In summary, our quantitative approach in determining precise numbers of molecules at CEV has unexpectedly revealed that A36 levels in the viral membrane are variable. It has also demonstrated that the velocity of virus movement depends on Nck rather than N-WASP which activates Arp2/3 complex dependent actin polymerization.
Very cool & elegant work!!!
verall, there appears to be a 4:2:1 ratio for the A36:Nck:N-WASP signalling network in MEFs.
Very cool! Do you think there are 2 Nck molecules binding to each A36 protein, or that half of the A36 proteins aren't binding Nck?
Loss of Grb2 or intersectin recruitment leads to even lower levels of N-WASP (245±26 and 276±66 respectively).
Could this be confirmed with a Grb2 or Intersectin mutant? Right now this claim is based on the A36 mutants, correct? But the A36 mutations could have many effects beyond loss of Grb2/Intersectin recruitment, right?
N-WASP
I think this text was left in on accident
Interestingly, however, in human cells, ~200 copies of the Arp2/3 complex are recruited during clathrin-mediated endocytic events (Akamatsu et al. 2020).
Are the cell lines in Akamatsu et al 2020 able to be infected by the virus? It'd be neat to one day have this direct molecule count to confirm the 2 NPFs per Arp2/3 complex in vaccinia motility!
The striking difference between these two mutant viruses is the level of Nck recruited to the CEV, with the numbers being 61% lower for the A36△NPF mutant compared to the wild-type virus. Nck and N-WASP are both essential for Vaccinia induced actin polymerization, but how their relative levels impact on the rate of actin-based motility is not established.
Is the entire CEV-Actin machinery known? While Nck is altered, there is probably a lot more happening, right?
1 (Figures 2F, 2G, S2F, S2G, and Movies S3, S4)
The Y-axes aren't consistent here so its hard to tell, but it seems like there is much stronger whole nucleus lamin signal in MA cells compared to control. Is this correct? Have y'all quantified the whole cell levels of lamin in MA & control cells?
low levels of γH2AX (<2 foci per nucleus),
I'm curious why <2 foci is the measurement here. It seems whole cell intensity or a full # of foci count would show greater variation between the conditions (based solely on the representative images). Sorry if this is a common assay that is frequently done.
To gain insight into how persistent migration might affect cell viability, we treated control and MA cells with methotrexate, a DNA biosynthesis inhibitor used in clinics against multiple human pathologies [26]. Interestingly, we found that MA cells showed higher resistance to apoptosis than control cells (Fig. 4E, 4F).
That is wild! Have y'all looked at other inducers of apoptosis? Is it possible that the methotrexate just isn't getting into the nucleus as much (since MA cells seem to have so much more lamin surrounding & throughout the nucleus).
Oh! I see you used staurosporine which is listed as an apoptosis inducer! Did y'all notice any changes in survival rate when treated with Staurosporine?
These results indicate that constrained migration alters the mechanical behavior of the nucleus, which might have functional implications on the capacity of MA cells to infiltrate tissues.
Interesting! So the MA cells will migrate more than control cells through collagen, but are less capable through animal tissue! Also it is incredibly strange that the Jurkat MAs are found significantly less in BM and Liver (but not Spleen), but the CCRF-CEM MA cells are significantly less in Spleen an Liver (but not BM), right!??!
In summary, our quantitative approach in determining precise numbers of molecules at CEV has unexpectedly revealed that A36 levels in the viral membrane are variable. It has also demonstrated that the velocity of virus movement depends on Nck rather than N-WASP which activates Arp2/3 complex dependent actin polymerization.
Very cool & elegant work!!!
The striking difference between these two mutant viruses is the level of Nck recruited to the CEV, with the numbers being 61% lower for the A36△NPF mutant compared to the wild-type virus. Nck and N-WASP are both essential for Vaccinia induced actin polymerization, but how their relative levels impact on the rate of actin-based motility is not established.
Is the entire CEV-Actin machinery known? While Nck is altered, there is probably a lot more happening, right?
Interestingly, however, in human cells, ~200 copies of the Arp2/3 complex are recruited during clathrin-mediated endocytic events (Akamatsu et al. 2020).
Are the cell lines in Akamatsu et al 2020 able to be infected by the virus? It'd be neat to one day have this direct molecule count to confirm the 2 NPFs per Arp2/3 complex in vaccinia motility!
Loss of Grb2 or intersectin recruitment leads to even lower levels of N-WASP (245±26 and 276±66 respectively).
