Heterotrimeric G Protein–RasGAP Coupling Drives Adaptation During Chemotaxis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors This manuscript studies the role of RasGAP C2GAP1 in the spatiotemporal dynamics of the actin-independent gradient-sensing module during Dictyostelium chemotaxis. By combining quantitative live-cell imaging, biochemical assays, gradient application experiments, FRET-based G-protein activation measurements, and AlphaFold3 structural modeling, the authors demonstrate that C2GAP1 binds activated Gα2, remains membrane-associated in an actin-independent manner, and locally attenuates both Ras activity and heterotrimeric G-protein signaling, thereby generating front-specific inhibition. The study presents an interesting dataset that supports a self-recruiting Gα2-C2GAP1 negative-feedback circuit for gradient sensing. The evidence for Gα2-C2GAP1 interaction and its biphasic dynamics defect in c2gapA- cells is compelling. However, the authors’ central claim that Gα2-C2GAP1 coupling constitutes adaptive module across the wide dynamic range requires more cautious discussion and additional validation. 
Major comments 1. As shown in Figures 1 and 2, c2gapA knockout cells are still able to establish clear intracellular gradients of PIP3 and PTEN along cAMP concentration gradients. This polarity forms at both low (100 nM) and high (10 µM) cAMP concentrations. These data indicate that Dictyostelium cells do not require C2GAP1 to perform gradient sensing or to generate front-back polarity across a wide concentration range. If C2GAP1 is truly essential for adaptation, the front-specific PIP3 polarization and rear PTEN recruitment should be lost or severely impaired at saturating concentrations, yet this separation persists in the mutants.      The finding that the initial transient PIP3 production and transient PTEN membrane dissociation immediately after cAMP stimulation are C2GAP1-dependent is indeed interesting. However, these early transient responses are unlikely to represent adaptation. They are more likely the “excitation” that occurs when an excitable system exceeds its threshold (Devreotes et al., 2017, PMID: 28793794 and related works). To date, no RasGAP has been shown to directly control this excitation step. The authors should clearly distinguish between the excitation and adaptation, and reconsider whether C2GAP1 primarily regulates adaptation or also modulates the initial excitation response.
2. The finding in Figure 6 that C2GAP1 negatively regulates heterotrimeric G-protein activation as demonstrated by FRET-based measurements is highly interesting. However, closer inspection of Figure 6D reveals that the effect of c2gapA deletion is clearly cAMP concentration-dependent: the enhancement of G-protein activation (greater FRET change) is pronounced at low cAMP concentrations, whereas the difference from wild-type cells becomes minimal or negligible at high (saturating) concentrations. The authors should present a full dose-response curve of FRET change across a broad range of cAMP concentrations. Such analysis would be expected to show a leftward shift of the curve in c2gapA- cells. If it is confirmed, this would indicate that C2GAP1 functions primarily to suppress G-protein activation specifically at the low cAMP concentration range, thereby involving in gradient sensing at lower chemoattractant levels. 
3. The data in Figures 4 and 8 suggest an intriguing possibility. In the high cAMP concentration range, C2GAP1 may retain “memory” of the previously sensed gradient and thereby increase cellular sensitivity to a new gradient applied in a different direction. To directly test this idea, the authors could additionally modify the Figure 4 protocol by removing the first gradient and then applying a uniform cAMP stimulus. This would reveal whether PIP3 preferentially accumulates at the original rear, indicating retained directional memory, or shows a random distribution.          Additionally, the c2gapA- response at the low cAMP range (100 nM) is noteworthy. These cells show almost no PIP3 response to the second identical stimulation. This suggests that C2GAP1 is required for proper PIP3 activation under low cAMP conditions. A plausible explanation is that in the absence of C2GAP1, G-protein activation is not suppressed properly after the first stimulus, resulting in depletion or desensitization of the downstream machinery. The authors should discuss this concentration-specific difference and consider additional experiments (for example, monitoring G-protein activation or Ras activity during the second low-dose stimulations).   Minor comments 4. line 20: front-specific specific inhibition; line 21: gra-dients. typo? 5. The sentence on line 403 seems to be incorrect.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Xu et. al. deals with the interesting phenomena of how chemoattractant gradient sensing is at the foundation of chemotaxis, the directional migration of cells in response to a chemoattractant, followed by cell polarity and ultimately migration. The authors mechanistically investigated the RasGAP, C2GAP1, as the integrating signaling molecule.  There are many ways the authors could have investigated these phenomena; they chose to focus on a cytoskeleton-free system that abolishes cell polarity and migration. There were many well-described experiments, but the data did not always match the interpretation.  This reviewer found this problematic for the following:

1. The author's title references chemotaxis, but there is no migration data presented in the manuscript, even as a 'proof of principle' that the signals are actually causing migration. Figure 8 discusses the migration observed, but the data are not actually shown or quantified.
2. It is hard to interpret all of the data presented in Figures 1-5 without a statistical analysis of the variance between WT/C2GAP.
3. Figures 1 and 2 ( including the videos) are hard to visualize and conceptualize;  maybe an arrow or a star indicating where the gradient sensing is occurring would help, as well as higher magnification. 
4. There are too many typos and missing characters, letters, and numbers to understand how the experiments were conducted. Example: Line 148 – 5.0  M of LatB? Is that accurate? Or four lines later, Line 152 – 5mM Latrunculin B is that accurate?

 

Author Response

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Reviewer 3 Report

Comments and Suggestions for Authors

The research article entitled “Heterotrimeric G Protein-RasGAP Coupling Drives Adaptation during Chemotaxis” by Xu et al., is a comprehensive study defining the role of RasGAP protein C2GAP1 in chemotaxis using a model organism Dictyostelium discoideum. The study shows the essential role of C2GAP1 in chemotactic gradient sensing, orientation towards chemotactic agent and adaptation in response to varied concentration of chemotactic agent.  I have raised following comments/concerns that may help to strengthen the manuscript.

1. Actin dynamics plays a key role in various cellular processes. Chemoattract gradient sensing, cell polarization toward the chemoattractant, and cell migration, all putatively interconnected processes one affecting/regulating one another. Therefore, are there any differences in PIP3 and PTEN dynamics at the plasma membrane in response to different concentrations of cAMP with intact actin cytoskeleton vs with depolymerized actin cytoskeleton by Latrunculin treatment? Similarly, is there any difference in plasma membrane targeting of C2GAP1 in the presence of intact actin cytoskeleton vs depolymerized actin?
2. Does chemoattract gradient sensing also coupled with spatial-temporal dynamics of cAMP receptor cAR1? The study of spatial-temporal dynamics of cAR1 may also be important for this study?
3. C2GAP1 possesses C2 domain, a well-known membrane targeting domain. Author may describe its role in this study.
4. References are missing in some parts of the manuscript. Please add appropriate references.
5. In Figure 3A, C2GAP1 labeling is missing.

Comments on the Quality of English Language

I am not qualified to judge english language.

Author Response

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Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have adequately addressed my previous concerns and their responses are convincing. I recommend its acceptance for publication.

Author Response

We thank Reviewer #1 for the positive response to our revised manuscript. 

Reviewer 2 Report

Comments and Suggestions for Authors

Figures look much better. My suggestion was not to remove the detailed methods, my suggestion was to reconcile the differences in the text to reflect the actual concentrations. Detailed methods provide an easier way for your data to be repeated. Please return, and simply provide the actual concentrations ( or range of concentrations).

Author Response

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Author Response File: Author Response.pdf