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Abstract
Cilia are microtubule-based cell extensions functioning in cell motility and signaling. The assembly of cilia and the trafficking of ciliary proteins require the intraflagellar transport (IFT) and other protein transport mechanisms. In this dissertation, I will analyze two transport events and their connections to ciliary functions using the biflagellate Chlamydomonas reinhardtii.First, we analyzed the transport of outer dynein arm (ODA), the multiprotein complex that drive flagellar beating. Based on genetic and biochemical analyses, ODAs preassemble in the cell body and then move into the flagellum by IFT. To study ODA transport in vivo, we expressed the essential intermediate chain 2 tagged with mNeonGreen (IC2-NG) in Chlamydomonas. IC2-NG moved by IFT dependent of ODA16 and IFT46 as expected; the transport was of low processivity and increased in frequency during flagellar growth. ODA16 is also responsible for recruiting ODAs to the basal bodies. Upon unloading from IFT, ODAs rapidly docked to the axoneme; but in the absence of the docking complex (DC), docking is transient. In full-length flagella, ODAs continued to enter and move inside cilia, the newly imported complexes frequently replaced axoneme-bound ODAs. We propose that the low processivity of ODA IFT contributes to flagellar maintenance by ensuring the availability of replacement ODAs along the length of flagella.
Second, we explored how a small GTPase, Arl13B, cooperate with the IFT/BBSome machinery. We discovered that the Chlamydomonas arl13b mutant have near normal IFTs and BBSomes, but a major biochemical defect in the ciliary membrane proteome. Among the proteins altered, phospholipase D (PLD), a BBSome cargo, is accumulated to a level similar to that observed in the bbs4-1 mutant. Live imaging revealed that PLD fails to move by IFT. We conclude that the association of PLD to the IFT/BBSome trains depends on Arl13B. Expression of tagged Arl13B (Arl13B-NG) rescued the biochemical defects of the arl13b mutant but IFT of Arl13B-NG was not observed indicating that Arl13B is not an adapter linking PLD to IFT during transport. In conclusion, Arl13B mediates ciliary export of PLD by enabling its binding to IFT/BBSome.