The Ciliary Kinase CNK2 Triggers Ciliary Disassembly

Background: Cilia (also known as flagella) are microtubule-based organelles that project from the surface of most eukaryotic cell types. Cilia are reabsorbed into the cell prior to mitosis and reassembled when cells enter G₀ phase. While the mechanisms of ciliary assembly and maintenance are well characterized, very little is known about the signals or mechanisms through which cilia are reabsorbed. We have discovered that the CNK2 kinase of the unicellular green algae Chlamydomonas reinhardtii provides an important signal for the initiation of ciliary reabsorption.  CNK2 is a member of the NIMA-related kinase family, a group of cell cycle kinases that have roles in coordinating cilia with the cell cycle. CNK2 has previously been shown to localize to cilia and participate in the regulation of ciliary length. We show that it acts by stimulating the process of reabsorption.  Methods: We characterized a new cnk2-1 mutant strain, which was compared to wild-type Chlamydomonas cells under the influence of a variety of chemical stimuli known to induce ciliary reabsorption, including 1 mM IBMX, 20 mM NaPPi, and pH shock. To observe pre-mitotic reabsorption, phototrophically-grown cells were synchronized on a 14h:10h light:dark cycle, then embedded in agarose and imaged during the 3^rd or 4^th dark cycle. Cells were imaged by DIC microscopy and flagellar lengths were measured using DeltaVision software. Results: The cnk2-1 mutant strain has an insertion in the first intron of the 5’ UTR that abolishes expression of CNK2 protein. The flagella of cnk2-1 cells are conditionally longer than WT, which led us to predict that cnk2-1 cells are defective in flagellar disassembly. Under conditions known to induce flagellar reabsorption in Chlamydomonas, WT cells reabsorb rapidly while cnk2-1 cells lose little or no length. During pre-mitotic reabsorption, WT cells undergo an extended phase of slow flagellar reabsorption followed by a short phase of rapid reabsorption. Consistent with the results of chemically induced reabsorption, cnk2-1 cells are defective in the initial slow phase of reabsorption. Conclusions: Taken together, these data indicate that CNK2 plays an important role in signals that initiate flagellar reabsorption. This is the first evidence that the two phases of pre-mitotic reabsorption are controlled independently, and that the rapid phase can proceed in the absence of normal slow phase reabsorption. The striking failure of cnk2-1 flagella to reabsorb in response to chemical stimuli indicate that this kinase plays a key role in triggering reabsorption. Our observed defects in the early, slow phase of pre-mitotic reabsorption are consistent with this interpretation.