Avalilação PREreview de A TRPV4-dependent calcium signaling axis governs lamellipodial actin architecture to promote cell migration

SUMMARY

This study makes a valuable contribution by uncovering a previously uncharacterized Transient Receptor Potential Vanilloid 4 (TRPV4)/Ca2+ driven signaling cascade that controls the assembly of actin-based lamellipodial protrusions that drive cell migration. By integrating ratiometric calcium imaging and FRET-based small GTPase sensors with gain- and loss-of-function assays in vitro and ex vivo, the authors show that TRPV4-mediated Ca2+ influx into lamellipodia activates calcium/calmodulin-dependent protein kinase II (CAMKII), the Rho-specific guanidine nucleotide exchange factor TEM4, and the small GTPase RhoA, promoting lamellipodial protrusions. While TRPV4/Ca2+ involvement in migration has been described before, this work is important in that it identifies key intermediate players linking calcium influx to actin assembly within lamellipodia. The study is thoughtfully designed, employs rigorous methodology with appropriate controls, and overall provides strong support for the central conclusions. A few of the conclusions could be further strengthened by straightforward additional experiments or by reanalyzing existing data, which could make the findings even more impactful.

MAJOR POINTS

1. (Fig. 4) The authors conclude that TRPV4/Ca2+ influx triggers actin assembly of lamellipodial protrusions via the RhoA/formin pathway. However, while the evidence involving Rho is compelling, the connection with formin is weak. For example, the reduction of actin intensity in lamellipodia upon formin inhibition is much less pronounced than TRPV4 or RhoA inhibition (Fig. 4G). It may be possible that formin acts redundantly with other factors downstream of Rho such as Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP). The authors may consider testing this or tone down the conclusion regarding formin.

2. (Fig. 5I-J) The authors conclude that RhoA activity is reduced in CaMKII-inhibited cells. However, CaMKII-inhibited cells seem to display more variability than TRPV4 inhibition in FRET-based RhoA activity across lamellipodial protrusions (compare Fig. 4B and Fig. 5J). It appears that RhoA activity was quantified using a single line scan for each cell. This measurement may not be representative of the overall RhoA activity across the entire length of the protrusion. To account for such variability, the authors could increase the number of scans across multiple protrusions and get an average per protrusion or per cell. This may provide a more robust quantification to support their conclusion.

MINOR POINTS

3. The authors should consider referring to the myosin they are working with as non-muscle myosin II (aka, NMII) to avoid confusion with the muscle myosin II. This clarification could be added when myosin II is first introduced (i.e., 4^th paragraph of the introduction).

4. In the first paragraph of the results section, the authors state that Thosea asigna virus 2A (T2A) self-cleavage allows near-equimolar expression. While it is reasonable to assume that the expression from a single transcript provides a consistent ratio, the open reading frame following T2A is often expressed at lower levels. To improve precision, the authors may consider rephrasing “near-equimolar expression” to something like “co-translational expression”.

5. (Fig. 1E) The dense data points make it hard to visualize the means in the bar plot. To improve readability, the authors could add some transparency to individual-cell data points. Alternatively, the authors could plot the mean value of each experiment as a data point. The same suggestions also apply to Fig. 7D-E.

6. (Fig. 6D) Although a western blot validation for TEM4 is displayed in Fig. S6E, the authors should consider including knockdown validations of the other RhoA regulators. This also applies to all other TRP channels knocked down in Fig. 1F. If a good antibody is not available, qPCR validation should be shown to demonstrate siRNA knockdown efficiency. Having this would clarify to what extent the observed reduced spreading speed correlates with the variability in knockdown efficiency.

7. (Fig. 7G-H) Mouse embryonic fibroblasts seem to assemble more filopodia-like protrusions in 3D. Is this also the case when TRPV4 is inhibited? Please include a representative image of TRPV4-inhibited cells next to the control for better visualization of cell morphology in both conditions. Along these lines, throughout the manuscript, the authors refer to lamellipodia as ‘protrusions’, which could be confused with filopodia. To avoid this, they should also consider describing filopodia in the introduction, which is relevant in this study as RhoA/formin are mostly known for assembling these protrusions.

8. (Fig. 7J) One possible caveat with using a TRPV4 inhibitor in skin explants is that it will target not only melanoblasts but all neighboring cells, which may trigger mechanical changes in the environment surrounding melanoblasts. For example, their claim that melanoblasts are more spherical could be the result of extrinsic mechanical changes rather than intrinsic factors such as reduced protrusion formation. While addressing this experimentally could be challenging, the authors should rephrase their interpretation in the text and acknowledge limitations of this experiment for clarity.

9. (Discussion) One study not included in the manuscript found that TRPV4 Ca2+ influx increased cell migration via an alternative pathway mediated by RhoA/myosin contractility in response to high-viscosity media (PMID: 36323783). It would be interesting if the authors included this in their discussion and compared those findings with their own.

10. (Discussion) To provide a broader perspective, the discussion could also touch on alternative effects of Ca2+ influx. For instance, is it possible that Ca2+ may also be contributing to the direct activation of actin-binding proteins? Many of them have Ca2+-binding motifs, for example, Ca2+ binding can activate the severing/capping activity of gelsolin, which might boost lamellipodial protrusions by increasing the number of barbed ends available for polymerization.

- The author is a cell biologist with experience in investigating actin-based networks, cell migration, and tissue morphogenesis.

Competing interests

The authors declare that they have no competing interests.