This study highlights the true complexity of protein processing, trafficking, and its ultimate function! I think it could benefit from some orthogonal methods of analysis - would be it be possible to try to isolate specific 'states' of processed protein (like you did with the CHX treatment) or using subcellular localization or IPs to identify exactly which glycosylation sites or cleavages have been made? Excited to see what comes next!

Some Lamp1 staining for lysosome localization could contribute to the above IF figures as well!

I wonder if there might be a more straightforward way to probe this interaction. It seems difficult to study the interaction between RNF13 and IDS because there are so many different precursors or processed forms of IDS. Perhaps a strategic approach could be to either isolate the process (in cells) for which the interaction is most important, and then determine which processed form of IDS is involved. Or maybe purify different forms of IDS and determine using binding assays, since IPs are less capable of identifying 'direct interactions'.

This is a clever way to probe protein processing! It might be useful to do some western blotting with this treatment to look at the impact on processing that way as well.

This is a very neat result! I'm wondering if it might be interesting to repeat an IP with different fractions - like a lysosome-enriched fraction vs a whole lysate. There are some pretty crude lysosome preps that could give some nice connections between forms that are trafficked, the maturation data you have, and the localization data you have below.

What is your hypothesis for this interaction, then? Does RNF13 interact with both species, throughout the whole processing stage? Or predominantly with one species?

It's really awesome that you developed these tools and processes to enable protein production in plants!

I wonder if you could also integrate a DSF or nano-DSF protocol to confirm folding/thermal stability of your protein as well. It could be another good indicator of successful protein production.

That's great!

this is a cool feature!

Given this is a membrane protein, did you have issues with solubility? Do you have data showing the protein is well folded after purification?

How long is this stable for? Do you have to make it fresh for each purification you do?

Are you treating with TEV in whole plant lysate?

I am not sure that co-localization of those tags is the best way to confirm that the protein integrity contains both tags. It is unlikely that you could see single proteins based on your image resolution - I think the stronger claim is the western blot you have that shows the size change upon cleavage (fig 2E,D)- and also just the fact that your protein is of expected size with the two tags.

It is super helpful that you included so many tag designs already in the construct. It really adds to the usability right off the bat.

It's really nice that this protocol is pretty agnostic to the source and doesn't require any modification of the natural biology!

This data isn't linked anywhere.

How do you control for lysosomal quality here, or intactness from your isolation protocol?

This is a really nice result! Do you have a way to measure 'lysosome concentration' or do you just base everything off of normalized protein concentration? I'm wondering if there is a way to be quantitative about experiments like these from one batch to another.

For all of these assays, is the assumption that most of your lysosomes are intact? How do you then measure enzyme activity from the intact lysosomes? Do you assume that the substrates can all pass through the membranes?

Do you have details on what speed and for how long?

This is a little unclear. Which step was repeated two more times? Just homogenization?

I don't agree with this statement. You do not need a packed column or a chromatography instrument to perform affinity chromatography; you can just bind to beads in batch and elute. It seems like the major improvement might just be cost effectiveness.

It would be super useful to see an outline of this cost versus buying magnetic beads for batch binding. This could include how many times you could reuse the beads with similar efficiency, etc.

Do you think that your beads are truly saturated? If you add more beads, can you recover what is lost in the Pass through? Or is it more of a binding efficiency issue?

It would be nice to see how the purity of these samples compared to one another.

How do you know this? Do you have a way to strip protein off of beads? Or are you just basing this off of estimated total protein concentration recovered?

Why not compare directly to 6x Ni-NTA magnetic beads? You can do this at very small scale and it would be nice to see the differences in capture efficiency and specificity.

Wouldn't you want to capture 100% of your purified protein? Is this just a matter of how many nanoparticles you add? Do you have a rough idea of the binding capacity of the nanoparticles, or is it pretty variable from one prep to the next?

How long can this powder be stored at 4C? Is it moisture sensitive?

