Jiang, Xue (2017-05). Eukaryotic Flagellar Assembly: Insights Learned from Intraflagellar Transport (IFT) and Small GTPase ARL3. Doctoral Dissertation.
Thesis
Eukaryotic cilia/flagella are the evolutionary conserved microtubule-based organelles. Projecting away from the cell surface, they perform a variety of sensing, signaling, and motility-based functions. In humans, defective cilia lead to an array of pleiotropic disorders, which are collectively referred to as ciliopathies. The aim of my study is to identify and characterize the factors that play roles in the ciliary/flagellar assembly by using a simple model organism, Chlamydomonas.
This study focuses on three flagellar assembly mutants, ift57-1, ift56-2, and arl3, which are identified from a mutant library using a PCR-based method called restriction enzyme cassette mediated PCR. ift57-1 and ift56-2 carry mutations in two subunits of intraflagellar transport complex B (IFT-B), IFT57 and IFT56, respectively. In many ciliopathies, mutations have been identified in components of IFT complexes, which are mainly used for delivering cargos in and out of cilia. The ift57-1 mutant expresses a reduced amount of IFT57 (<=5%) and assembles short motility-defective flagella with variable lengths. Although IFT57 is proposed to play an essential role in bridging two IFT-B subcomplexes, IFT-B1 and IFT-B2, our results show that IFT57 is not essential for the IFT-B assembly but rather functions to prevent IFT-B from degradation. Moreover, IFT57 may deliver specific motility-related cargos including an inner dynein arm subunit IC97. On the other hand, the null mutant ift56-2 possesses evenly shortened flagella. We verify that IFT56 is a bona fide IFT-B subunit. However, it is neither essential for the IFT-B assembly nor for the stability. Possible reasons leading to the shorter flagellar length in ift56 mutants, including precursor pool size, axonemal disassembly rate, and known length regulators, are explored but are found to play insignificant roles.
Recently, several small GTPases are reported to be involved in ciliogenesis. The small GTPase ARL3 mutant arl3 presents a variety of subtle flagellar defects. The mutant cells expressing the GTP-dominant form of ARL3 possess significantly shortened flagella, indicating that GTPase activity of ARL3 is involved in flagellar length control. Previous studies propose that ARL3 is important for the flagellar transport of lipid-modified peripheral membrane proteins. Our data show that a myristoylated protein CrPKG is likely to be affected in arl3. Using isolated pure flagella, we found that the amount of a few hydrophobic proteins, probably membrane proteins, are altered in the arl3 flagella, suggesting that ARL3 is involved in transport or retention of certain flagellar membrane proteins.
