- Aug 2024
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www.biorxiv.org www.biorxiv.org
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β-tubulin
Curious if you have data from other markers ...are these MTs acetylated? Are there motor proteins in this compartment?
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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You tested the ASD and CHD and ASD-CHD genes to identify those that impact survival or proliferation of NPCs in either direction. I was curious about the breakdown of genes based on direction of phenotype (increased vs decreased proliferation, increased vs decreased survival). For example, knock down of cilia-promoting genes (or different ones) having a positive impact on proliferation given the need for centrosomal networks being required for both cell division and ciliogenesis (so these being somewhat mutually exclusive).
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- Jul 2024
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www.biorxiv.org www.biorxiv.org
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One interesting use case would be to be able to detect cells on the pathway to growth arrest or cell death before a dramatic cellular phenotype as a way to evaluate phenotypes of only the "healthy" cells (if that is even a thing given we don't traditionally assay for more than a few dimensions). That said, even with the dramatic cellular phenotype, the shift is minor so perhaps early signals of poor cell health would not be detectable?
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www.biorxiv.org www.biorxiv.org
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T. brucei filamentous actin has not been visualised by phalloidin binding
I wonder if you’ve tried this in your hands or done so recently with phalloidin conjugated to brighter and more photostable fluorophores like Atto dyes. Even if filaments are short and dynamic, they may be concentrated here sufficiently to detect with phalloidin under appropriate conditions. I mention it because we were ultimately able to get this to work in alga for detecting an actin with even less homology to metazoan actin than trypanosomes when sufficiently concentrated (figure 7 of https://doi.org/10.1091%2Fmbc.E19-03-0141)
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- May 2024
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www.biorxiv.org www.biorxiv.org
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Super interesting result, particularly the clear demonstration that the site of mechanical deciliation is proximal to the transition zone.
One thing I’d be careful about is the claim that delay in regeneration is due to the need for protein synthesis of B9d1 on the basis of antibody staining with chx treatment. While it’s clear from figure 4 that this TZ component does increase its expression upon deciliation, antibody staining is likely insufficiently sensitive to make a claim of this protein being limiting for ciliary assembly in the precursor pool.
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www.biorxiv.org www.biorxiv.org
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Seriously this is such an incredibly cool result to be able to drive actin polymerization by a random protein that forms condensates when fused to an actin binding peptide! The implications for what might happen inside cells is wild!
Have you done any pyrene or seeded pyrene assays with your eps15-lifeact to see effects on actin nucleation or elongation?
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- Feb 2024
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52.53.155.43 52.53.155.43
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Super interesting results/model! Years ago, we saw nascent proteins near basal bodies (using a puromycin analog to label as well) in Chlamy, which doesn’t have a clear PCM-1 ortholog/centiolar satellites. So according to this, where there is protein synthesis at satellites whether they are (vertebrates) or are not (flies) near centrosomes/cilia, maybe there is different, satellite-free local protein synthesis at cilia/centrioles of a different pool of mRNAs. Or maybe your waystation hypothesis is correct rather than protein synthesis actually happening at satellites directly.
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- Jan 2024
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www.biorxiv.org www.biorxiv.org
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PTX failed to prevent the hyperosmotic shock-induced loss of primary cilia at any PTX concentration we tested
What is the percentage of ciliated cells and cilia length with PTX treatment in the iso-osmotic conditions? If hyperosmotic cilium changes are accompanied by excess MT formation, prevented by MT depolymerization, and both hyperosmotic and PTX treated cells promote cytoplasmic MT bundling/polymerization, what is the effect of PTX-associated MT polymerization (without hyperosmotic shock) on cilium frequency and length, and ODF2-positive cells under these experimental conditions? This would tell you if the excessive microtubules alone vs other factors caused by hyperosmotic shock were drivers of ciliary shortening/loss.
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- Dec 2023
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www.biorxiv.org www.biorxiv.org
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Treatment of HS for 2 hours is sufficient to severely decrease the number of ciliated cells but not the length of the remaining ciliated cells
Curious if the number of cells between pre- and post- heat shock is also decreased? It's clear that the the percentage of ciliated cells is decreased with or without TNF treatment but if there is a lot of concomitant cell death, the decrease in ciliated cell percentage could be tied to overall decreased cell health or other factors that are not cilia specific.
