Objective The binding of cell adhesive peptides(such as RGD)to integrins initiates the recruitment of cytoplasmic adaptor proteins(e.g.,vinculin)and the formation of focal adhesion(FA)complexes required for cell adhes...Objective The binding of cell adhesive peptides(such as RGD)to integrins initiates the recruitment of cytoplasmic adaptor proteins(e.g.,vinculin)and the formation of focal adhesion(FA)complexes required for cell adhesion.The ability to manipulate this ligand-mediated cell adhesion process is crucial for regulating cell migration,cell differentiation,injury healing,and immune response.Some recent studies reported the importance of the tether length/mobility of the cell adhesive ligands in regulating the traction force development of cells.In the native cellular microenvironment,such a dynamic change in the nanoscale tether length of bioactive ligands is often mediated by conformational changes of the structural proteins due to protein folding or degradation.However,no prior studies have demonstrated the modulation of the ligand tether mobility by controlling the intramolecular folding of polymeric linkers.Unfoldable synthetic macromolecules with easy synthetic routes and controllable structures,such as supramolecular host-vip single chain nanogels(SCNGs),are ideal candidates for mimicking the changes in the tether mobility of bioactive ligands via biorthogonal triggers.Methods S,S’-bis(a’a’-dimethyl-a’’-propargyl acetate)trithiocarbonate was first used to mediate the RAFT polymerization of N,N-dimethyl acrylamide,vinyl-adamantane and vinyl-β-cyclodextrin to yield the ADA@CD-SCNGs.The preparation of the unfoldable host-vip SCNGs was evidenced by the by gel permeation chromatography,proton nuclear magnetic resonance spectroscopy,atomic force microscopy and dynamic light scattering.Then the RGD peptide was conjugated to the alkynyl group on one end of the SCNGs before immobilizing the material on the substrate,which was confirmed by scanning electron microscopy(SEM).The regulation of cell behaviours by unfolding of the SCNG-RGD was confirmed by immunofluorescence staining of vinculin and Yes-associated protein(YAP).Results The preparation of ADA@CD-SCNGs was confirmed by GPC which showed a unimodal molecular weight distribution.DLS and AFM data also proved that the SCNGs had an average diameter of 12±3nm.SEM images showed that SCNGs were conjugated as a linker of RGD peptide to thiolated glass substrate at an average density of 162±11 particles/μm2.These particles disappeared after adding free competitive ADA vip molecules,indicating the triggered unfolding of the tether SCNGs.In addition,the unfolding of supramolecular ADA@CD-SCNGs was also evidenced by a decrease in the GPC elution time and a slight increase in the apparent molecular weight.These results show that the immobilized ADA@CD-SCNGs can be unfolded to tune the tether length and mobility of the conjugated RGD ligands.Then we investigated the regulation of the cell behaviors on the substrate by triggering the unfolding of SCNG linkers.A critical level of traction force is required to effectively initiate and maintain integrin-mediated formation of FA complexes and subsequent mechano-transduction signaling.An increased tether length in cell-adhesive ligands can lead to a diminished cell traction force as if cells are adhering to soft substrates.Here,the unfolding of the ADA@CD-SCNG-RGD triggered by the addition of free ADA led to disassembly of the mature focal adhesions in the cells as evidenced by the reduced vinculin and F-actin in staining.Subsequently,nuclear YAP also decreased significantly because of the impaired mechano-sensing and diminished cell cytoskeleton tension.In addition,the extensively spread cells gradually became round after the medium was supplemented with free competitive ADA to unfold the SCNG linker.These finding demonstrates that the substrates with the unfolded ADA@CD-SCNG-RGD only supported weak cell adhesions.In contrast,on the substrate conjugated with the nonunfoldable MBA-SCNG-RGD linker,the addition of free ADA resulted in no change in the spread cell morphology and protein expressions.These results indicate that the unfoldable host-vip ADA@CD-SCNG can be used to manipulate the nanoscale presentation of ligands to regulate cell behaviors.Conclusions We demonstrate the application of SCNGs as the supramolecular linker to tune the nanoscale ligand tether length.These findings demonstrate that the strategy of manipulating the tether mobility of bioactive ligands by using supramolecular SCNGs as linkers provides a highly tunable,biomimetic,and bio-orthogonal approach to study the dynamic events of cell adhesion.展开更多
