【作者】 杜婧； 陈晓飞； 马萧； 何利蓉； 徐佳； 冯西桥； 杨春；

【Author】 Jing Du,Xiaofei Chen,Xiao Ma,Lirong He,Jia Xu,Xiqiao Feng,Chun Yang*(Institute of Biomechanics and Medical Engineering,Engineering Mechanics Department,School of Aerospace,Tsinghua University,Beijing,100084,China

【机构】 清华大学航天航空学院工程力学系生物力学所；

【摘要】 Background:Mechanical environments have been increasingly recognized to play an important role in regulating cellular function and behavior,including proliferation,migration,apoptosis,and differentiation.More recently,emerging evidences demonstrate that the mechanical properties(e.g.,elasticity) of adhesion substrates manipulate stem cell fate in both two-demensional(2D)[1] and three-demensional(3D)[2] cultures.However,although the regulation of cell fate by extra-cellular matrix(ECM) stiffness has been reported to depend on cell traction,the connection between the mechanical properties of ECM and chemical signaling processes is not known.Evidences have been demonstrated that focal adhesion proteins activation change is closely associated with mechanical stimuli results in focal adhesion proteins activchanges,which facilitates remodeling of focal adhesion.Among these proteins,integrins are necessary elements for most mechano-sensing process and lie at the beginning of the sensing pathway.In the present study,we aim to explore the mechanism of stem cells sensing ECM elasticity,in which intergrin is vitally involved.Methods:Bone marrow mesenchymal stem cells(BMMSC) were cultured on collagen I-coated substrates with different elasticity(elastic modulus:Esoft ～ 0.1-1 kPa and Estiff～50-100 kPa).An antibody specifically recognizing the active conformation of β1 integrin was used to determine the levels of activated integrin two hours after cell seeding.Biotin labeling followed by capture-ELISA and western blotting was carried out to detach the integrin localization in the cells.Antibody labeling and flow cytometry assay were adapted to reinforce the integrin localization.Results:Two hours after cell seeding,activated integrin levels on soft substrate were observed to be significantly higher than which on stiff substrate,while total integrin levels were not affected by ECM elasticity.Similar results were obtained in BMMSC growing for 24 hr on substrates of different elasticity.This shocking result is conflict with the result of large focal adhesion size on stiffer substrate reported by other groups,which lead us to further investigate the integrin levels on cell membrane.Flow cytometric analysis,immunocytochemical staining and biotin labeling followed by capture-ELISA revealed that the cortical integrins,including active and inactive,remarkably decreased in BMMSC on soft substrate than that on stiff substrate,while total integrin was not changed.This result suggested a redistribution of integrin regulated by soft substrate.We then performed assays on integrin endocytosis and recycling.Endocytosis of activated β1 integrin was analyzed by antibody internalization assay and confocal microscopy.After 30 min of incubation,activated β1 integrin antibody were observed in characteristic vesicular structures in the cytoplasm.In contrast,no intracellular antibodies were detected in the control cells incubated at 4 ℃ to prevent endocytosis(data not shown).Soft substrate substantially increased the number of cytosolic vesicles containing internalized activated β1 integrin.Because antibody binding may cluster integrins on the cell surface and thus artificially promote their endocytosis,surface biotinylation assay was used to reinforce the steady-state internalization of β1 integrin.As a result,the measured internalization rates of both active and total integrin were higher on soft substrate than which on stiff substrate.BMMSC were then double-labeled using antibodies recognizing the integrin and clathrin or integrin and caveolin-1 to identify the internalization vehicle proteins.Integrin co-localized with caveolin-1;however,no detectable co-localization between clathrin and integrin was seen.By biotinylation assay of integrin internalization in the present of clathrin-mediated endocytosis inhibitor(monodansylcadaverine,MDC) or caveolae/raft inhibitor(methyl-b-cyclodextrin,MBCD),we found that inhibition of clathrin-mediated endocytosis did not affect the internalization of integrin on soft gel,while caveolae/raft inhibitor significantly repressed the internalization of integrin on soft gel.Conclusion:Taken together,we found that the activation and trafficking of integrin were vitally regulated by ECM elasticity.Since the function of integrin depends on its membrane location,the trafficking of integrin is also critical for cell responses to ECM,which may contribute the ECM elasticity-regulated cell fate specification.更多还原

