【作者】 王蕾；

【导师】 方靖； 张刚；

【作者基本信息】 青岛大学 ， 生物化学与分子生物学， 2022， 硕士

【摘要】 研究目的:真核生物的遗传物质复制后需要平均分配到两个子细胞中,该过程依赖于有丝分裂组装检查点(Spindle Assembly Checkpoint,SAC)对纺锤体微管与染色体着丝点正确结合的监控。着丝点蛋白Bub1是SAC的主要组成蛋白之一,在促进检查点激活方面发挥重要作用。本课题组及他人研究发现,Bub1也参与了染色体的移动排列,但是其具体分子机制尚不清楚。促进染色体移动与双极排列也意味着促进检查点沉默,那么,激活和沉默检验点这两个相反的功能是如何发生协同作用来保证细胞有丝分裂的正常进行呢?本研究根据Bub1相关功能域构建多个突变体并进行功能验证,确认了Bub1促进染色体移动与双极排列的分子机制,并进一步探究其与检查点的协同作用,为之后的细胞有丝分裂研究提供理论基础。研究方法:1.在HeLa细胞中共转染Bub1 siRNA和抗干扰的野生型YFP-Bub1质粒,免疫荧光染色以及活细胞成像观察有丝分裂细胞染色体移动和排列情况。2.根据Bub1的不同功能域进行突变质粒构建。在HeLa细胞中进行RNAi与回补实验,通过活细胞成像和免疫荧光检测不同突变体支持染色体排列和检查点的能力。3.通过活细胞成像和等温量热滴定法(ITC)确认Bub1中B56基序的生物学功能。将抗干扰的野生型YFP-Bub1质粒删除与BubR1结合的功能域,命名为Bub1DBubR1。以Bub1DBubR1为基础,选取BubR1的B56结合基序(649-697aa)分别替代Bub1与BubR1结合的结构域(266-311aa)和Bub1的B56结合基序的区域(634-686aa),分别命名为Bub1-B56N和Bub1-B56C。以不能结合B56的2A突变体(634-686aa)为阴性对照,命名为Bub1-B56 2A。利用免疫共沉淀检测突变体结合B56的能力以及Plk1介导的Bub1-B56N和Bub1-B56C上T680的磷酸化水平。在HeLa细胞中转染Bub1 siRNA以及抗siRNA干扰的Bub1-B56N和Bub1-B56C突变体,通过活细胞成像实验检测低剂量的nocodazole条件下有丝分裂细胞检查点强度。采用免疫荧光技术和Western blot检测两种突变体细胞中Bub1的荧光信号强度以及双磷酸化水平。4.将BubR1的假激酶功能域替换为Bub1的激酶功能域,通过活细胞成像和免疫荧光技术来探究Bub1与BubR1激酶功能分离对细胞有丝分裂的影响。研究结果:1.在HeLa细胞中转染Bub1 siRNA,细胞表现出严重的染色体排列错误,回补抗siRNA的野生型YFP-Bub1质粒后能够恢复染色体的正常排列。2.通过活细胞成像实验对一系列Bub1突变体的检测显示,Bub1DBubR1突变体显示出最强的染色体排列缺陷,免疫荧光定量显示出着丝点外端蛋白的磷酸化水平显著增加。另外两个具有轻度缺陷的突变体是Bub1激酶突变体和Bub1mut CENP-F突变体,它们在表型上高度相似。因此,Bub1主要通过与BubR1结合来影响染色体排列。3.通过活细胞成像实验发现Bub1本身的B56结合基序并不影响有丝分裂细胞检查点和有丝分裂的正常进行,ITC结果也表明了Bub1无法与B56蛋白结合。在Bub1 DBubR1基础上构建Bub1-B56N和Bub1-B56C突变体进行免疫共沉淀,相较于Bub1-B56N,Bub1-B56C结合的B56明显增多。使用Western blot以及免疫荧光定量发现,Bub1-B56C上的p T680信号明显高于Bub1-B56N。由于T680是Plk1磷酸化位点。因此,Plk1结合位点的距离决定了T680的磷酸化水平,进而影响了PP2A/B56的结合量。4.活细胞成像实验发现,表达Bub1-B56N的细胞可以在一定的时间内修复染色体排列缺陷,而超过50%的表达Bub1-B56C的细胞不能有效地激活有丝分裂检查点,在仍有一定数量的染色体未排列到赤道板的情况下细胞过早的进入有丝分裂后期。通过低剂量的nocodazole处理细胞来检测有丝分裂检查点强度,结果发现,Bub1-B56C检查点强度大幅度降低,而Bub1-B56N保持了一个正常的检查点强度。通过对Bub1 CD1结构域内S459/T461的磷酸化进行免疫荧光分析,结果显示表达Bub1-B56C的细胞着丝点上的双磷酸化减少了60%。以上结果表明,恢复Bub1与B56的结合能力破坏有丝分裂检查点。5.将BubR1的假激酶(732-1050aa)替换为Bub1的激酶区域(734-1085aa),野生型融合蛋白命名为Mad Bub WT,突变型融合蛋白命名为Mad Bub KD。转染Mad Bub WT和Mad Bub KD两种突变体进行活细胞成像或免疫荧光分析,结果显示,Mad Bub WT有丝分裂时间延迟。对相关蛋白的定位进行检测,发现部分蛋白无法全部定位于原位点。BubR1激酶活性的恢复影响了一些重要的有丝分裂参与者的着丝点定位,从而可能导致有丝分裂延迟。研究结论:Bub1通过结合BubR1来招募蛋白磷酸酶PP2A/B56促进染色体的移动和排列。原位恢复Bub1与B56的结合能力,导致Bub1的CD1结构域发生去磷酸化,进而破坏有丝分裂检查点。BubR1中激酶的重新激活破坏了一些着丝点蛋白的正确定位,可能影响有丝分裂的正常进程。因此,在Bub1-BubR1复合物之间分离激酶活性和磷酸酶活性能够确保适当的有丝分裂。更多还原

【Abstract】 Objectives:Spindle Assembly Checkpoint(SAC)monitors the correct binding of spindle microtubules to chromosome kinetochores..The centromeric protein Bub1 is one of the major components of SAC and plays an important role in promoting checkpoint activation.Our research team and others found that Bub1 is also involved in chromosomal alignment,but the specific molecular mechanism is still unclear.Interestingly,promoting chromosomal mobility and bipolar alignment also means promoting checkpoint silencing.How do the opposite