assembly. We found that Myh10 expression is positively regulated by the tubulin acetyltransferase Mec-17. Importantly, both Mec-17 and Myh10 gene expression are purchase AVE8062A induced by PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19683258 serum starvation conditions that activate cilium formation. In Mec-17deficient cells subject to serum starvation, Myh10 is not induced and ciliogenesis is 2 / 21 A Mec17-Myosin II Axis Controls Ciliogenesis deregulated. Conversely, pharmacological inhibition of HDAC6 increases microtubule acetylation, Myh10 expression and cilium formation. Our results suggest that Mec-17-dependent microtubule acetylation is coupled to the induction of Myh10, whose accumulation overcomes Myh9-dependent actin cytoskeleton stabilization and promote primary cilium assembly. Results Myh10 Is Required for Cilium Formation To investigate the role of the actin network in ciliogenesis, we focused on nonmuscle myosin II motors, which are known to regulate actin network dynamics and actinomyosin-microtubule crosstalk. We adopted the serum starvation protocol to induce cilium formation in RPE-Mchr1GFP cells, a retina pigment epithelial cell line expressing GFP-fused melanin-concentrating hormone receptor 1 protein. To study the role of myosin IIA and IIB in cilium formation, we first designed siRNA duplexes specifically targeting Myh10 or Myh9 mRNA. Western blot analysis and Q-PCR confirmed the effective 
knockdown of Myh10 and Myh9 expression by siRNAs. When RPE-Mchr1GFP cells were subjected to serum deprivation for 24 hours to induce ciliogenesis, Myh9 siRNA-treated cells were able to form cilia as efficiently as control siRNA group. In stark contrast, two different Myh10 siRNA duplexes both dramatically inhibited cilium formation. Knocking down Myh10, but not Myh9, in mouse IMCD3 cells also potently inhibited cilium formation in IMCD3 cells. Moreover, re-introducing a wild type human Myh10 restored cilium formation in Myh10-knockdown IMCD3 cells. These findings show that Myh10 is required for efficient ciliogenesis. It is also worthwhile to point out that Myh10 knockdown does not stop cells from proliferating before reaching confluency in our experiments, suggesting the effect of Myh10 on ciliogenesis is not an indirect consequence of altered cell cycle. Cilia are important for sonic hedgehog signaling. To confirm that Myh10 is required for cilia-mediated signal transduction, we stimulated cells with SAG, a small molecule agonist of Smoothened, to induce sonic hedgehog signaling in serum-starved cells. We measured Gli-1 mRNA induction, a downstream target of hedgehog signaling, by RT-PCR. As shown in Fig. 1D, Myh10 but PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19681941 not Myh9 knockdown strongly inhibited Gli-1 induction upon SAG stimulation, indicating that Myh10 is required for cilium-mediated sonic hedgehog signal transduction. These experiments collectively demonstrated that Myh10 is required for proper ciliogenesis. Myh10 Antagonizes Myh9 Activity in Cilium Formation To further ascertain the role of Myh10 in cilium formation, we determined whether an increase of Myh10 expression is sufficient to promote the growth of 3 / 21 A Mec17-Myosin II Axis Controls Ciliogenesis Fig. 1. Myh10 Is Required for Ciliogenesis. Myh10 knockdown inhibits cilium formation in RPE-Mchr1GFP cells. RPE-Mchr1GFP cells were transfected with control siRNA, Myh10 siRNA 1 and 2 and Myh9 siRNA duplex and then serum starved for 36 hours. Cells were fixed by methanol for indirect fluorescence staining using rabbit anti-glu-tubulin and mouse anti-c-tubulin. Nuclei 4 / 2