Tuesday, April 1, 2025
4/1/2025
Regulation of protein transport in cilia - Project Summary/Abstract How cells move proteins and organelles to create functionally specialized regions is a central question of cell biology. Often, this process involves molecular motors moving along cytoskeletal tracks. We study the assem- bly of cilia, which are thin cell projections with critical roles in cell motility and signaling. The axoneme, a com- plex microtubule-based machine, mediates ciliary bending to propel spermatozoa and generate fluid-flow above ciliated epithelia. The ciliary membrane is enriched in signaling proteins that achieve, for example, sens- ing of light, hormones, and mechanical cues. Cilia are widely distributed in the mammalian body and ciliary dysfunction causes many diseases ranging from male infertility to blindness. This has boosted an interest in how cilia are assembled and maintained. Because cilia lack protein synthesis, its 1000+ proteins need to be imported from the cell body. This involves the conserved intraflagellar transport (IFT) pathway, a bidirectional, motor-based motility of polymeric protein carriers, the IFT trains, along the axonemal microtubules. IFT shuttles proteins in and out of cilia but how it selects its cargoes and how the cargo load on the trains is regulated re- mains largely unknown. We will address these questions using the unicellular model Chlamydomonas rein- hardtii, which facilitates in vivo imaging of protein transport in its two 9+2 cilia with single-molecule sensitivity. We showed that IFT carries several proteins of the ciliary membrane and axoneme and that axonemal protein transport is sharply upregulated in short growing cilia, likely explaining the critical role of IFT in ciliary assem- bly. Over the next five years, we will investigate how IFT selects its cargoes, releases them in cilia and how the frequency of transport is regulated. A focus will be ARMC2, an adapter protein that mediates IFT of the radial spoke precursor complex. Pathogenic mutations in mammalian ARMC2 are linked to male infertility and re- duced lung function. We will determine ARMC2’s functional domains for radial spoke and IFT binding and in- vestigate whether autoinhibition and ciliary length-dependent activation of ARMC2 regulate the transport fre- quency. We showed that cells increase the cargo load on IFT trains when cilia are short but how they sense ciliary length is unclear. To this end, we will study Chlamydomonas LF5 kinase; loss of LF5 causes abnormally long cilia. LF5 is an ortholog of CDK-like5, which when mutated causes epileptic activity and impaired neurode- velopment. We will determine if LF5 reduces ciliary protein entry by IFT to restrain length. While IFT is the pre- dominant pathway to move ciliary building blocks into cilia, other proteins, especially of the ciliary membrane, use alternative ways of entry. Besides transmembrane proteins, ciliary signaling involves proteins that associ- ate with the membrane by lipidation. We studied how such proteins are removed from cilia and will now explore how they enter and accumulate in cilia, likely in an IFT-independent manner. We expect to gain insights into a novel pathway that cages such proteins in the ciliary membrane. Our broad goal is to understand how ciliary protein transport is regulated to ensure the assembly of cilia with the desired size, composition and function.
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