Studies On The Effect And Mechanism Of BIN2 Control Of Oocyte Development

Bin family includes BIN1,BIN2 and BIN3,they all have a conserved Bin/Amphiphysin/Rvs(BAR)domain,a dimerization module that binds membranes and detects membrane curvature.Previous studies showed that Bin proteins mainly regulate cell motility,fillapodia formation and endocytosisin in immune cells or the phagocytosis and release of neurotransmitter,however no study has been done in mouse oocytes.Our preliminary immunofluorescence(IF)and western blot showed that Bin2 is the only one of bins in oocytes and is much more predominant in oocytes than in granular cells,So we decided to focus on BIN2 in mice.And in this part,we carefully studied the functions of BIN2 in the control of female reproduction,and made some initial efforts to explore the potential mechanisms underlying the control by BIN2.Firstly we found that BIN2 localized in spindle and cell membrane by IF.Then we did comprehensive phenotype analysis after knocking down it with specific siRNA and found that loss of BIN2 caused significant meiosis delay mainly characterized by chromosome segregation problem at Anaphase I and chromosome congression problem at Metaphase II.Not surprisingly,IVF(in vitro fertilization)results showed significantly lower fertility and higher polypronucleus rate.Moreover,we also found that the microvilli on the membrane was severly abolished.Next we have characterized several important interacting proteins through IP-Maldi and established a pathway model that BIN2 interacts with y-catenin and Lck,gets activated and subsequently activates Rac1 and its downstream kinases,Lkb1 and Plkl to directly affect meiosis at protein level.Currently a series of co-IP and kinase activity test have already largely proved this model.We do some preliminary explorations on the potential mechanisms by which BIN2 control oocyte development.First we found there's no difference on y-catenin or p-y-catenin by western blot after knockdown of Bin2.Then we know that y-catenin can develop a dimer to transduct signal,so we deal with oocytes by DSS.After that,we surprisingly find that the treatment group has a deeper band in the double bigger than the ?-catenin by BIN2 IP sliver stain.Obviously we speculate that Bin2 activate y-catenin to dimer to play a role in signal transduction.In conclusion,this study suggests that membrane Bin2-triggered y-catenin signal is important for oocyte meiosis in vitro.We also did IP-phospho LCMS and characterize two adjacent phosphorylation sites-Thr424 and Ser425(phosphorylation possibility for both are 99.8%)in BIN2 and successfully made specific phospho antibody(p-BIN2),immunofluorescence showed that p-BIN2 concentrated at spindle more than BIN2,but Bin2 concentrated at membrane more than p-BIN2,suggesting that p-BIN2 may have no activity.Otherwise,the generation of Bin2 knockout mice is in process now.Taken together,BIN2 is a key protein that is specifically expressed in the oocyte and plays pivotal roles in the control of meiosis and developmental competence of oocytes.BIN2 controls oocyte development,thus affecting the quality of oocytes,by the dimerization of y-catenin rather than the phosphorylation of y-catenin.