Abstract
The length of cilia is controlled by a poorly understood mechanism that involves members of the conserved RCK kinase group, and among them, the LF4/MOK kinases. The multiciliated protist model, Tetrahymena, carries two types of cilia (oral and locomotory) and the length of the locomotory cilia is dependent on their position with the cell. In Tetrahymena, loss of an LF4/MOK ortholog, LF4A, lengthened the locomotory cilia, but also reduced their number. Without LF4A, cilia assembled faster and showed signs of increased intraflagellar transport (IFT). Consistently, overproduced LF4A shortened cilia and downregulated IFT. GFP-tagged LF4A, expressed in the native locus and imaged by total internal reflection microscopy, was enriched at the basal bodies and distributed along the shafts of cilia. Within cilia, most LF4A-GFP particles were immobile and a few either diffused or moved by IFT. We suggest that the distribution of LF4/MOK along the cilium delivers a uniform dose of inhibition to IFT trains that travel from the base to the tip. In a longer cilium, the IFT machinery may experience a higher cumulative dose of inhibition by LF4/MOK. Thus, LF4/MOK activity could be a readout of cilium length that helps to balance the rate of IFT-driven assembly with the rate of disassembly at steady state. We used a forward genetic screen to identify a CDK-related kinase, CDKR1, whose loss-of-function suppressed the shortening of cilia caused by overexpression of LF4A, by reducing its kinase activity. Loss of CDKR1 alone lengthened both the locomotory and oral cilia. CDKR1 resembles other known ciliary CDK-related kinases: LF2 of Chlamydomonas, mammalian CCRK and DYF-18 of C. elegans, in lacking the cyclin-binding motif and acting upstream of RCKs. The new genetic tools we developed here for Tetrahymena have potential for further dissection of the principles of cilia length regulation in multiciliated cells.
Highlights
The classical “long-zero” experiment in the green flagellate Chlamydomonas reinhardtii revealed that the length of cilia is sensed and actively maintained [1]
We find that cells lacking a LF4/MOK kinase of Tetrahymena, LF4A at the native (LF4A), have excessively long, and fewer cilia
We show that LF4A decreases intraflagellar transport, a mechanism that shuttles ciliary precursors from the cilium base to the tip
Summary
The classical “long-zero” experiment in the green flagellate Chlamydomonas reinhardtii revealed that the length of cilia is sensed and actively maintained [1]. When one of the two cilia of Chlamydomonas was removed, the intact cilium immediately started to shorten, while the amputated cilium started to regrow When both cilia reached about the same intermediate length, they continued to elongate at the same rate, to achieve an equal steady-state length [1]. During IFT, motor proteins move large protein complexes, IFT trains, that in turn ferry precursors of cilia, including tubulin, along axonemal microtubules [16,17,18]. In Chlamydomonas, the disassembly rate increases in cilia that are abnormally long [37] These observations suggest that there are mechanisms that sense and adjust cilium length by controlling the rates of cilia assembly (IFT) and disassembly
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