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- Nov 2023
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www.biorxiv.org www.biorxiv.org
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SMIFH2
It's less critical in this case since you see no appreciable effect, but just worth noting as you use this drug in the future that the concentration used is quite high, above the level known to inhibit myosin 2, not just formins (ref: PMC8121067)
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- Oct 2023
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www.biorxiv.org www.biorxiv.org
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The centrin antibody also stained fibrous structures which extend from the basal bodies towards both the cell centre and the cell cortex
This filamentous network of centric polymers is also seen in Chlamydomonas and forms a cage like structure around the nucleus. Curious if you ever see centrin filaments around the nucleus dinoflagellates or changes in the filamentous organization in response to calcium. While there have been many hypotheses about the nature and function of these central extending filaments and the function of calcium-mediated centrin filament contraction, to my knowledge, this hasn't been really settled.
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- Sep 2023
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www.biorxiv.org www.biorxiv.org
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Looks like your PYP ortholog detection started with a sequence identity-based search. Have you also done a protein structure based search, eg via foldseek, to identify potential orthologs that may have low sequence similarly but high structural conservation to uncover additional candidates with beneficial properties?
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- Aug 2023
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www.biorxiv.org www.biorxiv.org
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(Fig. 2D)
should this be 2F and 2G?
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(Fig. 2C)
should this read Fig. 2D, or 2E where this is quantified?
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This is a super interesting set of results, particularly the localization inside mitos! Do you think that the mitochondrial phenotype in PFN1 knockouts is due to spontaneous nucleation of actin filaments within mitos? Also I may have missed if you checked this but do you think the decreased f-actin in PFN1 KOs as shown in figure 4H is from degradation of actin as seen in this other system (https://doi.org/10.1073/pnas.1721935115) or is that already known for these PFN1 KOs in these cells?
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www.biorxiv.org www.biorxiv.org
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We generated a version of AK7 lacking the DPY30 domain and found that the ciliary localization is dramatically reduced, suggesting that this domain is important for its ciliary localization
Would be interesting to also have a control that lacks a different domain (the kinase, LID, or NMP domains) to see if the DPY30 domain containing deletion mutants can still localize to cilia to exclude the possibility of global misfolding, or other regions being responsible for ciliary localization.
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www.biorxiv.org www.biorxiv.org
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Our results strongly support that TBCCD1 loss of function compromised the symmetry of the MT centrosomal aster, causing both a dispersion of satellites in the cytoplasm, and a concomitant decrease in their pericentrosomal localization, supporting the previous evidence that the number of MTs anchored to the centrosome is lower in these cells.
I think this conclusion is the most difficult to support from the data shown in figure 1 a-c and B. The images seem of insufficient resolution in a-c to claim unanchored microtubules along with subtle differences in satellite distribution based on mean distance or percentage within a window.
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we show that these aggregates were in a distinct plane to that of the two centrioles that are separated
Seems like the radial pattern of control MTs may just be exacerbated due to physical/constraint proximity to the nucleus rather than a particular directional growth. It would also be be helpful to see cell morphology via brightfield/other in these images to see what the other membrane or cellular features might contribute to this polarization.
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- Jun 2023
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www.biorxiv.org www.biorxiv.org
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Thanks for sharing this very interesting and useful study! The ability to simultaneously visualize different actin isoforms with reduced effects on endogenous dynamics is fantastic and will no doubt lead to future discovery of differential functions.
The pitfalls of N-term actin tagging are well documented as you note, so strategies that allow for faithful binding to endogenous nucleators would indeed be beneficial. However, the preferred internal 229/230 tag still shows no greater co-localization (and perhaps reduced co-localization, as I am unsure of the statistical difference in figure 1C) with total f-actin/phalloidin staining relative to N-terminal tagging. This suggests that there are indeed additional effects of the internal tag on dynamics (likely driven by affected ABP binding) despite largely not identifying those defects in your assays. I would have also therefore have been interested to see the N-term tagged control for figure 3 alongside the internal tags. This control wouldn’t be quantitatively comparable of course but I can’t remember if formin binding is affected for n-term tagged actins or just getting through the formin ring.