Could this be confirmed with a Grb2 or Intersectin mutant? Right now this claim is based on the A36 mutants, correct? But the A36 mutations could have many effects beyond loss of Grb2/Intersectin recruitment, right?
verall, there appears to be a 4:2:1 ratio for the A36:Nck:N-WASP signalling network in MEFs.
Very cool! Do you think there are 2 Nck molecules binding to each A36 protein, or that half of the A36 proteins aren't binding Nck?
N-WASP
I think this text was left in on accident
These results indicate that constrained migration alters the mechanical behavior of the nucleus, which might have functional implications on the capacity of MA cells to infiltrate tissues.
Interesting! So the MA cells will migrate more than control cells through collagen, but are less capable through animal tissue! Also it is incredibly strange that the Jurkat MAs are found significantly less in BM and Liver (but not Spleen), but the CCRF-CEM MA cells are significantly less in Spleen an Liver (but not BM), right!??!
To gain insight into how persistent migration might affect cell viability, we treated control and MA cells with methotrexate, a DNA biosynthesis inhibitor used in clinics against multiple human pathologies [26]. Interestingly, we found that MA cells showed higher resistance to apoptosis than control cells (Fig. 4E, 4F).
That is wild! Have y'all looked at other inducers of apoptosis? Is it possible that the methotrexate just isn't getting into the nucleus as much (since MA cells seem to have so much more lamin surrounding & throughout the nucleus).
Oh! I see you used staurosporine which is listed as an apoptosis inducer! Did y'all notice any changes in survival rate when treated with Staurosporine?
low levels of γH2AX (<2 foci per nucleus),
I'm curious why <2 foci is the measurement here. It seems whole cell intensity or a full # of foci count would show greater variation between the conditions (based solely on the representative images). Sorry if this is a common assay that is frequently done.
1.8-fold change in 639
Wow! That's a big change in 3 days!!! I'd be interested in seeing images of more than just the nucleus. Are there cell shape changes in the MA cells compared to the control?
Also, I'd be interested to see a comparison between control, ORM, and MA cells! Since the ORM cells can revert back to "normal" cells, it'd be interesting to compare the expression in revertable and non-revertable cells to get more insight into the genes responsible for this reversion!
1 (Figures 2F, 2G, S2F, S2G, and Movies S3, S4)
The Y-axes aren't consistent here so its hard to tell, but it seems like there is much stronger whole nucleus lamin signal in MA cells compared to control. Is this correct? Have y'all quantified the whole cell levels of lamin in MA & control cells?
o investigate the effect of persistent migration on the nuclear changes of MA cells, we determined their lamin B1 distribution and found that MA cells showed an aberrant distribution of lamin B1
Were the MA cells in suspended culture for at least 24 hours (like the recovered ORM cells)?
expanded
Very cool! I'm guessing "expanded" means the cells were split/passaged, right? It could be helpful if this is clearly defined since expand could be interpreted as expansion microscopy preparation which would lead to larger nuclei. It is super cool that these phenotypes persist!
urkat and CCRF-CEM
I really appreciate the consistency of phenotype across both cell types! I'm curious if a similar phenotype would occur in non-cancerous cells as well!
then culturing them in the absence of further mechanical stimuli
Very cool process! Have you taken the MA cells through several passages an they continue to retain this phenotype?
by allowing cells to migrate three–rounds across rigid filter membranes
Interesting! Is this something that would be happening in living tissue? I'd be curious to hear if y'all tried different membrane materials or numbers of passages!
(A) Phenotypes of 5-day-old atm1-1 plants with reduced root length compared to wild type on 0.5 X Murashige and Skoog (MS) medium without or with 15 mM Sucrose (Suc) at 45 μmol m-1 s-1 LD conditions. (B) Quantitative analysis of root growth in Col-0 and atm1-1 seedlings. n=10; ***P<0.001, two-way ANOVA and Tukey’s multiple comparison test. (
Is this just redundant w/ Fig 2 BC? I see that the values are a lot higher in Fig 2 BC and those were 6-day old, instead of the 5-day old plants shown here. Sorry if it's an ignorant question.
nt center: ***
The first image in Panel E has 2 asterisks instead of the 3. Not sure if intentional.
ATM1 promoter is expressed during post-embryogenic root organogenesis in Arabidopsis thaliana.
What is marked in red? is that propidium iodide like in Fig 2D?
Compared to WT, under sugar-free conditions, the layer of columella stem cell daughter cells (CSCDs) was absent in atm1-1 (Fig. 4E), suggesting that the competence of these cells to properly differentiate is altered. Under sugar supplementation conditions, the size of the CSCDs and the fully differentiated columella cells (DCCs) were reduced in atm1-1 compared to WT plants but not the number of cells (Fig. 4E).
