| Nuclear envelope(NE)is composed of outer nuclear membrane(ONM),inner nuclear membrane(INM),nucleopore and lamina.INM is distinct and enriched with unique transmembrane proteins,named INM protein.Studies have revealed crucial roles for INM proteins in genome organization and stability,mechanotransduction,transcriptional regulation and cell signaling,enzymatic and so on.Gene mutations of some INM proteins also lead to serious diseases.Although more than thirty INM proteins have been identified in proteomic studies,only a few of them have been well characterized.The function characterization of many other INM proteins remains challenging.TMEM201(Transmembrane protein 201)ia a novel INM protein encoded by the TMEM201 gene.The human homolog is a 666 amino acid protein with five predicted transmembrane segments.TMEM201 is conserved throughout metazoans and down to Danio rerio.In addtion,TMEM201 is widely expressed in different tissues,and the expression is higher in specific organs such as brain,testis and ovary.Although TMEM201 is identified in a number of proteomic studies,whereas its function remains to be defined.This paper mainly discussed TMEM201 function and the underlying mechanism on endothelial cell(EC)migration and angiogenesis.Angiogenesis is a critical process that occurs during development and is also activated in wound healing.Reconstructing vascular networks is a complex process involving multiple processes and various kinds of cell,including the degradation of vascular basement membrane,EC activation,proliferation,migration and blood vessel reconstruction.To investigate the regulatory effect of TMEM201 on EC migration and angiogenesis,we established sh RNA-mediated TMEM201 knockdown in human umbilical vein endothelial cell(HUVEC),followed with Transwell and wound healing assays.Results showed that TMEM201 knockdown impaired HUVEC migration and polarity,with disorganized nulceus and Golgi apparatus in wound healing.Moreover,TMEM201-knockdown lead to a striking failure of HUVEC tube formation.And the results of fibrin gel beads sprouting assay indicated that TMEM201 expression is positively correlated with EC sprouting ability.To further address TMEM201 function on angiogenesis,Tmem201 knockout mouse and tmem201 knockout zebrafish were generated by CRISPR/Cas9 technology.Tmem201 knockout mice exhibited a delay in the development of the retinal vascular plexus.In addition to physiological angiogenesis,TMEM201 function on pathological angiogenesis was also investigated.Results showed that TMEM201 deficiency caused impaired angiogenesis in oxygen-induced retinopathy model and matrigel plug assay.The zebrafish embryo model was used to further address the proangiogenic role of TMEM201.As expected,loss of TMEM201 lead to the defective intersegmental vessels development in zebrafish embryo.Re-expressed TMEM201 can partially rescue vessel development and angiogenesis in tmem201 knockout zebrafish.The INM localization of TMEM201 limits the pathway it may be involves in.TMEM201 is likely to regulate cell projections by interacting with proteins localized in the NE or nuclear periphery.An APEX2-mediated proximity labeling technique with high temporal resolution and spatial specificity was applied to identify TMEM201 interaction partners.In the standard method,TMEM201 was fused with APEX2,the following proteomic results identified a number of proteins in other subcellular organelles such as endoplasmic reticulum.Therefore,modified rapamycin and APEX2-dependent identification of proteins(RAPID)proximity labeling method was developed.In the RAPID method,TMEM201 was fused with the FKBP-rapamycinbinding(FRB)domain.APEX2 was fused with 12-k Da FK506-binding protein(FKBP12)and the nuclear localization signal,which restricts APEX2 activity to the nucleus.FRB combined with FKBP12 under the rapamycin treatment,so APEX2 was recruited in close proximity to TMEM201.The following proteomic results showed that RAPID specifically identified proteins located in the NE or nucleus.In the proteomics results,we found SUN2 and Lamin-A/C,which two are the component of LINC complex,is close to TMEM201.LINC complex is formed by a trans-luminal direct interaction between SUN-domain proteins in the INM and KASHdomain protein nesprins spanning the ONM.The LINC complex binds to the actin cytoskeleton and contributes to establishing cell polarity prior to migration,so the intact and functional LINC complex is important for cell migration,as well as EC function and homeostasis.In our study,TMEM201 was found interacting with LINC complex component,SUN2 and Lamin-A/C in HUVEC,and the N-terminus was identified for the interaction.In HUVEC,TMEM201 truncation which lacks N-terminus(TMEM201△N)can not promote EC migration and tubeformation.In zebrafish,TMEM201△N can not rescue vessel development in tmem201 knockout zebrafish,whereas trunction keeps N-terminus can rescue the phenotype.There are several hypotheses about the detailed mechanisms,which would be explored in the future.In addtion to the function and underlying mechanism studies of TMEM201 in EC migration and angiogenesis,this paper also utilized Tmem201-knockout mice.In Tmem201 knockout mice,we found that loss of TMEM201 caused female infertility.TMEM201 is highly expressed in the ovary and oocytes,while TMEM201 deficiency caused defects in follicular maturation and ovulation.In summary,firstly,this study reported TMEM201 function and underlying mechanism on EC migration and angiogenesis for the first time.TMEM201 interacts with LINC complex and the specific domain was defined,which is required for EC migration and angiogenesis.Secondly,we established the proximity labeling method to identify TMEM201 interaction partners,which provide references and resource matrials for further studies regarding TMEM201 and other INM proteins.Last but not the least,the analysis of Tmem201 knockout mouse phenotype,suggested that TMEM201 functions on follicular development.This finding provide clues for further studies of TMEM201. |
PDF Full Text Request