Thesis (Ph.D.). / Written for the Institute of parasitology. Title from title page of PDF (viewed 2009/06/10). Includes bibliographical references.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 19, 2010). Includes bibliographical references.

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.

Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 3 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 4 uppsatser.

The outer dynein arm-docking complex (ODA-DC) specifies the outer dynein arm-binding site on the flagellar axoneme. The ODA-DC of Chlamydomonas contains equimolar amounts of three proteins termed DC1, DC2, and DC3 (Takada et al., 2002). DC1 and DC2 are predicted to be coiled-coil proteins, and are encoded by ODA3 and ODA1, respectively (Koutoulis et al., 1997; Takada et al., 2002). Prior to this work, nothing was known about DC3. To fully understand the function(s) of the ODA-DC, a detailed analysis of each of its component parts is necessary. To that end, this dissertation describes the characterization of the smallest subunit, DC3. In Chapter II, I report the isolation and sequencing of genomic and full-length cDNA clones encoding DC3. The sequence predicts a 21,341 D protein with four EF hands that is a member of the CTER (Calmodulin, Troponin C, Essential and Regulatory myosin light chains) group and is most closely related to a predicted protein from Plasmodium. The DC3 gene, termed ODA14, is intronless. Chlamydomonas mutants that lack DC3 exhibit slow, jerky swimming due to loss of some but not all, outer dynein arms. Some outer doublet microtubules without arms had a "partial" docking complex, indicating that DC1 and DC2 can assemble in the absence of DC3. In contrast, DC3 cannot assemble in the absence of DC1 or DC2. Transformation of a DC3-deletion strain with the wild-type DC3 gene rescued both the motility phenotype and the structural defect, whereas a mutated DC3 gene was incompetent to rescue. The results indicate that DC3 is important for both outer arm and ODA-DC assembly. As mentioned above, DC3 has four EF-hands: two fit the consensus pattern for calcium binding and one contains two cysteine residues within its binding loop. To determine if the consensus EF-hands are functional, I purified bacterially expressed wild-type DC3 and analyzed its calcium-binding potential in the presence and absence of DTT and Mg2+. As reported in Chapter III, the protein bound one calcium ion with an affinity (Kd) of ~1 x 10-5 M. Calcium binding was observed only in the presence of DTT and thus is redox sensitive. DC3 also bound Mg2+ at physiological concentrations, but with a much lower affinity. Changing the essential glutamate to glutamine in both EF-hands eliminated the calcium-binding activity of the bacterially expressed protein. To investigate the role of the EF hands in vivo, I transformed the modified DC3 gene into a Chlamydomonas insertional mutant lacking DC3. The transformed strain swam normally, assembled a normal number of outer arms, and had a normal photoshock response, indicating that the E to Q mutations did not affect ODA-DC assembly, outer arm assembly, or Ca2+-mediated outer arm activity. Thus, DC3 is a true calcium-binding protein, but the function of this activity remains obscure. In Chapter IV, I report the initial characterization of a DC3 insertional mutant having a phenotype intermediate between that of the DC3-deletion strain and wild type. Furthermore, I suggest future experiments that may help elucidate the specific role of DC3 in outer arm assembly and ODA-DC function. Lastly, I speculate that the ODA-DC may play a role in flagellar regeneration.

Thesis (Ph. D.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).