Analysis of the gliding machinery in the green alga, Chlamydomonas reinhardtii

This dissertation explores two different types of motility associated with the flagella of the unicellular, biflagellate green alga, Chlamydomonas reinhardtii. The first part of the dissertation concerns the flagellar-surface dependent gliding motility and encompasses two chapters. Chapter 1 provides the reader with essential background information. Chapter 2 describes biochemical approaches initiated to characterize a cargo for the retrograde IFT motor, cytoplasmic dynein 1b (DHC1b). Chapter 2 is divided into two sections. In the first section, proteomic results are presented from pull down assays of a Chlamydomonas flagellar extract using recombinant, MBP-tagged heavy chain of cytoplasmic dynein 1b (MBP-DHC1b). These pull downs yielded a set of proteins, distinct from IFT particle proteins, among which the most prominent were the major f&barbelow;lagellar m&barbelow;embrane g&barbelow;lycoprotein 1B, FMG-1B, and the f&barbelow;lagellar-a&barbelow;ssociated p&barbelow;rotein 12 (FAP12) of unknown function. These findings suggest that the same retrograde motor used for IFT, cytoplasmic dynein 1b, also has a second functional role in flagella-dependent gliding motility. In the second section of Chapter 2, results are presented from pull down assays using a FMG-1B immuno-affinity resin. These reverse pull downs identified a set of FMG-1B-associated proteins that could represent novel components of the algal gliding machinery. Among them were FAP12 and a small fraction of the DHC1b motor. Taken together, the biochemical results presented in Chapter 2 suggest that FAP12 may be directly or indirectly associated with the transmembrane gliding receptor, FMG-1B. A careful analysis of FAP12 is presented in three parts in Chapter 3 beginning with a biochemical characterization of FAP12. A polyclonal antibody was generated against full length FAP12 and used to localize FAP12 as distinct puncta along the length of the flagellum and to show that it was absent from the cell body, suggesting a uniquely flagellar function. Moreover, FAP12 was localized to the flagellar membrane and requires a non-ionic detergent for solubilization, consistent with the fact that FAP12 is known to be myristoylated. Quantification of FAP12 led to an estimate of 1770 copies per flagellum. In the second part of this chapter, an immunoblot analysis of whole cell protein extracts showed the absence of FAP12 in bald mutants, which do not assemble any flagella, and a reduced level in mutants with short flagella. In part three, artificial microRNA interference (amiRNAi) was used to reduce the expression of the FAP12 gene. FAP12 knockdown strains with the greatest depletion of FAP12 were able to attach to a glass slide but were largely unable to assume the characteristic 180° gliding configuration. Gliding velocities were not affected in the FAP12 knockdown cells. To show that this gliding phenotype was due to the loss of FAP12, recombinant His6-FAP12 protein was electroporated into the FAP12-deficient cells rescuing the wild-type gliding phenotype. These findings suggest that FAP12 is more important for mediating the adhesion function of FMG-1B to the solid substrate than it is for affecting the velocity of the purported gliding motor, cytoplasmic dynein 1b. Chapter 3 concludes with a model suggesting how FAP12 (which contains a lipase domain) participates in the early signaling events associated with the gliding motility.;The second part of this dissertation focuses on i&barbelow;ntraf&barbelow;lagellar t&barbelow;ransport (IFT), a form of motility occurring in the compartment between the flagellar membrane and the axoneme. IFT is characterized as the bidirectional movement of large protein complexes, called IFT trains, along the length of axonemal outer doublet microtubules. IFT trains are constructed from two protein complexes, designated A and B, containing six and thirteen distinct protein subunits, respectively. In Chapter 4, a method is presented which allows the isolation of the intact IFT complex B. The studies reported here show that the IFT complex B is sensitive to centrifugal shear forces. When centrifuged at lower speeds, the complex B remains intact, whereas higher centrifugal forces cause the IFT172 protein subunit to partially dissociate from the complex B. This experiment reveals the fragility of complex B and shows that the association of the IFT172 subunit with other components of complex B is weak and sensitive to various physical and chemical treatments.