Investigation of signal networks that control dynamic cell shape changes

Abstract

Cells process information via complex signal networks that include multiple components. Knowledge, about the spatio-temporal organization of these components and their activity state is critical to understand how these signal networks process information. Signal networks involving Rho GTPases play a key role in the spatio-temporal coordination of cytoskeletal dynamics during cell migration. Previous studies that directly investigated the crosstalk between the major Rho GTPases Rho, Rac and Cdc42 revealed a strong increase in Rho activity after Rac1 activation. This Rac1-Rho crosstalk might play a role in mediating the tight spatio-temporal coupling between cell protrusions and retractions that are typically observed during mesenchymal cell migration. To address this question, improved sensors were developed in this thesis to measure the activity state of endogenous small GTPases of the Ras, Rap, Rac, and Rho families in living cells. Using these sensors, Rac activation was observed to be tightly and precisely coupled to local cell protrusions, followed by Rho activation during retraction. In a screen for potential crosstalk mediators, a subset of the Rho activating Lbc-type GEFs, Arhgef11 and Arhgef12, were found to be enriched at the cell periphery during protrusions-retractions cycles. Furthermore, via an optogenetic approach, these Lbc GEFs were observed to recruit to the plasma membrane by active Rac1, suggesting that they indeed might link the cell protrusion signal Rac and the cell retraction signal Rho. Furthermore, depletion of these GEFs via RNA interference impaired cell protrusion-retraction dynamics, which was accompanied with a decrease in migration distance and an increase in migration directionality. These results show that Arhgef11 and Arhgef12 facilitate effective exploratory cell migration by coordinating the cell morphogenic processes of cell protrusion and retraction by coupling the activity of the associated small GTPases Rac and Rho. Typical activity sensor designs including the improved small GTPase activity sensors described above are limited in the number of readouts that can be combined simultaneously inside a single cell. To extend this number, a new programmable single stranded mRNA-based sensor design was developed. These sensors enable parallel measurements of multiple protein-protein interactions that are dependent on signal network activity states inside a single living cell. As a proof-of-concept, protein kinase A activity sensors were developed, which allowed to distinguish activity dynamics of their different regulatory subunits in parallel inside living cells. Furthermore, RNA scaffold- based activity sensors for small GTPases were developed which enabled monitoring of the activity kinetics of Ras and Rap during pharmacological perturbations. RNA scaffolds, which were functionalized with dominant positive Rac or Rho GTPases enabled a method to evaluate the specificity of effector molecules in parallel inside living cells. These results show that the application of programmable RNA scaffolds can provide critical information about signal network components inside individual, living cells, which cannot be obtained via available standard methods. Such information can be critical to decipher the spatio- temporal organization of complex signal networks inside cells.