Controlling Cell Adhesion And Migration By Light-responsive Proteins

Remote,spatiotemporally resolved,and reversible control of cell adhesion and migration is highly desirable for many biotechnological applications and cell-based therapy.Here,We demonstrated that light-regulated cell adhesion and migration could be achieved on the surface of LOV2,a light-responsive protein modified with Arg-Gly-Asp(RGD)ligand.Blue light illumination causes the structural change of LOV2 and expose the RGD sequence,leading to enhanced protein adhesion and migration.Placing the surfaces back to dark conditions results in re-caging the ligands and detachment of the cells.The adhesion and migration can be reversibly regulated for multiple cycles.By precisely controlling the position of light,directional migration of cells can be achieved.The reversible and spatiotemporal control of cell adhesion and migration used photo-switchable protein structure-caged ligands hold great promise for precisely defining and regulating cell-materials interactions in vitro and in vivo.The first chapter introduces the background knowledge,materials and experimental methods of the article.Firstly,we briefly introduced the light-responsive protein and its application,and also introduced the LOV2 protein used in this project.We mentioned its source and properties under light regulation.Then we introduced the basic situation of cell adhesion,its biological significance and molecular basis.We focused on the role of integrin and RGD sequence in cell adhesion.In the second chapter,the design and experimental methods of LOV2 mutant protein are introduced in detail,and its properties are preliminarily tested.We selected two RGD insertion sites on the Ja-helix of LOV2 protein,which had the least effect on the structure of LOV2 protein.In addition,RGD sequence was linked to the C-terminal of LOV2 protein as a positive control and RGE as a negative control.The six mutant proteins have the same light regulation properties as the original LOV2 proteins.Their color changes under light and dark conditions can be directly observed by naked eyes.Their UV,fluorescence and CD spectra are also measured.In the third chapter,we designed an ELISA-based binding ELISA and competitive ELISA to verify the photocontrolled unfolding function of LOV2 protein,and whether the unfolded RGD sequence can be combined with integrin protein to achieve the desired protein function.The results showed that the binding of mutant proteins to integrins was in line with our expectations.The binding of mutant proteins to integrins was good in light and weak in dark.In Chapter 4,we introduce that light-regulated cell adhesion and migration can be achieved by culturing cells on the surface of medium modified with LOV2 mutant protein.The RGD sequence of LOV2 mutant protein can be exposed by blue light irradiation,which can bind integrins to the Ja-helix of LOV2 mutant protein,so that cells can adhere to it.In dark state,the structure of mutant protein changes,the unfolded part folds back into Ja-helix,RGD sequence is hidden,and cells are separated from the mutant protein.Using this property,we use a moving optical fiber to irradiate the surface to regulate the movement of cells,which move toward the site of light irradiation.We also observed the morphological changes of cells in the process of adhesion and their recoverability in isolation.Finally,we summarize the above work and put forward the prospect of further research.