Light-Modulated Cell Adhesion to Control Cell and Tissue Morphogenesis

Abstract

Cell adhesion through integrin receptors to the extracellular matrix is essential for pattern formation during tissue morphogenesis. For example, cell adhesion differences correlate with epithelial branching, and stem cells often reside in microenvironments essential for stem cell maintenance and differentiation. Tissue engineering relies on the ability to replicate in vivo pattern formation to grow complex multicellular tissues in vitro, a largely unresolved challenge. Although cell growth can be patterned by immobilizing matrix proteins by photolithography or microcontact printing, such techniques are not easily adaptable to a physiological 3D environment and most importantly do not allow dynamic control of cell adhesion during in vitro organogenesis. To address this challenge, we started to develop a system by which cell-matrix interactions can be reversibly modulated by patterned 470 nm illumination utilizing a novel short peptide, Zdk, that binds to the dark-state of oat LOV2 with high affinity. Aminosilane-coated coverslips were reacted with NHS-PEG-biotin conjugates, and a biotinylated LOV2 domain attached through neutravidin. As proof-of-principle, we bound mCherry-Zdk-RGD fusion proteins to biotin-LOV2-coated coverslips, and demonstrate rapid, reversible and patterned light-induced release of mCherry-Zdk-RGD from the surface. We are investigating how light-triggered release of cell matrix attachment controls focal adhesion dynamics and cell migration. Rapid rebinding of mCherry-Zdk-RGD is due to the photochemical properties of LOV2, which rapidly reverts to the dark state after photoactivation. Because this may be too fast to manipulate cell-matrix adhesions that turn over at a substantially slower timescale, we carried out a mutagenesis screen of LOV2 variants generated by error-prone PCR. Using fluorescence recovery of the LOV2-bound flavin molecule as readout, we identified numerous novel mutations near the flavin-binding site that revert to the dark state at much slower rates, presumably by stabilizing the flavin-cysteinyl adduct.