Effect And Significance Of MTORC1 On The Stability Of Microtubules

Objective:mTOR(mechanistic target of Rapamycin)is an atypical serine/threonine kinase that is highly conserved in eukaryotes.It is formed of two complexes known as mTORC1 and mTORC2,which are involved in numerous biological processes in cells.In lower organism cells,the function of mTOR in regulating cytoskeleton has been confirmed.However,whether it regulates cytoskeleton in mammalian cells is still controversial.This study aims to investigate the effect of mTORC1 on the stability of microtubule and the effect of Rapamycin combined with microtubule target chemotherapeutic drugs on microtubules of tumor cells.Methods:1.Immunofluorescence technique was used to evaluate the stability of microtubules by comparing the ability of Rapamycin pretreated and untreated cells to resist Nocodazole.2.Western blotting was used to the ability of microtubule target chemotherapeutic drugs combined with Rapamycin to induce tumor cell apoptosis.3.Crispr-Cas9 technology and shRNA technology were used to constructe ATG5-deficient cell lines.4.Immunofluorescence technique was used to evaluate the resistance of ATG5 normal group and defective group cells to Nocodazole.5.The expression of microtubule-associated proteins such as Stathmin,KIF2 A and CLIP-170 under different activation levels of mTORC1 was detected by Western blotting.Results:1.The ability of Nocodazole to depolymerize microtubules significantly reduced in Rapamycin pretreated HeLa cells compared with control group.2.After using Rapamycin to stabilize microtubules,the ability of Taxol and Nocodazole to induce apoptosis in tumor cells was reduced,whereas the ability of Etoposide to induce tumor apotosis did not decrease.3.In ATG5 knockdown and knockout cells,there was no significant difference in the ability of Nocodazole to depolymerize the microtubule structure compared with the control group.Conclusions:1.Rapamycin could enhance the stability of microtubules and mTORC1 could negatively regulate microtubule stability.2.Rapamycin-stabilized microtubules could inhibit the apotosis of microtubul e-targeted chemotherapy drugs to tumor cells.3.The ability of Rapamycin to stabilize microtubules is not related with cell autophagy.The occurrence of autophagy required stable microtubules,but the stabilization of microtubules did not require the involvement of autophagy.