Research On The Role And Mechanism Of Rab34 In The Ciliogenesis And Hedgehog Signaling Pathway

Primary cilia are one kind of cilia that exist on the surface of eukaryotic cells,small in size but complex in structure.They are highly conserved,nonmobile organelle,which play important sen-sory roles in sensing mechanical,chemical,and biological signals in the environment and assisting the conduction into cells to cause cellular responses.The length of cilia is about 5 to 10?m and the diameter is about 0.25?m.The cilia extend from the basal body?from the mother centriole of the centrosome?and consist primarily of central axoneme,ciliary membrane surrounding it,and ciliary matrix.The axoneme is the skeletal structure of the cilia,consisting of 9 groups of circularly ar-ranged doublet vascular bundles and their accessory proteins.The ciliary membrane is a two-layer lipid membrane attached to the cell membrane,but the membrane protein component is different.It is rich in specific signaling receptors and ion channels,enabling the cilia to detect changes in the extracellular environment and transmit extracellular signals.To regulate multiple cellular processes during development and maintain tissue homeostasis.The cilia matrix consists mainly of soluble proteins for cilia assembly and turnover.The process of ciliogenesis is complex and orderly.When the mitotic cycle of the cell ends and progresses into a G0 quiescent state,assembly of the primary cilia begins.The mother centriole is transformed into a basal body,and ciliary vesicles formed by successive fusion of cilia pre-vesicles in the vicinity thereof are recruited and further extended into axoneme.The mother centriole has a distal appendage consisting of a variety of proteins recruited by the distal centriolar proteins;they are essential for anchoring the mother centrosome to the ciliary membrane and evolve into a transi-tion fiber in the matrix.The final vesicles fuse with the plasma membrane to expose the cilia to the extracellular environment.Proteins cannot be synthesized in cilia,so the self-assembly,maintenance and disassembly of primary cilia must rely on intraflagellar transport?IFT?to regulate the bidirec-tional transport of substances in the cilia.The IFT system is evolutionarily conserved and consists of two stable complexes?IFT-A and IFT-B?that are used to link the cilia targeting protein to motor molecules responsible for bidirectional transport along the microtubule axoneme.The IFT-A com-plex is responsible for the reverse transport of substances in the cilia,mediated by cytoplasmic dynein 2,while IFT-B is responsible for forward transport,and is mediated by the kinesin family member Kinesin 2.Mutations in any of the proteins in the IFT system can result in changes or deletions in primary cilia morphology.Studies have shown that primary cilia are critical in regulating the signaling pathways of intra-cellular Ca²⁺levels as well as the Hedgehog?Hh?signaling pathway and the planar cell polarity?PCP?pathway.Among them,the Hedgehog signaling pathway is used for intercellular communi-cation and plays an important role in the development of the central nervous system,limbs and embryonic structures,controlling the"destiny"and proliferation of cells.Unlike other core devel-opmental signaling pathways,vertebrate Hh signaling is completely dependent on highly special-ized organelles,primary cilia.Studies have shown that cilia play an important role in the transduc-tion of Sonic Hedgehog?Shh?signaling pathways.The majority of proteins in the Hh signaling pathway are localized on primary cilia,including the seven-transmembrane protein Smoothened?Smo?,the 12-transmembrane protein Patched?PTCH1?,the transcription factor Gli1-3,and the negative regulator Sufu.The membrane receptor Ptc is located on the base of the cilia and inhibits Hh signaling by preventing Smo from entering the cilia in the absence of ligand Hh.At this time,the full-length transcription factor Gli3^FL was cleaved into the inhibitory factor Gli3^Rep.When bound to the ligand Hh,Ptc moves out of the cilia,Smo is activated and accumulates in the cilia,allowing the downstream Gli transcription factor to be modified to activate,inducing transcription and ex-pression of the target gene.Deletion of Hh signaling activity leads to severe congenital malfor-mation,primary cilia is essential for Hh signaling,and abnormalities in Hh signaling pathway can cause a variety of tumors,which provides a new idea for the treatment of tumor diseases.Studies linking cilia/basal body/centrosome proteins to human genetic diseases have also shown that impaired cilia function may have profound effects on cellular homeostasis.Defects in primary cilia structure and function are associated with a wide range of dysplasia and metabolic diseases,and can affect many different organs during embryonic development and after birth,in-cluding open brain injury,forebrain non-cracking deformity,polysyndactyly,small eye disease,pol-ycystic kidney disease,retinal degeneration and obesity are often referred to as"ciliopathies".Pa-tients with ciliopathies often have multiple symptoms,such as chronic sinusitis and respiratory in-fections,translocation of the heart and other organs,thoracic deformity,reduced fertility,pelvic dysplasia,visual,auditory and olfactory abnormalities,cartilage abnormalities,multi-finger?toe?malformation,abnormal brain development,polycystic hyperplasia of liver and kidney,and so on.Therefore,in-depth exploration of the regulation mechanism of cilia is of great significance to the development of basic biological theory and the development of new therapies for the treatment of human ciliopathies.Rab34 is a small GTPase associated with the Golgi apparatus.Previous studies have shown that the absence of Rab34 leads to an abnormal phenotype of polysyndactyly in mice.Rab protein is one of the six