GTPase activating protein DLC1 spatio-temporally regulates Rho signaling

Joint Public Review:

Summary:

The manuscript of Heydasch et al. addresses the spatiotemporal regulation of Rho GTPase signaling in living cells and its coupling to the mechanical state of the cell. They focus on a GAP of RhoA, the Rho-specific GAP Deleted in Liver Cancer 1 (DLC1). They first show that removing DLC1 either by a CRISPR KO or by downregulation using siRNA leads to increased contractility and globally elevated RhoA activity, as revealed by a FRET biosensor. This result was expected, since DLC1 is deactivating RhoA its absence should lead to increasing amounts of active RhoA. To go beyond global and steady levels of RhoA activity, the authors developed an acute optogenetic system to study transient RhoA activity dynamics in different genetic and subcellular contexts. In WT cells, they found that pulses of activation lead to an increased RhoA activity at focal adhesions (FA) compared to plasma membrane (PM), which suggests that FAs contain less RhoA GAPs, more RhoA, or that FAs involve positive feedback implying other GEFs for example. In DLC1 KO cells, they found that the RhoA response upon pulses of optogenetic activation was increased (higher peak) both at FA and PM, which could be expected since less GAP should increase the amount of active RhoA. But surprisingly, they observed a higher rate of RhoA deactivation in DLC1 KO cells, which is counterintuitive: less GAP should result in a slower rate of deactivation. Less GAP should also lead to a lower rate of observed RhoA activation (smaller koff) and delayed peak. From the data, it seems hard to conclude on these two expectations since the initial rates (slopes right after the activation) and times at peak appear similar in both WT and DLC1 KO cells. Further on, the authors study the dynamics of DLC1 on FAs depending on the mechanical state and nicely show a causal decrease of DLC1 enrichment at FA upon FA reinforcement, hereby probing a positive feedback where RhoA activation is further amplified as the force exerted at FA is increasing.

Strengths:

- Experiments are precise and well done.
- Technically, the work brings original and interesting data. The use of transient optogenetic activation within focal adhesions together with a biosensor of activity is new and elegant.
- The link between DLC1 and global contractility/RhoA activity is clear and convincing.
- The surprisingly higher rate of RhoA deactivation in DLC1 KO cells is convincing, as well as the differences in the dynamics of RhoA between focal adhesions and plasma membrane.
- The correlation between DLC1 enrichment and focal adhesion dynamics is very clear.

Weaknesses:

- There is no explanation for the higher rate of RhoA deactivation in DLC1 KO cells.
- For the optogenetic experiments, it is not clear if we are looking at the actual RhoA dynamics of the activity or at the dynamics of the optogenetic tool itself.
- There is no model to analyze transient RhoA responses, however, the quantitative nature of the data calls for it. Even a simple model with linear activation-deactivation kinetics fitted on the data would be of benefit for the conclusions on the observed rates and absolute amounts.