The Disassembly Of Mitotic Spindles Is Governed By Hierarchy Redox Signals On A Cysteine-rich Protein CRIPT

Background: proliferation has to be tightly controlled to guarantee the fidelity of chromosome segregation into daughter cells during the cell cycle,in which the assembly and disassembly of spindles is highly dynamic and organized.Although much has been learned about how spindles assemble,how spindles rapidly and irreversibly disassemble is completely unknown.On the other hand,recent studies found that cellular ROS levels could function as ‘‘second messengers' ' regulating cellular proliferation,whereas it is still waiting to be studied whether redox signals work on the spindle movement.Here we identified a cysteine-rich protein CRIPT as a key molecule controlling the progression of spindle disassembly through a redox-sensing pathway.Objective: Main research content1.The effect of CRIPT on the mitosis process2.Distribution of CRIPT in cells3.Effects of different CRIPT mutants on spindle dissociation and chromosome segregation in mitosis4.Exploring which proteins interact with CRIPT and possible pathways5.Whether CRIPT has tumor suppressor function Method: 1.By knocking out and silencing the CRIPT gene,the effect of deletion of CRIPT protein on mitosis is examined,and observation of whether it can eliminate this effect when restoring its expression2.The changes of spindle in mitotic cells transfected with C3 Y mutant vector were recorded by time-lapse microscopy to understand the process of affecting spindle movement.3.Mutant fluorescent vectors were constructed by mutating cysteine at different positions,and the effect of the transfected cells on the movement of the spindle was observed to determine what different phenomena would occur in the mutation of cysteine residues at different positions.4.Identification of interacting proteins associated with CRIPT by immunoprecipitation,GST-pull down,and mass spectrometry5.A tumor model was made and the exosomes transfected with the mutant vector were used to verify whether the mutated protein could prevent the proliferation of tumor cells and inhibit tumor growth.Results: Through a series of experimental analyses,it is concluded that CRIPT plays an important role in the regulation of cell proliferation.It is mainly located in the nucleolus of the nucleus during the interphase,and interacts with the cytoskeleton to localize in the cytoplasm during the cleavage phase.It interacts with the backbone protein and the mitotic protein CKAP1,which may form a complex involved in the dissociation of the spindle.The CRIPT mutant can arrest cells in the metaphase by inhibiting the dissociation of the spindle in mitosis,and may regulate the movement of the spindle by a redox pathway.The mutant can inhibit the cells in the middle stage and has the same tumor suppressing mechanism as paclitaxel.We envisage whether or not to inhibit the proliferation of the tumor.Therefore,by constructing a tumor model on nude mice,the exosomes transfected with the mutant plasmid are injected.At the tumor site,it was observed to prevent further tumor growth and even varying degrees of necrosis.Therefore,it can be concluded that CRIPT mutant protein may be a potential antitumor drug.