The role of primary cilia in mechanical transmission of osteocyte based on a 3D finite element model

The primary cilium,as a mechanical receptor of osteocytes,participates in the regulation of osteocyte mechanosensitivity.However,how the length of osteocyte primary cilia changes with fluid shear stress(FSS)are unclear,and how the mechanical transmission within osteocytes altered by primary cilia is not well understood yet.Therefore,the ciliary length changes of osteocyte under 15dyn/cm2 of FSS were experimentally detected,and then 3D finite element models of osteocyte primary cilia containing the basal body and axoneme were built.The results showed that(1)The ciliary length of the CON group,FSS 1h,and FSS 6h were 3.71±1.34μm,3.79±1.04μm,and 1.24±0.73μm respectively,indicating the different durations of FSS might lead to the adaptive changes of cilium length.The calculations showed(2)when the ciliary length became shorter with the ciliary angle stayed the same,the deformation and stress of the cell membrane and membrane skeleton was increased.However,the deformation and stress of the cilia membrane,basal body,the rotation angles of basal body were decreased,and those of cytoplasm,cytoskeleton,actin cortex and nucleus were also decreased;(3)With the decrease of the ciliary angle,the deformation and stress of the cilia membrane,basal body,as well as the rotation angles of basal body were increased.Those of the cytoplasm,cytoskeleton,actin cortex,and nucleus were also increased except the cell membrane and membrane skeleton.The calculation results suggested the length and angle of the primary cilia,the deformation and stress of intracellular structures in osteocyte were altered with ciliary basal body,indicated the connection between the basal body and cytoskeleton may be a key factor that affected the mechanical transport in osteocytes across the cell membrane.This finally promoted the adaptive change of ciliary length under FSS.

Primary cilia mediate Klf2-dependant Notch activation in regenerating heart

Unlike adult mammalian heart,zebrafish heart has a remarkable capacity to regenerate after injury.Previous study has shown Notch signaling activation in the endocardium is essential for regeneration of the myocardium and this activation is mediated by hemodynamic alteration after injury,however,the molecular mechanism has not been fully explored.In this study we demonstrated that blood flow change could be perceived and transmitted in a primary cilia dependent manner to control the hemodynamic responsive klf2 gene expression and subsequent activation of Notch signaling in the endocardium.First we showed that both homologues of human gene KLF2 in zebrafish,klf2a and klf2b,could respond to hemodynamic alteration and both were required for Notch signaling activation and heart regeneration.Further experiments indicated that the upregulation of klf2 gene expression was mediated by endocardial primary cilia.Overall,our findings reveal a novel aspect of mechanical shear stress signal in activating Notch pathway and regulating cardiac regeneration.

Primary cilia in hard tissue development and diseases

Bone and teeth are hard tissues.Hard tissue diseases have a serious effect on human survival and quality of life.Primary cilia are protrusions on the surfaces of cells.As antennas,they are distributed on the membrane surfaces of almost all mammalian cell types and participate in the development of organs and the maintenance of homeostasis.Mutations in cilium-related genes result in a variety of developmental and even lethal diseases.Patients with multiple ciliary gene mutations present overt changes in the skeletal system,suggesting that primary cilia are involved in hard tissue development and reconstruction.Furthermore,primary cilia act as sensors of external stimuli and regulate bone homeostasis.Specifically,substances are trafficked through primary cilia by intraflagellar transport,which affects key signaling pathways during hard tissue development.In this review,we summarize the roles of primary cilia in long bone development and remodeling from two perspectives:primary cilia signaling and sensory mechanisms.In addition,the cilium-related diseases of hard tissue and the manifestations of mutant cilia in the skeleton and teeth are described.We believe that all the findings will help with the intervention and treatment of related hard tissue genetic diseases.

