We had designed three specific aims that lead step by step to our goal, control of collective cell dynamics using optogenetics:
Aim 1 – Develop spatial and temporal control over single-cell mechanics using optogenetics
The three GTPases RhoA, Rac1, and CDC42 are involved in cell motility, both in-vitro and in-vivo. Subcellular concentration gradients of these molecules ultimately guide cells. So far, the three GTPases had been linked to different qualities of cell motion –cell contraction, lamellipodium formation, filopodia formation. In this aim, we wanted to find out which Rho-GTPases had the highest potential usefulness for the collective cell guidance which we explore in the later aims 2 and 3.
We thus used a set of three optogenetic epithelial MDCK cell lines in which Rac1, RhoA and CDC42 (respectively) are photoactivatable. In single cells, Rac1 activation made cells form a lamellipodium, and CDC42 activation was even more reactive than Rac1. This let us to the decision that we wanted to use Rac1 and CDC42 activation for control of collective dynamics in aims 2 and 3.
Aim 2 – Control the flocking dynamics of migrating clusters
The results from this work package then confirmed that CDC42 activation can cause outgrowth of fingers from islands, something which had not yet been achieved, to our knowledge. This could lead to an in-vitro directed migration of a cell cluster, which might later be used as a basis to navigate cell systems through more complex “landscapes”, as they are encountered in many biological systems.
Aim 3 – Investigate the dynamics of jamming and fluidization in confluent cell monolayers.
Cell jamming and unjamming have been observed in a variety of confluent tissues. In general, there is a lack of experimental data on traction dynamics or optogenetic control of jamming. We thought that the biggest scientific novelty was to be found here, which is why we focused on this.
We thus photoactivated Rac1 and CDC42 first in circular regions in confluent cell monolayers. We found that CDC42 often produced drastic responses, such as collective cell streams, unlike Rac1, which often showed no appreciable response. We then proceed to test distinct patterns of CDC42 activation and different parameters to optimize the process and to determine to what extent the monolayer can be fluidized (unjammed) or jammed. For instance, we studied how monolayer dynamics varies with the size of the photoactivated regions.
Taken together, we found that this new tool allows to fluidize jammed epithelial monolayers, enabling us to measure cell flows and traction force dynamics as a function of the illumination patterns. These results will allow us to define and control trajectories between states in a jamming diagram.