What are the advantages of the dot blot over a conventional sds-page analysis? SDS-PAGE likely takes the same amount of time as a dot blot, but potentially gives you more information about each sample.

This might just be an artifact of biorxiv, but I don't see a table description anywhere. What were the adverse reactions for the SDS-PAGE? Did the gel just not run straight or stain evenly? Also, what are the units for purification yield?

what volume of extraction buffer do you use here? Information on the ratio of 'dried pellet material' to extraction buffer would be helpful here.

It makes sense that you use the auto-induction for more high throughput purposes. After finding an optimal condition and scaling up, would you recommend switching to IPTG? Would you expect any changes?

If you are growing the cells in a single flask, at which step do you split up the pellet into a 96-well plate?

It would be very illuminating to study how these different isoforms may differentially impact Rac1 activity in vitro. If their expression levels are similar in certain cell types, does one directly activate rac1 while the other indirectly activates Rac1? If the Lpd isoform doesn't bind directly to Rac1, how does it enhance Rac1 activity?

It might be useful to comment on why your structural predictions above (of just the csRAPH domain) gave you a different hypothesis than predicting the structure of the full Lpds in complex with Rac1. What do you get for structural predictions when you just look at Lpds alone?

It would also be interesting to examine expression patterns of all of these isoforms.

This is a really nice pulldown! It would be cool to see binding curves for these interactions, too.

What was your rationale for this construct in particular? Why not just express the RA-PH, or why not the full-length construct?

I think this only suggests that Lpds doesn't heterodimerize, but Lpds could still associate with itself as a dimer to facilitate interaction with Rac1. It would be interesting if you could purify it and determine its tendency to dimerize that way.

I would be careful with conclusions here - just because something coIPs with a protein doesn't mean it binds directly. There could be other proteins in the mix that facilitate this interaction/complex.

Why do you think that there wasn't the clear distribution along the periphery in Fig 5g like you saw for your 2uM His-actinin control? Does this distribution have any impact on multiple bleb formation?

What relevance does this have to a physiological process? Do you think it is just that there are multiple mechanisms for blebs to form, or do you think they are initiated in certain ways dependent on context?

This is a very interesting idea. You nicely demonstrated that blebs can form from basic elements of the cytoskeleton. It seems that an idea that follows is that more 'control' or determination on whether or where to form the bleb comes from higher-level signaling and localization.

Why do the blebs fade or resolve at t31 or t26 in figs 3e and 3f, respectively? The actin network seems to become even more concentrated with this event.

But the distribution was significantly different at 2uM of each. Do you have concerns about the distribution at these lower concentrations?

This seems mostly due to the methylcellulose, correct? I'm wondering if there is a way to determine the actual number of anchor points in the liposome? Perhaps some staining against the His tag? It might be interesting to see where deformations lie in relation to clusters of anchor points.

This is a really clever approach! I love how you separated out variables like this.

Do you also have the Kd of untagged actinin for F-actin? It could be nice to know if the tag has any impact on binding. I'm also curious if the membrane tethered actinin has a different affinity for actin filaments compared to free-floating actinin.

This is a super beautiful figure! Very clear distinction.

What do you think is in the bottom right corner of the liposome in figure 1b? It looks like that's where there is a cluster of actin filaments - is that a premature bleb or something? It shows up right at the beginning, before myosin contractility.

How much do the anchor points actually move within the membrane? Is it enough to generate just local changes in force, or larger scale changes in the overall relationship between the cortex and the membrane?

I'm really curious how broadly this extends to general movements of the body. Like - if you have a cyclist or runner who mainly use their legs in movement patterns, are they going to be more 'efficient' at generic movements that require the legs, and closer to a non-expert at movements that require the arms? Or is being an 'expert' at some motor skill enough? How about other non-sport motor skill experts, like musicians?

This suggests that tremors negatively impact mastery of a movement. Are these inefficiencies true barriers to mastery, or is efficiency just a byproduct of mastering a movement?

Very interesting, do you have any hypothesis for what might be going on there?