【Abstract】 Background:Mechanical environments have been increasingly recognized to play an important role in regulating cellular function and behavior,including proliferation,migration,apoptosis,and differentiation.More recently,emerging evidences demonstrate that the mechanical properties(e.g.,elasticity) of adhesion substrates manipulate stem cell fate in both two-demensional(2D)[1] and three-demensional(3D)[2] cultures.However,although the regulation of cell fate by extra-cellular matrix(ECM) stiffness has been reported to depend on cell traction,the connection between the mechanical properties of ECM and chemical signaling processes is not known.Evidences have been demonstrated that focal adhesion proteins activation change is closely associated with mechanical stimuli results in focal adhesion proteins activchanges,which facilitates remodeling of focal adhesion.Among these proteins,integrins are necessary elements for most mechano-sensing process and lie at the beginning of the sensing pathway.In the present study,we aim to explore the mechanism of stem cells sensing ECM elasticity,in which intergrin is vitally involved.Methods:Bone marrow mesenchymal stem cells(BMMSC) were cultured on collagen I-coated substrates with different elasticity(elastic modulus:Esoft ～ 0.1-1 kPa and Estiff～50-100 kPa).An antibody specifically recognizing the active conformation of β1 integrin was used to determine the levels of activated integrin two hours after cell seeding.Biotin labeling followed by capture-ELISA and western blotting was carried out to detach the integrin localization in the cells.Antibody labeling and flow cytometry assay were adapted to reinforce the integrin localization.Results:Two hours after cell seeding,activated integrin levels on soft substrate were observed to be significantly higher than which on stiff substrate,while total integrin levels were not affected by ECM elasticity.Similar results were obtained in BMMSC growing for 24 hr on substrates of different elasticity.This shocking result is conflict with the result of large focal adhesion size on stiffer substrate reported by other groups,which lead us to further investigate the integrin levels on cell membrane.Flow cytometric analysis,immunocytochemical staining and biotin labeling followed by capture-ELISA revealed that the cortical integrins,including active and inactive,remarkably decreased in BMMSC on soft substrate than that on stiff substrate,while total integrin was not changed.This result suggested a redistribution of integrin regulated by soft substrate.We then performed assays on integrin endocytosis and recycling.Endocytosis of activated β1 integrin was analyzed by antibody internalization assay and confocal microscopy.After 30 min of incubation,activated β1 integrin antibody were observed in characteristic vesicular structures in the cytoplasm.In contrast,no intracellular antibodies were detected in the control cells incubated at 4 ℃ to prevent endocytosis(data not shown).Soft substrate substantially increased the number of cytosolic vesicles containing internalized activated β1 integrin.Because antibody binding may cluster integrins on the cell surface and thus artificially promote their endocytosis,surface biotinylation assay was used to reinforce the steady-state internalization of β1 integrin.As a result,the measured internalization rates of both active and total integrin were higher on soft substrate than which on stiff substrate.BMMSC were then double-labeled using antibodies recognizing the integrin and clathrin or integrin and caveolin-1 to identify the internalization vehicle proteins.Integrin co-localized with caveolin-1;however,no detectable co-localization between clathrin and integrin was seen.By biotinylation assay of integrin internalization in the present of clathrin-mediated endocytosis inhibitor(monodansylcadaverine,MDC) or caveolae/raft inhibitor(methyl-b-cyclodextrin,MBCD),we found that inhibition of clathrin-mediated endocytosis did not affect the internalization of integrin on soft gel,while caveolae/raft inhibitor significantly repressed the internalization of integrin on soft gel.Conclusion:Taken together,we found that the activation and trafficking of integrin were vitally regulated by ECM elasticity.Since the function of integrin depends on its membrane location,the trafficking of integrin is also critical for cell responses to ECM,which may contribute the ECM elasticity-regulated cell fate specification.更多还原