LF4/MOK and a CDK-related kinase regulate the number and length of cilia in Tetrahymena
Fig 2
LF4A-GFP localizes to the basal bodies and along cilia, is transported by IFT and affects IFT.
(A-B) A wild-type (negative control non-GFP expressing) cell (A) and a cell expressing LF4A-GFP under the native promoter (B) were subjected to simultaneous fixation/permeabilization (using a mixture of paraformaldehyde and Triton X-100) and stained with the anti-GFP antibodies (red) and DAPI (blue). In the negative control, the red background (anti-GFP) signal is in the cell body and occasionally at the tips of cilia (A inset). Specific LF4A-GFP signal is present near the basal bodies and along the shafts of locomotory cilia (B inset compare to A inset). (C) An LF4A-GFP expressing cell that was first permeabilized (with Triton X-100) and then fixed (with paraformaldehyde) prior to immunofluorescence. Compared to (B), the LF4A-GFP signal remained strong near the basal bodies but is decreased in cilia. (D) A TIRFM image of a live LF4A-GFP-expressing cell. Arrowheads indicate several basal bodies within a locomotory row, the box highlights an example of a ciliary shaft. (E) Five representative examples of kymographs obtained from the TIRFM videos of LF4A-GFP cells. In most cilia, LF4A-GFP particles are stationary and some move with velocities similar to IFT velocities (arrows) or diffuse (an example of a diffusion event is marked with a red line on the duplicate of the bottom right kymograph). (F) The IFT velocities (left) and IFT event frequencies (right) in cilia of either wild-type or LF4A-KO cells that express DYF1-GFP, an IFT reporter. (G) The IFT velocities (left) and event frequencies (right) in cilia of either wild-type or GFP-LF4A-overexpressing cells. Both strains were exposed to Cd2+ for 3 hours. In (F) and (G), the sample sizes are indicated, asterisks mark significant differences (one way Anova p<0.01), error bars represent SDs. (H) Examples of kymographs of GFP-DYF1 in cilia of either wild-type or LF4A-KO cells corresponding to the data shown in panel F. (H’) Examples of kymographs of GFP-DYF1 in either wild-type or GFP-LF4A overproducing cells corresponding to the data shown in panel G. Abbreviations: oa, oral apparatus; PFA, paraformaldehyde.