Year: 2021
Type: dissertation
Abstract: Primary cilia are surface-exposed organelles that function as signaling hubs. Photoreceptor cells harbor a hyperspecialized cilium termed the outer segment (OS). Defects in trafficking out of the OS cause photoreceptor degeneration in Bardet Biedl Syndrome (BBS). We know that the BBSome, a complex of BBS proteins, carries unwanted proteins out of the OS and past work in cultured cells hinted at a function of ubiquitin (Ub) in BBSome-mediated trafficking. Using a panel of specific detection reagents, I found that Ub chains with linkages at lysine 63 (K63) and at the N-terminus (M1) accumulate in the OSs of Bbs mutant mice. I combined isolation of mouse OS with quantitative mass spectrometry to profile the proteins bearing UbK63 chains that accumulate in BBSome-deficient OS. Besides presynaptic proteins and inner segment proteins, identified hits include resident outer segment proteins, thus suggesting that the BBSome may remove mistargeted as well as damaged proteins out of the OS. Tax1bp1 is a Ub-binding protein that was thought to be highly enriched in Bbs mutant OS, making it a potential candidate for the adaptor that bridges ubiquitinated cargoes to BBSome. However, my imaging and biochemical studies show that Tax1bp1 does not function as a Ub-adapter for BBSome. The intraflagellar transport complex B (IFT-B) powers intraciliary trafficking via the microtubule motors kinesin and dynein. Surprisingly, the IFT-B subunit IFT172 is mutated in BBS and retinitis pigmentosa (RP), and IFT172 participates in anterograde ciliary trafficking as well as turnaround of IFT trains at the cilia tip. My proteomics studies revealed that dynein light chain 1, a negative regulator of dynein-2’s motor activity, interacts with free IFT172, suggesting that the rearrangement of anterograde IFT complexes at the ciliary tip may enable a switch from kinesin to dynein. In conclusion, my proteomics and imaging work has revealed novel, actionable insights into the basic mechanisms of ciliary trafficking.
DOI: 10.17077/etd.006339
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W4283827744