functions of the activation and silencing test work together to ensure proper mitosis?In this study,multiple mutants were constructed according to the relevant functional domains of Bub1 and their functions were verified to confirm the molecular mechanism of Bub1 promoting chromosome movement and bipolar arrangement,and further explore its synergistic effect with checkpoint,providing a theoretical basis for subsequent cell mitosis research.Methods:1.HeLa cells were co-transfected with Bub1 siRNA and wild-type YFP-Bub1 plasmid,which is resistant to RNAi.Immunofluorescence staining and live cell imaging to observe the chromosome movement and arrangement of mitotic cells.2.Construct mutant plasmids based on different functional domains of Bub1.RNAi and rescue experiments were performed in HeLa cells to detect different mutant’s ability to support chromosome alignment and checkpoints through live cell imaging and immunofluorescence.3.The role of the B56 motif in Bub1 was investigated by live cell imaging.It’s ability to bind B56 protein was also examined by isothermal calorimetric titration(ITC).The anti-interference wild-type YFP-Bub1 plasmid was deleted from the functional domain binding to BubR1 and named Bub1DBubR1.Based on Bub1DBubR1,the B56 binding motif of BubR1(649-697aa)is selected to replace the domain binding Bub1 and BubR1(266-311aa)and the B56 binding motif of Bub1(634-686aa)respectively.Named Bub1-B56N and Bub1-B56C.A 2A mutant(634-686aa)that cannot bind to B56 was designated Bub1-B56 2A as a negative control.Bub1-B56N and Bub1-B56C mutants were constructed on the basis of Bub1DBubR1,and Bub1-B56 2A was used as the negative control.Immunoprecipitation of the YFP-tagged protein was performed to detect B56 binding as well as Plk1-mediated phosphorylation of T680 on Bub1-B56N and Bub1-B56C.4.HeLa cells were co-transfected with siRNA against Bub1 and RNAi-resistant Bub1-B56N and Bub1-B56C constructs.The time each cell spent in mitosis in the presence of low amount of nocodazole was recorded by live cell imaging.Immunofluorescence and Western blot were also performed to detect the fluorescence signal intensity and dual phosphorylation levels of Bub1.5.The pseudokinase of BubR1 was replaced with the genuine kinase of Bub1,and the effect of functional separation of Bub1 and BubR1 on cell mitosis was investigated by live cell imaging and immunofluorescence.Results:1.Bub1 siRNA was transfected into HeLa cells,cells showed severe chromosome alignment errors,which were fully rescued by co-transfection of RNAi-resistant YFP-tagged wild-type Bub1 plasmid.It shows Bub1 promotes accurate alignment of chromosomes.2.The Bub1DBubR1 mutant showed the strongest chromosomal alignment defect when tested on a series of Bub1 mutants using live cell imaging assay,Quantitative immunofluorescence showed a significant increase of the phosphorylation