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Armed and Disordered: Dissecting Radial Spoke Transport in Chlamydomonas
Cargo adapters link various large ciliary building blocks to intraflagellar transport (IFT) trains to ensure transport into cilia. How such adapters interact with IFT and their cargoes and how these interactions are regulated remains largely unknown. To this end, we analyzed the functional domains of Chlamydomonas ARMC2, an obligate adapter for IFT of the radial spoke precursor complex (RS-PC), an 11-subunits complex assembled in the cell body. ARMC2 is conserved in organisms with motile cilia, its loss causes paralyzed flagella in Chlamydomonas and pathogenic mutations in mammals cause male infertility and reduced lung function. ARMC2 consists of two principal domains, an N-terminal ~400 residue intrinsically disordered region (IDR) and a Cterminal region encompassing multiple armadillo (ARM) repeats forming an α-solenoid. The IDR of ARMC2 (e.g., ARMC2Δ412-1053 lacking the entire C-terminal α-solenoid) is sufficient for transport by IFT. However, the construct and most constructs with partial deletions of the ARM domain fail to restore motility and radial spoke assembly in the armc2 mutant, suggesting that the ARM domain is required for RS-PC transport. Fusion of the IDR of ARMC2 directly to radial spoke protein 3 (RSP3), a central component of the RS-PC, restores radial spoke transport and cell motility in an ARMC2 and RSP3-deficient double mutant, circumventing the need of ARMC2 for RS-PC transport. Alphafold predicts that the ARM domain of ARMC2 could bind its own IDR as well as the N-terminal IDR of RSP3. Indeed, RSP3(1-156)-GFP moves by IFT in an ARMC2- dependent manner, indicating that this region of RSP3 mediates docking of the RS-PC to ARMC2. The transport frequencies of ARMC2 and the RS-PC are strongly regulated by ciliary length: While ARMC2 frequently moves by IFT during ciliary assembly, its transport is rarely observed in fulllength cilia and western blots detect only trace amount of tagged ARMC2. Remarkably, the IDR alone, i.e., truncated ARMC2Δ412-1053, and shorter IDR fragments (i.e., ARMC21-150) display high frequency transport by IFT in full-length cilia. This indicates that the ARM domain negatively regulates IFT of ARMC2/IDR and suggests that changes in ARMC2 rather than in IFT critically regulate IFT-ARMC2 interaction. We propose an auto-inhibitory model, in which the cargo binding ARM domain negatively regulates binding of the IDR to IFT. After activation by a currently unknown signal, ARMC2 unfolds, allowing the ARM domain to bind its RS-PC cargo and the IDR to IFT trains, initiating transport into cilia.
Speakers
Karl Lechtreck