, 2007, http://www.jcb.org/cgi/content/full/176/5/653. Chapter IV was originally published in Molcular Biology of the Cell, 15:4382-4394, 2004, http://www.molbiolcell.org/cgi/content/full/15/10/4382.
Hou, Yuqing, "Identification and Characterization of Components of the Intraflagellar transport (IFT) Machinery: a Dissertation" (2007). University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 323.