Wow! This is a very striking and exciting finding! Was the cell/layer count consistent across 100% of roots? It'd be great to see quantification of that as well. the Recovered number when sucrose is added is super cool!
(Fig. S1)
Is there a reason the +Suc propidium iodide staining is so much brighter than +Mock WT? is Propidium iodide staining just pretty inconsistent in these cells?
florescence
Just a minor point. I think y'all meant fluorescence.
Transcriptomic analysis of atm1-1 seedlings in response to sugars
Was there altered expression of other myosins? or cytoskeletal proteins?
Discussion
Very cool work! I really enjoyed reading through this preprint! Thank you for sharing your work!
VLP
Very cool! It'd be neat to see if cdc42 is upregulated in covid patients to a similar degree!
Disruption of filopodia inhibits SARS-CoV-2 VLP invasion.
Very cool results! With the # of VLP internalized, did you make any distinctions between surface-bound VLPs and Filopodia-dependent internalization? Could these results be related to decreased endocytic efficiency?
Of note, VLP infection can also induce longer filopodia formation and increase the movement of filopodia, but cannot increase the dynamics of filopodia in SMIFH2-treated cells
But internalization of VLP in SMIFH2 treated cells is still reduced compared to untreated cells, so do the dynamics of the filopodia not matter on viral entry?
ore dendritic morphology
It looks like blebbistatin is having a similar effect as the other drugs on filopodia length, number, and dynamics, but isn't reducing VLP internalization? If that is the case, the length & dynamics of filopodia don't matter for viral entry? Or do you think that the reduced internalization of VLP is because the overall cell morphology is so messed up in Blebbistatin treated cells?
SMIFH2 (15 μM for 1 h)
Worth noting the lack of specificity of SMIFH2 for formins
https://doi.org/10.1242/jcs.253708
Still a useful drug to test with though!
while some VLPs continually move laterally on the plasma membrane and take substantially longer time for entry
Very cool! Have you ever observed VLPs moving back up a filopodia? or is endocytosis at the base of filopodia so frequent that it would be internalized before reaching the filopodia?
SARS-CoV-2 VLPs enter host cells via filopodia in two patterns
Did the particle ever attach midway to the filopodia or is it always starting at the tip? Also, how often are these behaviors observed? I see the % surfing vs % grabbing, but of all of the VLP entries, what percent are dependent on these 2 mechanisms?
Reciprocally, inhibition of Cdc42 significantly reduced the number of entered VLPs, while activation of Cdc42 reinforced the number of entered VLPs (Fig 1L-O). Together, these results confirm that VLP infection enhanced the formation and dynamics of filopodia in host cells by activating Cdc42.
Did Cdc42 inhibition reduce the number of / length of filopodia, or just the viral entry?
Nevertheless, VLP infection has no influence on protrusion angle of filopodia (Fig 1E), suggesting that the overall sensing orientation of filopodia is not specifically changed upon infection.
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.
silanized
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?
Interestingly, co-alignment of actin fibers with microtubules in CLASP-depleted cells (Figure 4F,G) was reduced as compared to controls (Figure 4E), suggesting a diminished coordination of these filaments, consistent with our in vitro result
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).
Video 2
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?
However, using a low-speed co-sedimentation approach, we did not find any evidence that CLASP2α can bundle F-actin (Supplemental Figure S2B-D).
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
Furthermore, we found that F-actin also robustly co-localized with microtubules in the presence of GFP-L-TOG2-S (PCC = 0.88 ± 0.04, SD, N = 9 FOVs over 3 independent experimental days) (Figure 1B,D).
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?
Taken together, we conclude that CLASP2α directly crosslinks F-actin to microtubules.
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?
Arpc5
Also ARPC3 disappeared, based on the STRING figure
(a)
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?
In the case of overnight drug treatments for TNT counting and co-culture experiments, cells first adhered for 4–6 hr and were then treated with 50 μM CK-666 or 1 μM IMM-01 for 16–18 hr.
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?
we observed fewer TNTs
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:
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
Following CK-666 addition, we observed an increase of IRSp53 fluorescence at the plasma membrane
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.
. (e)
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.
Eps8 and IRSp53 are recruited to form longer protrusions upon Arp2/3 inhibition.
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
cytoplasmically connect cells
Will a TNT fuse to another cell's TNT or will it attach directly to the membrane?
Here, we present AlphaPulldown, a Python package that streamlines protein-protein interaction screens and high-throughput modelling of higher-order oligomers using AlphaFold-Multimer.
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.