levels of on the out kinetochore proteins.Two other mutants with mild defects are the Bub1 kinase mutant and the Bub1mut CENP-F mutant,which are highly similar in phenotype.Thus,Bub1 affects chromosome alignment mainly through binding to BubR1.3.Live cell imaging revealed that Bub1’s B56 binding motif did not affect mitotic cell checkpoints and mitosis,and ITC results also showed that Bub1 could not bind to B56protein.Bub1-B56N and Bub1-B56C mutants were constructed on the basis of Bub1DBubR1 for immunoprecipitation.Compared with Bub1-B56N,Bub1-B56C binds significantly more B56 as shown by immunoprecipitation experiments.Western blot analysis and quantitative immunofluorescence showed a significant increase of p T680singals on Bub1-B56C than Bub1-B56N.T680 is the Plk1 phosphorylation site,the distance to Plk1 binding site determines the phosphorylation levels of T680 which further affects the amount of PP2A/B56 binding.4.Live cell imaging experiments showed that Bub1-B56N repaired chromosome alignment defects after a delay,while more than 50%of Bub1-B56C cells entered anaphase before aligning all the chromosomes without a mitotic delay which indicates the failure to activate the checkpoint.Mitotic checkpoint strength was measured by treating cells with a low dose of nocodazole and it was found that Bub1-B56C checkpoint strength was significantly reduced while Bub1-B56N maintained a normal checkpoint strength.Immunofluorescence analysis of phosphorylation of S459/T461 in the Bub1CD1 domain revealed a 60%reduction in phosphorylation at the kinetochore of Bub1-B56C expressing cells.These results suggest that restoring the binding ability of Bub1 to B56 disrupts the mitotic checkpoint.5.The pseudokinase of BubR1(732-1050aa)was replaced with the kinase region of Bub1(734-1085aa),with the wild-type fusion protein named Mad Bub WT and the mutant fusion protein named Mad Bub KD.Mad Bub WT and Mad Bub KD mutants were transfected for live cell imaging or immunofluorescence analysis,and the results showed that Mad Bub WT had delayed mitosis time.The localization of related proteins was detected and it was found that some proteins could not all be located in the original site.Restoration of BubR1 kinase activity affected centromeric localization of some key mitotic players which may result in delayed mitosis.Conclusions:Bub1 binds to BubR1 to recruit the protein phosphatase PP2A/B56 to facilitate chromosome movement and alignment.In situ restoration of Bub1’s binding ability to B56 resulted in dephosphorylation of Bub1’s CD1 domain,which in turn disrupted the mitotic checkpoint.Reactivation of the kinase in BubR1 disrupts the correct localization of some kinetochore proteins and may affect the normal course of mitosis.Thus,separation of the kinase activity and phosphatase activity between Bub1-BubR1complex ensures proper mitosis.更多还原