文摘Objective The binding of cell adhesive peptides(such as RGD)to integrins initiates the recruitment of cytoplasmic adaptor proteins(e.g.,vinculin)and the formation of focal adhesion(FA)complexes required for cell adhesion.The ability to manipulate this ligand-mediated cell adhesion process is crucial for regulating cell migration,cell differentiation,injury healing,and immune response.Some recent studies reported the importance of the tether length/mobility of the cell adhesive ligands in regulating the traction force development of cells.In the native cellular microenvironment,such a dynamic change in the nanoscale tether length of bioactive ligands is often mediated by conformational changes of the structural proteins due to protein folding or degradation.However,no prior studies have demonstrated the modulation of the ligand tether mobility by controlling the intramolecular folding of polymeric linkers.Unfoldable synthetic macromolecules with easy synthetic routes and controllable structures,such as supramolecular host-vip single chain nanogels(SCNGs),are ideal candidates for mimicking the changes in the tether mobility of bioactive ligands via biorthogonal triggers.Methods S,S’-bis(a’a’-dimethyl-a’’-propargyl acetate)trithiocarbonate was first used to mediate the RAFT polymerization of N,N-dimethyl acrylamide,vinyl-adamantane and vinyl-β-cyclodextrin to yield the ADA@CD-SCNGs.The preparation of the unfoldable host-vip SCNGs was evidenced by the by gel permeation chromatography,proton nuclear magnetic resonance spectroscopy,atomic force microscopy and dynamic light scattering.Then the RGD peptide was conjugated to the alkynyl group on one end of the SCNGs before immobilizing the material on the substrate,which was confirmed by scanning electron microscopy(SEM).The regulation of cell behaviours by unfolding of the SCNG-RGD was confirmed by immunofluorescence staining of vinculin and Yes-associated protein(YAP).Results The preparation of ADA@CD-SCNGs was confirmed by GPC which showed a unimodal molecular weight distribution.DLS and AFM data also proved that the SCNGs had an average diameter of 12±3nm.SEM images showed that SCNGs were conjugated as a linker of RGD peptide to thiolated glass substrate at an average density of 162±11 particles/μm2.These particles disappeared after adding free competitive ADA vip molecules,indicating the triggered unfolding of the tether SCNGs.In addition,the unfolding of supramolecular ADA@CD-SCNGs was also evidenced by a decrease in the GPC elution time and a slight increase in the apparent molecular weight.These results show that the immobilized ADA@CD-SCNGs can be unfolded to tune the tether length and mobility of the conjugated RGD ligands.Then we investigated the regulation of the cell behaviors on the substrate by triggering the unfolding of SCNG linkers.A critical level of traction force is required to effectively initiate and maintain integrin-mediated formation of FA complexes and subsequent mechano-transduction signaling.An increased tether length in cell-adhesive ligands can lead to a diminished cell traction force as if cells are adhering to soft substrates.Here,the unfolding of the ADA@CD-SCNG-RGD triggered by the addition of free ADA led to disassembly of the mature focal adhesions in the cells as evidenced by the reduced vinculin and F-actin in staining.Subsequently,nuclear YAP also decreased significantly because of the impaired mechano-sensing and diminished cell cytoskeleton tension.In addition,the extensively spread cells gradually became round after the medium was supplemented with free competitive ADA to unfold the SCNG linker.These finding demonstrates that the substrates with the unfolded ADA@CD-SCNG-RGD only supported weak cell adhesions.In contrast,on the substrate conjugated with the nonunfoldable MBA-SCNG-RGD linker,the addition of free ADA resulted in no change in the spread cell morphology and protein expressions.These results indicate that the unfoldable host-vip ADA@CD-SCNG can be used to manipulate the nanoscale presentation of ligands to regulate cell behaviors.Conclusions We demonstrate the application of SCNGs as the supramolecular linker to tune the nanoscale ligand tether length.These findings demonstrate that the strategy of manipulating the tether mobility of bioactive ligands by using supramolecular SCNGs as linkers provides a highly tunable,biomimetic,and bio-orthogonal approach to study the dynamic events of cell adhesion.
