Couldn't there still be active actin polymerization by formins and the like in other parts of the cell? I think you could add that the active actin polymerization is Arp2/3 complex-mediated.

Does loss of Arf6 cause differences in Arp2/3 complex recruitment to clathrin sites?

Is this the actin-related protein Arp2 that is a part of the Arp2/3 complex? I think this is an important distinction that you might take advantage of and discuss a little bit more here.

In figure 1D, it looks like the width of the bleached region didn't change, but it looks like the overall intensity of the signal around the whole GUV deceases - do you think this is from cycling through depolymerization, then monomeric actin, then repolymerizing at the bleached spot?

I think this FRAP experiment is really cool, and definitely important to show that the actin at the cortex turns over. Also, really cool that the turnover is close to that of living cells even without debranching, severing, disassembling proteins! Can you distinguish between filamentous actin and monomeric actin with your labelling technique? (It might be worth mentioning how you're labelling the actin here.) I'm curious what the ratio of G-actin to F-actin looks like and how this might affect turnover. You have a really great paragraph about this in discussion, but might be useful to mention briefly here.

This is a really interesting paper with a really cool technique, some beautiful images, and a ton of data that tells us a lot about the mechanics of cell protrusions. Additionally, their GUVs with minimal actin cortices could be a super useful tool for better understanding what different proteins and combinations of proteins are doing at the cortex.

Did you do any experiments where you polymerized actin filaments with the Arp2/3 complex, but with non-membrane anchored VCAs?

Interesting.. I'd expect these structures to be really dynamic. Is the turnover of actin at the membrane decreased in these deflated GUVs?

I was still kind of questioning if the GUV misshaping was really due to the actin vs just having extra membrane around (even though you had the bare GUVs being more spherical), but this experiment and the CytoD experiment are really convincing. Although I wonder if you could include a quantification of roundness or a similar parameter pre and post ablation?

How often did you see these different morphologies? I'm curious if some are more common than others.

Really clever way to drive formation of an actin cortex at the membrane of the GUVs. But obviously VCAs aren't attached to the membrane like this in biological systems, so are there biological consequences that might affect some of this data? I think it would be interesting to hear a little bit about this in the discussion maybe?

I think it would go a long way to support your concluding hypotheses if you did a quantification of branching for Figure 6C because it is a little hard to make out with the density of the filaments and because it is so important for your conclusions.

It would be interesting to look at direct binding between these proteins, especially because they are mutants that might not behave quite as expected.

This is a very interesting paper looking out how different actin binding proteins work together to regulate actin in specific functions!

I think it would be useful to talk again about the effect of the different constructs that you used in the context of what that means biologically in the cell.

I am interested what this would look like in cells! Maybe a next step?

Actin + Arp2/3 + VCA + delta-mod (cyan) is interesting - it looks like nucleation is delayed, but then is able to rapidly at least approach the same level as Arp2/3 + VCA. I'd love to hear some more thoughts about this.

These are amazing!

I think this is obviously quite visible in the images, but it would be interesting to see a quantification of filament length in each case and also the occurrence of these bright bundle-like regions. I know you do this more precisely in the next section but I think this would also help set that section up a little bit.

It would be super cool to see some images along with these graphs looking at single filament elongation.

I find this interesting also in terms of the effects of ERK3 knockdown on the morphology of the cells and the specific actin structures. You mentioned previously that Rac1 leads to the formation of lamellipodia and Cdc42 leads to the formation of filopodia, and looking at the images of your cells, it looks like filopodia are more affected than lamellipodia. Is that something you noticed looking through your images?

You might specify that this is true for human isoforms. There are lots of weird actins out there in other organisms that are well under that 95%

It might be useful to quantify protrusions for all of 6F because it seems like there are definitely some big differences!

Woah! Curious if the kinase activity (that you test in the next section) is required for this change in actin polymerization?

It would be interesting to see this in ERK3 knockdowns as well to see if the localization of Cdc42 changes with loss of ERK3. I'm also curious about the localization of Rac1.

Very cool! Did you check other subunits or did they not precipitate?

Are there stats for Figure 2F? It seems like there's more variance with the Rac1 blot quantifications than with the Cdc42 blot quantifications.

Super interesting! Do these mutants have motility defects too?

By eye, I agree that it looks like cell shape is altered in Figure 1A, but it would be cool to include a quantification (maybe like how round the cells are or something). I also think that Figures 1H-J are relevant here to help connect some dots for this conclusion and show that the loss of ERK3 does cause changes in specific actin dynamics that result in shape and motility defects, but you don't talk about Fig 1H-J until later. Finally, I think the conclusion about polarized migration is more relevant in the next section than at the end of this section.

I really enjoyed reading this paper - what a great story! It really highlights how little we still know about actin regulation (even in human cells!), but it also does a great job filling in some of that knowledge with this pathway involving this atypical MAPK.

Do we know that SMIFH2 broadly inhibit all of the formins in Arabidopsis?

Interesting, is the hypothesis that some of these formins that nucleate filaments off the side of existing filaments are responsible for these specific branched filaments?

I think you do a great job here and in the next figure validating CK-666 (definitely showing that there isn't added effect in the Arp2/3 complex mutants is great!), but you might consider adding CK-689 as an inactive control just to cover all your bases.

What a cool paper! I think papers dealing with different proteins interacting with a single pool of actin are so interesting. Also super awesome that you validate CK-666 in these cells and start dealing with potential off-target effects of SMIFH2 in these cells - so much added value to the field!

There are a couple of these graphs where we're looking at the distribution of data and there's mention that there's more of a skew in a sample compared to another, but sometimes this is really difficult to see in the graphs. It might be useful to split out arp2 mutants vs arpc2 mutants so that we're comparing 2 instead of 4. It might also be useful if you could include some measure of skew, especially for the ones where you think there might be a difference in skew.

It would be interesting to see specific data points here instead of bars for these graphs. Based on the representative images, it looks like there could be a couple population with different lengths, especially in the arpc2 hypocotyls. If that's the case, it would also be interesting to hear why that might be.

Maybe i'm missing this, but I don't see Eps8 in this map. Might just be worth discussing in the text why it's not there?

Interesting that ARPC5 disappeared, but not ARPC5L if I'm reading that correctly?

I noticed in figure 4A the tubulin loading control isn't super consistent. Did you normalize to your loading control? Might also be worth doing total protein as your loading control instead of tubulin, because it is possilbe that tubulin is being affected since you're probing other cytoskeleton related proteins.

This experiment is very cool, but I wonder if you could show a similar thing with naturally forming TNTs? Do you see more actin in natural TNTs when the Arp2/3 complex is inhibited? I worry that stretching the cell like this could be causing other things to happen in the cell and isn't fully representative of a TNT forming on its own.

I really like this data with the formin agonist!

It would be really great to see representative images of the data quantified in Figure 2C.

To really illustrate this it would be helpful to show a bivariate analysis between like number of lamellipodia per cell and number of TNTs per cell for 2B. Then you could actually graphically show this anti-correlation and provide a correlation coefficient to strengthen this conclusion. It would also be interesting to show this with CK-666 treatment as I wondered while reading this if the increase in TNTs/filopodia with CK-666 treatment was accompanied by a decrease in lamellipodia.

What a cool paper! Beautiful images and I love papers that deal with this concept of a limited pool of actin in the cell that is directed to its different functions by its interactions with actin binding/interacting proteins.