1. Evaluation Summary:

    This paper is of interest for cell biologists and biophysicists that work on eukaryotic cell motility and the dynamics of the actin cytoskeleton. The authors combine a series of clever biological approaches to fuse small Dictyostelium cells together into 'giant cells' that make it much easier to spatially resolve actin wave dynamics with and without electrical stimulation when cultured on smooth or nano-textured surfaces. Sophisticated and methodical computational approaches are used to analyze these images and relate the data to actin polymerization and wave dynamics parameters using optic flow and associated techniques. This study is mostly descriptive, a full mechanistic explanation of the results remains open, but this compelling experimental system opens up possibilities for the field to analyze the molecular subtleties involved in these cytoskeletal reorganizations.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  2. Reviewer #1 (Public Review):

    This manuscript describes an elegant system of electro-fused cells where multiple simultaneous actin waves can be visualized and analyzed. For the study of electrotaxis, the advantage of this system is to decouple reorganization of actin networks under application of electric fields from other cellular events that can complicate this analysis. The authors use electro-fused cells to study propagation of actin waves in 2D on flat surfaces, or in 1D on nanotopographic surfaces. This study is mostly descriptive, but this compelling experimental system opens up possibilities for the field to analyze the molecular subtleties involved in these cytoskeletal reorganizations. Authors achieved their aims, conclusions seems to be supported by the results, although few additional controls are probably required.

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  3. Reviewer #2 (Public Review):

    In this manuscript, Yang and coworkers investigate the impact of electric fields on the dynamics of cortical actin waves. Their experiments were performed with giant Dictyostelium discoideum cells that were produced by electric pulse-induced fusion and expressed a fluorescence marker for filamentous actin. Due to the large sizes of these fusion products, unconfined waves dynamics could be observed in the giant cells by fluorescence microscopy. Besides freely moving waves, the authors also focused on small wave-like patches that exhibit shorter lifetimes and emerge if cells are placed on nanopatterned substrates. The recordings show that electric fields increase the area, duration, and speed of the cortical waves. In particular, their results convincingly demonstrate that the direction of wave propagation is guided to preferentially align with the direction of the electric field and follows switches in the orientation of the field, with fast switches in direction in the case of the small patches on nanoridges and slower u-turns in the case of freely moving waves. An inhomogeneous intracellular distribution of waves in the presence of an electric field furthermore reveals subcellular polarization with different time scales of wave initiation and inhibition at the front and back of the cell.

    Taken together, the results are a timely contribution to current research on the dynamics and functional role of cortical actin waves in motile cells.

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  4. Reviewer #3 (Public Review):

    Summary of what the authors were trying to achieve:

    The authors set out to relate electric field stimulation to cortical actin wave dynamics, which they do largely by fusing small together into large cells that make the spatiotemporal elements of these elusive waves much more tractable to analyze. This is quite clever and clearly effective. They try to further relate wave dynamics to specific nanostructured surfaces and to an excitable systems framework called STEN-CEN. Despite some correctable flaws, this paper was a pleasure to consider and explore.

    Major strengths and weaknesses:

    This is a detailed study with some real technical accomplishments and experimental finesse. There is no doubt that electric fields do alter cortical actin wave dynamics. The imaging and data are novel in this regard. However, there were also a number of choices in how the data were presented and discussed that made it difficult for this reviewer to grasp the larger picture. The need for the nano topography is lost until the end, the statistics are a bit hard to follow, the STEN-CEN concept and cartoons are interesting but don't quite come together as currently written, and some key methodology and statistical metrics need to be addressed in the figures and methods section.

    Did the authors achieve their aims and support their conclusions:

    All of that said, the authors presented clear and novel data. Their core conclusion is irrefutable-field stimulation caused changes to cortical actin waves, with field direction biasing the wave direction and dynamics. Some of the subsidiary claims related to STEN-CEN, signaling pathways, and the specific role of the field could be better supported, but this is certainly feasible.

    A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community:

    These data are particularly exciting because the field of bioelectric stimulation (non-neural) is in dire need of live imaging of key downstream processes related to the actual physical mechanisms and machinery in cells that respond to such stimulation, and this paper beautifully illustrates that. Very few studies in this field have any sort of live imaging of sub cellular processes, and these data were a pleasure to go through.

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