GTPase activating protein DLC1 spatio-temporally regulates Rho signaling
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This study presents a valuable finding on how the GAP DLC1, a deactivator of the small GTPase RhoA, regulates RhoA activity globally as well as at Focal Adhesions. Using a new acute optogenetic system coupled to a RhoA activity biosensor, the authors present solid evidence that DLC1 amplifies local Rho activity at Focal Adhesions. Nevertheless, the proposed mechanism could be further supported by a deeper analysis of the data.
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Abstract
Tightly regulated spatio-temporal Rho GTPase activity patterns regulate morphogenetic processes such as cell migration. Emerging evidence suggests that binding of Guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) to the cytoskeleton or adhesions mediate feedback regulation to spatio-temporal Rho GTPase activation. To explore such feedback regulation, we study the Rho specific GAP Deleted in Liver Cancer 1 (DLC1) which binds to focal adhesions (FAs) through mechanosensitive interactions. Using a FRET biosensor, we show that DLC1 loss of function leads to global increase in Rho activity and contractility throughout the cell without affecting a striking lamellar RhoA activity band in fibroblasts. To interrogate the Rho GTPase signaling flux, we build a genetic circuit consisting of an optogenetic actuator to control Rho activity, and a Rho activity biosensor. In spreading cells at steady state, optogenetic manipulation of Rho activity reveals that DLC1 controls the rate of Rho activation rather than duration, both at FAs and at the plasma membrane (PM). Local and reversible optogenetic control of contractility shows that DLC1 associates/dissociates with FAs during their reinforcement/relaxation. This might provide positive feedback that locally increases the rate of Rho activation at FAs that experience local tension to facilitate FA disassembly. Our results indicate that DLC1 operates both at the PM and at FAs to regulate global Rho activity levels at steady state, or to amplify local Rho activity at FAs experiencing a strong mechanical input, presumably to induce robust FA disassembly. This provides new insights in the complexity of spatio-temporal Rho GTPase signaling.
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eLife assessment
This study presents a valuable finding on how the GAP DLC1, a deactivator of the small GTPase RhoA, regulates RhoA activity globally as well as at Focal Adhesions. Using a new acute optogenetic system coupled to a RhoA activity biosensor, the authors present solid evidence that DLC1 amplifies local Rho activity at Focal Adhesions. Nevertheless, the proposed mechanism could be further supported by a deeper analysis of the data.
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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 …
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. -