Regardless, I’ll emphasize the importance of additional tools and information such as what you present here. The extensive interactions of actin with hundreds of binding proteins with myriad functions throughout cells highlights extensive combinatorial complexity that benefits from the availability of a full suite of actin labels so that the right labeling strategy can be selected based on application. This is therefore a very welcome addition to that suite of strategies!
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www.biorxiv.org www.biorxiv.org
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Reassessment of exosome biogenesis
I think the cytoplasm/extracellular facing portion of receptors depicted in Figure 7 don't seem to preserve topology? For example if you follow the purple receptor, the terminal yellow tail is sometimes cytoplasmic facing, sometimes extracellular/lumen facing. I only mention it because made the trafficking dynamics in the diagram a bit unclear.
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- Apr 2023
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www.biorxiv.org www.biorxiv.org
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(Extended data Fig. 10)
I think this should say Extended data Fig. 9
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Interestingly, visualization of actin dynamics in Chlamydomonas using Lifeact-NeonGreen, showed a similar response to osmotic stress with an increase in skewness
The control image here looks quite a bit different than typical actin localization in a wild-type cell (with a more prominent localization to what I am assuming is the cell apex relative to mid-cell actin). I'm wondering if this is specific to this/a cw strain or whether this is a non-representative image. Also curious what the larger number of cells looks like in NaCl as skewness is measured but the clear phenotype from this image appears to be an increase in actin bundling, just as in Arabidopsis. If this is representative, is there an increase in actin bundler expression or expression of formins in your Chlamy dataset? Lastly, for visualization, fixed cell phalloidin staining of these cells might give you better resolution to identify the specific phenotypic consequences of osmotic stress with respect to the cytoskeletal rearrangement.
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Early cellular responses to osmotic stress involve shifts in the balance between endocytosis and exocytosis of proteins important for growth, such as cellulose synthase complexes
Using lifeact in this particular Chlamy strain, do you typically see apical actin puncta in untreated cells (which we think are endocytic pits, see https://doi.org/10.1101/2020.11.24.396002) and if so, are these quantifiably different in NaCl?
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www.biorxiv.org www.biorxiv.org
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Competition between actin binding proteins for stabilizing different actin populations is a super interesting phenomenon. I always imagined this being due to some actin binding proteins acting as a sink for monomers dropping local actin concentration available for other ABPs or sequestering monomers to other spatial domains (something we’ve observed in other systems). However, I found it really interesting that an actin bundler forming presumably linear actin tunneling nanotubes that seems to be antagonistic to branched actin formation by ARP2/3 directly binds components of the ARP2/3 complex. Do you imagine that the higher branching ARP2/3 complex isoforms are also sequestered from the rest of the complex where lamellipodia would form? Are you able to localize those Eps8-interacting ARP2/3 components to nanotubes?
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www.biorxiv.org www.biorxiv.org
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It looks like each iteration involves permeabilization. How necessary is this step in each iteration rather than simply rounds of primary and secondary antibodies with washes in between?
Also we’ve found detection of actin with fluorescent phalloidin to require improved signal and reduced background, particularly in photosynthetic cells with a great deal of chlorophyll autofluorescence. This requires 1) low concentrations, 2) reduced incubation time, and 3) brighter fluorophore such as Atto compared to Alexa dyes. For your antibodies, you mention low concentrations also result in low signal, so this is suboptimal so I’m still wondering if we could improve phalloidin staining for difficult samples with iteration. I know you don’t find similar improvements in phalloidin staining with iteration but I’m wondering if your phalloidin staining protocol had been optimized as much as you’ve optimized the antibody concentrations for your 1X designations to minimize signal to noise before performing phalloidin iterations to increase signal. Further, it could be that the absolute target abundance matters for the changes in signal to noise ratio with iteration. So by comparing iteration for low abundance antibody targets with iteration for high abundance non-antibody labeling, you may not see comparable improvements for a non-antibody label. If you were to use this protocol on cells where the f-actin signal is harder to detect than in these cells, might you see improvements with iterative labeling? Or alternatively if you compared antibody iteration with a non-antibody label against a target of lower abundance in the same cells, perhaps the signal to noise difference would change more with iteration? It would be of general interest if iterative staining could also improve signal to noise for low signal or low abundance non-antibody labels.
