Purpose
Primary cilia is a microtubule-based sensory organ and its malfunction, known as ciliopathy, is associated with photoreceptor degeneration. Previous studies have reported that drugs or proteins that induce change in actin cytoskeleton affect ciliogenesis. However the mechanism of the link between actin cytoskeleton and ciliogenesis is not fully understood. The aim of this study is to find actin-related key molecules that control ciliogenesis and to determine the exact mechanism of cilia assembly.
Methods
A high-throughput assay using siRNA was performed to evaluate the functional impact of protein kinases that regulate actin dynamics in ciliogenesis. RPE1 cells stably expressing Smo-EGFP were transfected with 10 nM siRNAs. For indirect immunofluorescence, cells were fixed in methanol for 4 minutes at −20°C. Primary and secondary antibodies were applied for 1 hour at room temperature. Cytochalasin D (actin depolymerizing agent) was applied to the cells at 200 nM.
Results
We found that TESK1, which inactivates cofilin that severs actin filaments, is a potent negative regulator of ciliogenesis. TESK1 knockdown increased the number of ciliated cells compared with the nonspecific siRNA control (33.8% vs 2.1%). Double knockdown of TESK1 and cofilin did not induce ciliogenesis, which indicates that cofilin is a downstream effector of TESK1 in cilia assembly. When TESK1 was knockdowned, more ciliary vesicles that carry Smo-EGFP accumulated around the centrosome than that of the nonspecific siRNA control (90.2% vs 21.6%), suggesting that up-regulated actin dynamics by TESK1 knockdown facilitates the directional trafficking of ciliary vesicles to the centriolar compartment. CEP164 is a molecule that is known to be essential for ciliary vesicle docking to the basal body. In the condition of its knockdown, an increased CP110 cap removal, an important step in ciliogenesis initiation, was observed when treated with Cytochalasin D versus without treatment (46.1% vs 13.3%). This suggests that although ciliary vesicle docking does not occur, actin dynamics up-regulation can remove the CP110 cap in a significant number of cells.
Conclusions
Up-regulation of actin dynamics through TESK1 knockdown facilitates ciliogenesis by directional ciliary vesicle trafficking to the centrosome and CP110 cap removal. TESK1 could therefore be exploited as a potential therapeutic target for retinal ciliopathies.
Keywords: 695 retinal degenerations: cell biology •
648 photoreceptors •
537 gene screening