Reviewer #2 (Public review):

Sasaki et al. use a combination of live-cell biosensors and patch-clamp electrophysiology to investigate the effect of membrane potential on the ERK MAPK signaling pathway, and probe associated effects on proliferation. This is an effect that has long been proposed, but convincing demonstration has remained elusive, because it is difficult to perturb membrane potential without disturbing other aspects of cell physiology in complex ways. The time-resolved measurements here are a nice contribution to this question, and the perforated patch clamp experiments with an ERK biosensor are fantastic - they come closer to addressing the above difficulty of perturbing voltage than any prior work. It would have been difficult to obtain these observations with any other combination of tools.

However, there are still some concerns as detailed in specific comments below:

Specific comments:
(1) All the observations of ERK activation, by both high extracellular K+ and voltage clamp, could be explained by cell volume increase (more discussion in subsequent comments). There is a substantial literature on ERK activation by hypotonic cell swelling (e.g. https://doi.org/10.1042/bj3090013, https://doi.org/10.1002/j.1460-2075.1996.tb00938.x, among others). Here are some possible observations that could demonstrate that ERK activation by volume change is distinct from the effects reported here:
(i) Does hypotonic shock activate ERK in U2OS cells?
(ii) Can hypotonic shock activate ERK even after PS depletion, whereas extracellular K+ cannot?
(iii) Does high extracellular K+ change cell volume in U2OS cells, measured via an accurate method such as fluorescence exclusion microscopy?
(iv) It would be helpful to check the osmolality of all the extracellular solutions, even though they were nominally targeted to be iso-osmotic.

(2) Some more details about the experimental design and the results are needed from Figure 1:
(i) For how long are the cells serum-starved? From the Methods section, it seems like the G1 release in different K+ concentration is done without serum, is this correct? Is the prior thymidine treatment also performed in the absence of serum?
(ii) There is a question of whether depolarization constitutes a physiologically relevant mechanism to regulate proliferation, and how depolarization interacts with other extracellular signals that might be present in an in vivo context. Does depolarization only promote proliferation after extended serum starvation (in what is presumably a stressed cell state)? What fraction of total cells are observed to be mitotic (without normalization), and how does this compare to the proliferation of these cells growing in serum-supplemented media? Can K+ concentration tune proliferation rate even in serum-supplemented media?

(3) In Figure 2, there are some possible concerns with the perfusion experiment:
(i) Is the buffer static in the period before perfusion with high K+, or is it perfused? This is not clear from the Methods. If it is static, how does the ERK activity change when perfused with 5 mM K+? In other words, how much of the response is due to flow/media exchange versus change in K+ concentration?
(ii) Why do there appear to be population-average decreases in ERK activity in the period before perfusion with high K+ (especially in contrast to Fig. 3)? The imaging period does not seem frequent enough for photobleaching to be significant.

(4) Figure 3 contains important results on couplings between membrane potential and MAPK signaling. However, there are a few concerns:
(i) Does cell volume change upon voltage clamping? Previous authors have shown that depolarizing voltage clamp can cause cells to swell, at least in the whole-cell configuration: https://www.cell.com/biophysj/fulltext/S0006-3495(18)30441-7 . Could it be possible that the clamping protocol induces changes in ERK signaling due to changes in cell volume, and not by an independent mechanism?
(ii) Does the -80 mV clamp begin at time 0 minutes? If so, one might expect a transient decrease in sensor FRET ratio, depending on the original resting potential of the cells. Typical estimates for resting potential in HEK293 cells range from -40 mV to -15 mV, which would reach the range that induces an ERK response by depolarizing clamp in Fig. 3B. What are the resting potentials of the cells before they are clamped to -80 mV, and why do we not see this downward transient?

(5) The activation of ERK by perforated voltage clamp and by high extracellular K+ are each convincing, but it is unclear whether they need to act purely through the same mechanism - while additional extracellular K+ does depolarize the cell, it could also be affecting function of voltage-independent transporters and cell volume regulatory mechanisms on the timescales studied. To more strongly show this, the following should be done with the HEK cells where there is already voltage clamp data:
(i) Measure resting potential using the perforated patch in zero-current configuration in the high K+ medium. Ideally this should be done in the time window after high K+ addition where ERK activation is observed (10-20 minutes) to minimize the possibility of drift due to changes in transporter and channel activity due to post-translational regulation.
(ii) Measure YFP/CFP ratio of the HEK cells in the high K+ medium (in contrast to the U2OS cells from Fig. 2 where there is no patch data).
(iii) The assertion that high K+ is equivalent to changes in Vmem for ERK signaling would be supported if the YFP/CFP change from K+ addition is comparable to that induced by voltage clamp to the same potential. This would be particularly convincing if the experiment could be done with each of the 15 mM, 30 mM, and 145 mM conditions.

(6) Line 170: "ERK activity was reduced with a fast time course (within 1 minute) after repolarization to -80 mV." I don't see this in the data: in Fig. 3C, it looks like ERK remains elevated for > 10 min after the electrical stimulus has returned to -80 mV

Figures and data

Mitotic activity is upregulated by membrane depolarization.

ERK is activated upon high K+ perfusion.

ERK activity depends on membrane voltage

The molecular mechanism by which regulate depolarization-induced mitosis.