Regulation Of KIF11 In Ciliogenesis And Hedgehog Signaling Pathway

Cilia are hair-like organelles composed of microtubule structures that protrude from the surface of many types of eukaryotic cells.Cilia play important roles in many biological processes,such as environmental perception and cell movement.In general,motile cilia are mainly involved in cell movement and fluid transport,whereas non-motile cilia or primary cilia are involved in a series of signal transduction.The assembly and length control of cilia are essential for cilia homeostasis maintenance,organ development and many human diseases.It is very important to explore the molecular mechanism of ciliary assembly and length control for a deeper understanding of various ciliopathies,the development of relevant medicine and the exploration of effective treatment schemes.However,the molecular mechanisms underlying cilia assembly and length control are still unknown.To explore the molecular mechanisms of ciliary assembly and length control,we used a combination of approaches,including western blotting,immunofluorescence,super-resolution microscopy and centrosome fractions isolation,and characterized the key role of kinesin family member 11(KIF11)in ciliary formation and Hedgehog signaling pathway.In this study,firstly,we isolated and analyzed centrosome fractions and found that the protein level of KIF11 protein in centrosome fractions was significantly increased upon ciliogenesis.Meanwhile,immunofluorescence and three-dimension structured illumination microscopy(3D-SIM)showed that KIF11 localization in centrosome was increased in serum-free cultured human retinal pigment epithelial(RPE1)cells,suggesting that KIF11 likely plays an important role in ciliogenesis.Then we used two si RNA targeting KIF11 to knockdown the protein expression,and found that both the ciliary length and the percentage of ciliated cells were decreased,while overexpression of KIF11 partially recovered ciliated abnormalities caused by KIF11 depletion.Moreover,we also treated RPE1 cells with monastrol,an inhibitor of KIF11 at different concentrations,and observed the same ciliary defect induced by KIF11 depletion.This suggests that the regulation of KIF11 in ciliary homeostasis depends on its motor activity.We then explored whether KIF11-mediated ciliogenesis is involved in cilia-dependent signaling pathway.Hedgehog is known to be the most classical extracellular signaling pathway dependent on ciliary integrity,and smoothened(Smo)proteins and glioma-associated oncogene homolog I(Gli)are downstream component signals of the Hedgehog signaling pathway.We treated serum-starved RPE1 cells with the Shh-conditioned medium and examined the location of Smo and Gli,Immunofluorescence results showed that Smo and Gli1,Gli2 were activated by ligand Shh stimulation,while the nuclear localization of Gli1 and Gli2 was weakened and the ciliary localization of Smo were significantly weakened upon si RNA-mediated KIF11 depletion or inhibition with monastrol.We also overexpressed KIF11 proteins in KIF11 depleted RPE1 cells,and found the abnormal Hedgehog signaling pathway could be partially recovered.The above data indicate that KIF11-mediated ciliogenesis is involved in the regulation of the Hedgehog signaling pathway.In conclusion,this study identifies a novel ciliary protein-KIF11,and illustrates the role of KIF11 in ciliogenesis and Hedgehog signaling pathway.This provides a new perspective for a deeper understanding of cilia-related functions,as well as a new theoretical basis and research strategy for the diagnosis and treatment of ciliopathies.