major families of regulated small molecule GTP-binding proteins.It regulates its activated state by binding to GTP/GDP and is an important molecular switch for cell signaling.Rab protein is involved in the formation of cilia in cells cultured in vitro,but has not been found in vivo.Rab34 is a member of the Rab family and participates in intracellular vesicle trafficking with effec-tors.A recent large-scale CRISPR screening study reported that Rab34 is associated with cilia and Hh signaling in cultured cells,however,no in vivo studies and mechanisms have been proposed in this study.Therefore,our study aims to further understand the molecular and cellular mechanisms by which Rab34 affects mouse development in mice.Given that many cilia gene mutations affect the limb development pattern by reducing the Gli3processing that produces the Gli3 repressor,we explored the possibility of Rab34 involvement in ciliogenesis.Firstly,we designed two independent sgRNAs that specifically recognized Rab34.The Crispr-cas9 gene editing technique was used to clone the DNA oligonucleotides corresponding to Rab34 into the pLenti-Crispr-v2 vector.Then,by packaging,transfecting and collecting the lentivi-rus to further infect the cell lines of interest,we constructed Rab34^-/-stable cell lines of cultured in vitro.We found that ciliogenesis in the Rab34^-/-cell line significantly decreased by immunofluores-cence staining.In order to explore whether the deletion of Rab34 in mice affects the formation of cilia,we constructed Rab34 knockout mice using gene targeting technology.The obtained chimeric mice were crossed with wild type C57BL/6J mice to obtain first generation?F1?heterozygous mice.F1hybrid heterozygous mice were crossed with each other,and Rab34 gene homozygous embryos of different embryonic stages were collected and their genetic phenotypes were observed.Statistics of mouse embryo genotypes showed that about 25%of the E18.5 embryos were homozygous and all alive;however,no newborn homozygous mice were alive,indicating that the Rab34 mutant mice died at birth.Embryos of different embryonic stages were obtained by dissection.By observing mouse embryos,it was found that Rab34^-/-mouse embryos showed abnormal features of polydactyly,cleft lip and cleft palate.To determine whether ciliogenesis was affected in Rab34 mutant mice,we collected embryos from E10.5 Rab34 mutant mice and wild type mice and prepared frozen tissue sections.The immunofluorescence staining method was used to detect whether the formation of cilia of the mutant homozygous embryonic neural tube and limb buds was normal.We found that in the Rab34 mutant,the number of cilia was significantly reduced.This indicates that in mice,the deletion of Rab34 affects cilia formation.In addition,by immunofluorescence staining,we tested the effects of Rab34 gene on centriolar and ciliary proteins such as Rab8,Rab11,Rabin8,BBS4,Ehd1,IFT20,and Cp110.Compared with wild-type cells,the location and expression of such proteins in Rab34-deficient cells did not change significantly,which indicated that the above ciliogenesis-related proteins were independent of Rab34.At the same time,in order to determine the subcellular localization of Rab34,a stable cell line overexpressing Rab34 was constructed.We firstly used the mouse cDNA library as a template to construct LAP-Rab34,as well as two mutants of LAP-Rab34,namely LAP-Rab34-T66N?inhibited state?and LAP-Rab34-Q111L?activated state?.Co-immunostaining with GFP and acetylated tubulin?Ac-Tub?was used to detect the expression of Rab34 and its mutants.It was found that the endocytosis protein Rab34 localized to the cell cilia.By overexpressing the Rab34 protein in NIH3T3 cells,we found that it neither promoted nor inhibited the ciliogenesis of NIH3T3 cells.In order to better understand the molecular mechanism of polysyndactyly phenotype formation in Rab34 mutant mouse embryos,we utilized Western blotting to detect the expression levels of Gli2 and Gli3 proteins in embryos.The results showed that the expression level of the repressor Gli3^Rep in Gli3 processing in Rab34 mutant embryos decreased,while the expression lev-els of Gli2^FL and Gli3^FL were significantly increased.Besides,the RNA levels of Patch1 and Gli1in WT and Rab34 mutant embryo cells before and after SAG activator\activation were detected by RT-qPCR.After SAG activation,the RNA levels of Patch1 and Gli1 in WT cells were 6-13 times than earlier,but not in Rab34 mutant embryo cells.This result indicates that the Hh signaling is impaired in the Rab34 mutant cells.Therefore,Rab34 is a type of protein necessary for early for-mation of ciliary vesicles and Hh signaling in vivo.We observed cilia in neuroepithelial cells in the neural tube near the forelimb region of E10.5WT and Rab34^-/-embryos by transmission electron microscopy?TEM?.We found that there was significant accumulation of ciliary vesicles in Rab34^-/-embryos.Phenomenon.Furthermore,the Rab34 mutant was anchored to the plasma membrane and was less surrounded by vesicles than the wild type?1.2%vs.11.3%?.Thus,Rab34 is a protein necessary for the fused vesicles from the pre-ciliary to the formation of ciliary vesicles and for the migration of the mother centrioles from the perinuclear region to the plasma membrane.Taken together,this study reveals for the first time that in mice,the Rab34 gene plays an im-portant role in the process of ciliogenesis.It is mainly involved in the early formation of ciliary vesicles;Rab34 mutation affects the processing of Gli3 protein and the function of Gli2protein,thereby inhibiting the transduction of Hedgehog signaling pathway.The gene knockout technique provides powerful technical support for the study of the regulation of ciliogen-esis and the study of gene-related phenotypes.The study laid the foundation for intervention and treatment of primary ciliogenesis diseases.