Regulation of Cilium Length and Intraflagellar Transport by the RCK-Kinases ICK and MOK in Renal Epithelial Cells

Abstract

Primary cilia are important sensory organelles. They exist in a wide variety of lengths, which could reflect different cell-specific functions. How cilium length is regulated is unclear, but it probably involves intraflagellar transport (IFT), which transports protein complexes along the ciliary axoneme. Studies in various organisms have identified the small, conserved family of ros-cross hybridizing kinases (RCK) as regulators of cilium length. Here we show that Intestinal Cell Kinase (ICK) and MAPK/MAK/MRK overlapping kinase (MOK), two members of this family, localize to cilia of mouse renal epithelial (IMCD-3) cells and negatively regulate cilium length. To analyze the effects of ICK and MOK on the IFT machinery, we set up live imaging of five fluorescently tagged IFT proteins: KIF3B, a subunit of kinesin-II, the main anterograde IFT motor, complex A protein IFT43, complex B protein IFT20, BBSome protein BBS8 and homodimeric kinesin KIF17, whose function in mammalian cilia is unclear. Interestingly, all five proteins moved at ∼0.45 µm/s in anterograde and retrograde direction, suggesting they are all transported by the same machinery. Moreover, GFP tagged ICK and MOK moved at similar velocities as the IFT proteins, suggesting they are part of, or transported by the IFT machinery. Indeed, loss- or gain-of-function of ICK affected IFT speeds: knockdown increased anterograde velocities, whereas overexpression reduced retrograde speed. In contrast, MOK knockdown or overexpression did not affect IFT speeds. Finally, we found that the effects of ICK or MOK knockdown on cilium length and IFT are suppressed by rapamycin treatment, suggesting that these effects require the mTORC1 pathway. Our results confirm the importance of RCK kinases as regulators of cilium length and IFT. However, whereas some of our results suggest a direct correlation between cilium length and IFT speed, other results indicate that cilium length can be modulated independent of IFT speed.

A role for primary cilia in aortic valve development and disease

“…We focus on cilia length due to the recognition that length is a major predictor of proper cilia function. Inappropriate elongation of primary cilia can hinder the biological processes in which cilia function, and several ciliopathies have been associated with defects in cilia length (Niggemann et al, ; Yuan et al, ; Broekhuis et al, ; Broekhuis et al, ). Our data demonstrate that primary cilia are present on interstitial cells of the developing outflow tract cushions at E11.5 and E13.5, while being rarely observed on the valve endocardium (Fig.…”

An essential role of intestinal cell kinase in lung development is linked to the perinatal lethality of human ECO syndrome

“…Recently, ICK localization was reported on primary cilia of mammalian cells [7, 9, 10, 18–20]. Using the primary cilium marker Arl13B (Fig.…”

HIF Stabilization Weakens Primary Cilia

“…Signaling pathways known to be involved with dynamic control of cilium length include cAMP-dependent protein kinase A (PKA) activation [34, 35] and ros cross-hybridizing kinases (RCKs) [36]. While measurements of cilium lengths are subject to fixation errors [37] as many fixatives grossly deform cilia morphology, few reports of measured cilium lengths using live cilia exist.…”