Centrosome positioning and primary cilia assembly orchestrate neuronal development

Establishment of axon and dendrite polarity, migration to a desired location in the developing brain, and establishment of proper synaptic connections are essential processes during neuronal development. The cellular and molecular mechanisms that govern these processes are under intensive investigation. The function of the centrosome in neuronal development has been examined and discussed in few recent studies that underscore the fundamental role of the centrosome in brain development. Clusters of emerging studies have shown that centrosome positioning tightly regulates neuronal development, leading to the segregation of cell factors, directed neurite differentiation, neuronal migration, and synaptic integration. Furthermore, cilia, that arise from the axoneme, a modified centriole, are emerging as new regulatory modules in neuronal development in conjunction with the centrosome. In this review, we focus on summarizing and discussing recent studies on centrosome positioning during neuronal development and also highlight recent findings on the role of cilia in brain development. We further discuss shared molecular signaling pathways that might regulate both centrosome and cilia associated signaling in neuronal development. Furthermore, molecular determinants such as DISC1 and LKB1 have been recently demonstrated to be crucial regulators of various aspects of neuronal development. Strikingly, these determinants might exert their function, at least in part, via the regulation of centrosome and cilia associated signaling and serve as a link between these two signaling centers. We thus include an overview of these molecular determinants.

Inhibition of Ciliogenesis Enhances the Cellular Sensitivity to Temozolomide and Ionizing Radiation in Human Glioblastoma Cells

Objective To investigate the function of primary cilia in regulating the cellular response to temozolomide(TMZ)and ionizing radiation(IR)in glioblastoma(GBM).Methods GBM cells were treated with TMZ or X-ray/carbon ion.The primary cilia were examined by immunostaining with Arl13 b andγ-tubulin,and the cellular resistance ability was measured by cell viability assay or survival fraction assay.Combining with cilia ablation by IFT88 depletion or chloral hydrate and induction by lithium chloride,the autophagy was measured by acridine orange staining assay.The DNA damage repair ability was estimated by the kinetic curve ofγH2 AX foci,and the DNAdependent protein kinase(DNA-PK)activation was detected by immunostaining assay.Results Primary cilia were frequently preserved in GBM,and the induction of ciliogenesis decreased cell proliferation.TMZ and IR promoted ciliogenesis in dose-and time-dependent manners,and the suppression of ciliogenesis significantly enhanced the cellular sensitivity to TMZ and IR.The inhibition of ciliogenesis elevated the lethal effects of TMZ and IR via the impairment of autophagy and DNA damage repair.The interference of ciliogenesis reduced DNA-PK activation,and the knockdown of DNA-PK led to cilium formation and elongation.Conclusion Primary cilia play a vital role in regulating the cellular sensitivity to TMZ and IR in GBM cells through mediating autophagy and DNA damage repair.

Characterization of two novel knock-in mouse models of syndromic retinal ciliopathy carrying hypomorphic Sdccag8 mutations

Mutations in serologically defined colon cancer autoantigen protein 8(SDCCAG8)were first identified in retinal ciliopathy families a decade ago with unknown function.To investigate the pathogenesis of SDCCAG8-associated retinal ciliopathies in vivo,we employed CRISPR/Cas9-mediated homology-directed recombination(HDR)to generate two knock-in mouse models,Sdccag8^(Y236X/Y236X) and Sdccag8^(E451GfsX467/E451GfsX467),which carry truncating mutations of the mouse Sdccag8,corresponding to mutations that cause Bardet-Biedl syndrome(BBS)and Senior-L?ken syndrome(SLS)(c.696T>G p.Y232X and c.1339-1340ins G p.E447GfsX463)in humans,respectively.The two mutant Sdccag8 knock-in mice faithfully recapitulated human SDCCAG8-associated BBS phenotypes such as rod-cone dystrophy,cystic renal disorder,polydactyly,infertility,and growth retardation,with varied age of onset and severity depending on the hypomorphic strength of the Sdccag8 mutations.To the best of our knowledge,these knock-in mouse lines are the first BBS mouse models to present with the polydactyly phenotype.Major phototransduction protein mislocalization was also observed outside the outer segment after initiation of photoreceptor degeneration.Impaired cilia were observed in the mutant photoreceptors,renal epithelial cells,and mouse embryonic fibroblasts derived from the knock-in mouse embryos,suggesting that SDCCAG8 plays an essential role in ciliogenesis,and cilium defects are a primary driving force of SDCCAG8-associated retinal ciliopathies.