Orthogonal Blue and Red Light Controlled Cell–Cell Adhesions Enable Sorting-out in Multicellular Structures

Abstract

The self-assembly of different cell types into multicellular structures and their organization into spatiotemporally controlled patterns are both challenging and extremely powerful to understand how cells function within tissues and for bottom-up tissue engineering. Here, we not only independently control the self-assembly of two cell types into multicellular architectures with blue and red light, but also achieve their self-sorting into distinct assemblies. This required developing two cell types that form selective and homophilic cell–cell interactions either under blue or red light using photoswitchable proteins as artificial adhesion molecules. The interactions were individually triggerable with different colors of light, reversible in the dark, and provide noninvasive and temporal control over the cell–cell adhesions. In mixtures of the two cells, each cell type self-assembled independently upon orthogonal photoactivation, and cells sorted out into separate assemblies based on specific self-recognition. These self-sorted multicellular architectures provide us with a powerful tool for producing tissue-like structures from multiple cell types and investigate principles that govern them.

Spatiotemporal Regulation of Cell–Cell Adhesions

“…For example, when cells expressing Vivid or Cph1 at their cell surface were mixed and illuminated with either blue or red-light clusters of cells with Vivid or Cph1 cells formed, respectively. When both blue and red light was used self-isolated clusters containing either Vivid or Cph1 cells were observed (Figure 4) [57]. That means that the adhesive force for Vivid and Cph1 is lower than that for the homodimers formed for each system due to the specific protein-protein interactions.…”

“…In these optogenetic approaches, complementary light-dependent binding partners are expressed in the surfaces of different cell types by transfecting these proteins along with a plasma membrane localization sequence and a membrane anchoring sequence. Following translation, the localization sequence ensures that the protein is exported to the cell membrane where the extracellular portion operates as a bioartificial cell adhesion receptor [51,52,57]. For instance, the proteins Cryptochrome 2 (CRY2) from Arabidopsis thaliana and its blue light-dependent binding partner cryptochrome-interacting basic helix-loop-helix (CIBN) protein, were expressed on the surfaces of MDA-MB-231 cells, which do no form native cellcell adhesion.…”

“…When colloidial particles are labled with the iLID/Nano, nMag/pMag, or nMagHigh/pMagHigh clusters of particles can be seen to form with respect to the kinetics of the system (adapted from Müller et al [62]). In cellular systems utilizing the vivid (VVD) and Cph1 systems descrete clusters are observed rather than any intermixing (adapted from Rasoulinejad et al [57]).…”

Red Light Optogenetics in Neuroscience

“…They can be based on homodimerization (i.e., actuator-actuator interaction) or heterodimerization (i.e., actuator-binding partner interaction). Red light-controlled homodimerization has been mainly based on a cyanobacterial phytochrome Cph1 ( Schmidl et al, 2019 ; Sentürk et al, 2019 ; Rasoulinejad et al, 2020 ). Homodimerization systems only need one vector to be transported to the target, which decreases the complexity of the study.…”

“…As another example, the idea of a light-responsive cell sorting system could be used to manipulate neuronal development. In an in vitro application by Rasoulinejad et al (2020) , cells expressed either homodimerizing blue light-activated VVD or homodimerizing red light-activated phytochrome Cph1. Light-activated dimerization of both systems gathered the cells into two separate multicellular tissue-like structures ( Rasoulinejad et al, 2020 ).…”