Interesting finding! How many filopodia angles were measured? Panels B-D, F-G all have n= cells or filopodia, but Panel E has n = 3 experiments but doesn't specify the number of filopodia.

Just curious, is silanization essential for these assays to work? I recently saw some literature that silica nanoparticles can mess up actin dynamics / localization (https://doi.org/10.1007/s00204-020-02694-6 ; doi:10.1021/nn103344k). I know that basically all actin dynamics have been measured in the presence of glass/silica, and the 30 min on silanized glass likely wouldn't impact anything substantially, and there really aren't any good alternatives to silica-based coverslips, so definitely not expecting anything else, but I was just curious what the extra silanization does?

It might be cool to see the rearrangement of the MTs in a single channel (or w/ DAPI) like Actin is shown in A-C. It looks like the Tubulin in F-G is whacky compared to the control, but its hard to tell if this is true with the actin overlay. From the whole cell view, it looks like MTs are localized more centrally in the knockdowns, compared to the cortical localization in the Control, but i didn't see any mention of this in the text (but I might have missed it and I'm not up to date on the CLASP-literature so it might already be known).

This is amazing!!! I'd love to see if this is happening with the truncated CLASP2a as well! Since the 2 actin binding TOGs are right next to each other, do you think both of those are both binding actin monomers helping to form stable dimers? helping to initiate non-spontaneous actin nucleation? Maybe even TOG3 binds actin?

Is it possible that microtubules are required for CLASP2a's F-actin bundling abilities? You show it can bind F-actin in the absence of MTs (Sup Fig 1) but its binding & function could be different in the presence of MTs

Very cool!!!! Strangely, it looks like the signal in the MT and F-actin channels are comparable between Full Length and truncated CLASP2as, but it looks like there is less GFP signal in the truncated version, compared to full-length. This can be seen with the increased blue signal in the merged channel. While the colocalization quantifications are awesome and needed, it could be nice to have some intensity quantifications / linescans as well, like you did for F-actin signal in Figure 2

This is weird, right? Is truncated CLASP2a better at binding F-actin (and maybe worse at binding MTs) than FL? Even though you cite a paper saying that TOG1 (which is deleted in this construct) also binds to F-actin?

If you add purified CLASP2a-GFP to Phalloidin-stabilized F-actin, will the CLASP bind & coat F-actin? and if so, could you then add the MTs and see if they coat the stable F-actin filament? Basically, does this go both ways? or does it need to be MT -> CLASP2a -> Actin?

Also ARPC3 disappeared, based on the STRING figure

The TNTs in the WT here look drastically different from the WT/control TNTs in previous figures In both the GFP/mCherry and the Eps8-IRSp53, the TNT actin looks thinner when treated with CK-666 compared to Mock/DMSO. Have you done any diameter measurements of the TNTs?

Have you done any experiments where you pre-treat with CK-666 before adhering the cells? Its possible the branched actin networks are necessary for the initial membrane deformation, but after the TNT is formed, Arp2/3 complex inhibition frees up more G-actin which goes the the linear filaments in the TNT?

It might be a good idea to also cite Fig 2C here since Fig 1e shows 0% TNTs so 'fewer' TNTs was a bit confusing

Fig 4 C & F) does the 00:00 time point indicate the time when the cells were plated? In the earlier experiments, cells were allowed to adhere for 4-6 hours before treatment for 16-18 hours. Do TNTs form within 30 min of plating?

Also, minor point, but C is in hh:mm:ss time stamp, but F is hh:mm time stamp. Since all of the timepoints in C end it 00 sec it would be good to make C/F consistent as hh:mm (or hh:mm:ss)

Also, after 30 min, CK-666 is added and the extended protrusions form shortly after. For the control quantified in d and g, was DMSO added at the 30 min mark? It would be nice too see the control images as well. The force from the flow of added liquid could cause morphological changes

In Fig 4 C,F) It would be helpful to have these fluorescent images merged with a brightfield image so we can see the outline of the TNTs and where these fluorescent proteins are localized in the cell.

It would be helpful to include a heat map gradient/scale/legend under subpanels in C. It took me a while to realize that it was a heat map LUT and not a blue marker and a magenta marker.

If I understand correctly, the figure shows that IPSp53 and Eps8 interact and localize to these extension, but not that they are responsible for forming the longer protrustions, right? The figure text suggests they are responsible which isn't shown until Fig 5

Will a TNT fuse to another cell's TNT or will it attach directly to the membrane?

Would it be possible to develop a similar pipeline that uses alphafold to predict protein-drug interactions? Meaning I have a chemical structure and I want to determine which proteins it might bind to in a species.