A network of interacting ciliary tip proteins with opposing activities imparts slow and processive microtubule growth

Abstract

Cilia are essential motile or sensory organelles found on many eukaryotic cells. Their formation and function rely on axonemal microtubules, which exhibit very slow dynamics, however the underlying biochemical mechanisms are largely unexplored. Here, we reconstituted in vitro the individual and collective activities of the ciliary tip module proteins, CEP104, CSPP1, TOGARAM1, ARMC9 and CCDC66, which interact with each other and with microtubules, and, when mutated, cause ciliopathies such as Joubert syndrome. CEP104, a protein containing a tubulin-binding TOG domain, is an inhibitor of microtubule growth and shortening that interacts with EBs on the microtubule surface and with a luminal microtubule-pausing factor CSPP1. Another TOG-domain protein, TOGARAM1, overcomes growth inhibition imposed by CEP104 and CSPP1. CCDC66 and ARMC9 do not affect microtubule dynamics directly but act as scaffolds for their partners. Cryo-electron tomography showed that together, ciliary tip module members form plus-end-specific cork-like structures which reduce protofilament flaring. The combined effect of these proteins is very slow processive microtubule elongation, which recapitulates axonemal dynamics in cells.

Ccdc66 regulates primary cilium stability, disassembly and signaling important for epithelial organization

“…To directly examine the role of CCDC66 in microtubule stabilization, we performed in vitro experiments using MBP-mNeonGreen-CCDC66, which was expressed and purified from insect cells as described previously [40]. MBP-mNeonGreen, which does not bind to microtubules, and the first two TOG domains of TOGARAM1, which bind free tubulin but not the microtubule lattice and promote microtubule polymerization but not stability in vitro , were used as controls [50, 57]. We polymerized rhodamine-labeled microtubules from 2 µM tubulin mixture in the presence of GTP and glycerol at 37°C for 30Lmin.…”

“…CSPP1, a component of the ciliary tip module, was shown to inhibit microtubule growth and shortening, as well as stabilize damaged microtubule lattices [77]. A recent study showed that CCDC66, along with CEP104, CSPP1, TOGAGRAM1 and ARMC9, forms a complex at the ciliary tip which stabilizes microtubules by promoting slow and processive growth of the microtubule plus ends [50]. This study also found that CCDC66 does not affect the frequencies of catastrophes and rescues, nor did it induce pausing, suggesting that CCDC66 acts as a scaffold for the recruitment of the axonemal tip MAPs involved in regulating microtubule dynamics [50].…”

“…A recent study showed that CCDC66, along with CEP104, CSPP1, TOGAGRAM1 and ARMC9, forms a complex at the ciliary tip which stabilizes microtubules by promoting slow and processive growth of the microtubule plus ends [50]. This study also found that CCDC66 does not affect the frequencies of catastrophes and rescues, nor did it induce pausing, suggesting that CCDC66 acts as a scaffold for the recruitment of the axonemal tip MAPs involved in regulating microtubule dynamics [50]. Notably, our findings that CEP104 cannot restore axonemal stability in CCDC66-depleted IMCD3 cells support this proposed mechanism.…”

“…Moreover, CCDC66 is part of a ciliary tip module that includes other MAPs such as CEP104, CSPP1, TOGARAM1 and ARMC9, which are mutated in the neurodevelopmental ciliopathy known as Joubert syndrome [48, 49]. A recent in vitro reconstitution study showed that these proteins collectively stabilize microtubule plus ends and promote their slow and persistent elongation, reflecting axonemal tip dynamics observed in cells [50]. Although functions and mechanisms of CCDC66 in cilium assembly are well-characterized in human retinal pigmental epithelial (RPE1) cells, its potential roles in different ciliogenesis pathways and other stages of the cilium cycle, specifically in the maintenance and disassembly of the cilium, remain unknown.…”