Thanks for publishing this interesting study!
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Abstract
Re: figure 4 and the associated claim, is acetylated alpha-tubulin normalized by the total alpha tubulin intensity less? Or is it proportional to reduced microtubes generally in vim-/- MEFs? Given PCM defects and impaired re-assembly of mictotubules after nocodazole treatment, basal turnover and the total mictotubule network may also be affected in vim-/- MEFs, but if I’m not mistaken, that’s never quantified here. Depending on the normalized outcome, the reduced acetylation rates may be independently reliant upon vimentin or be a consequence of globally reduced microtubules.
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www.biorxiv.org www.biorxiv.org
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This is an incredibly interesting and novel result identifying a role for the CEP290 ciliary transition zone protein in cytoplasmic microtubule dynamics and adhesion. One immediate question given the work is done in IMCD3 cells that are competent to form cilia is whether there is expression and a role for CEP290 in cells that are not, such as Jurkat T cells that express many ciliary proteins for use in the immunological synapse but never form a cilium. Those are suspension cells so the adhesion phenotypes may not be evident, but given the more generalized role in microtubule dynamics shown here, I wonder whether CEP290 is expressed in this cell type, when during the cell cycle it might be expressed in a cilium-incompetent cell type such as this, and if there are phenotypes associated in its knockdown in such a context. The last certainly is out of scope of the current work but perhaps existing publicly available datasets would contain the information about expression and cell cycle-associated (and therefore microtubule reorganization associated) timing of CEP290 expression in truly cilium-independent context.
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This is an incredibly interesting and novel result identifying a role for the CEP290 ciliary transition zone protein in cytoplasmic microtubule dynamics and adhesion. One immediate question given the work is done in IMCD3 cells that are competent to form cilia is whether there is expression and a role for CEP290 in cells that are not, such as Jurkat T cells that express many ciliary proteins for use in the immunological synapse but never form a cilium. Those are suspension cells so the adhesion phenotypes may not be evident, but given the more generalized role in microtubule dynamics shown here, I wonder whether CEP290 is expressed in this cell type, when during the cell cycle it might be expressed in a cilium-incompetent cell type such as this, and if there are phenotypes associated in its knockdown in such a context. The last certainly is out of scope of the current work but perhaps existing publicly available datasets would contain the information about expression and cell cycle-associated (and therefore microtubule reorganization associated) timing of CEP290 expression in truly cilium-independent context.
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- Feb 2023
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Abstract
Re: figure 4 and the associated claim, is acetylated alpha-tubulin normalized by the total alpha tubulin intensity less? Or is it proportional to reduced microtubes generally in vim-/- MEFs? Given PCM defects and impaired re-assembly of mictotubules after nocodazole treatment, basal turnover and the total mictotubule network may also be affected in vim-/- MEFs, but if I’m not mistaken, that’s never quantified here. Depending on the normalized outcome, the reduced acetylation rates may be independently reliant upon vimentin or be a consequence of globally reduced microtubules.
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- Dec 2022
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www.biorxiv.org www.biorxiv.org
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Early cellular responses to osmotic stress involve shifts in the balance between endocytosis and exocytosis of proteins important for growth, such as cellulose synthase complexes
Using lifeact in this particular Chlamy strain, do you typically see apical actin puncta in untreated cells (which we think are endocytic pits, see https://doi.org/10.1101/2020.11.24.396002) and if so, are these quantifiably different in NaCl?
-
Interestingly, visualization of actin dynamics in Chlamydomonas using Lifeact-NeonGreen, showed a similar response to osmotic stress with an increase in skewness
The control image here looks quite a bit different than typical actin localization in a wild-type cell (with a more prominent localization to what I am assuming is the cell apex relative to mid-cell actin). I'm wondering if this is specific to this/a cw strain or whether this is a non-representative image. Also curious what the larger number of cells looks like in NaCl as skewness is measured but the clear phenotype from this image appears to be an increase in actin bundling, just as in Arabidopsis. If this is representative, is there an increase in actin bundler expression or expression of formins in your Chlamy dataset? Lastly, for visualization, fixed cell phalloidin staining of these cells might give you better resolution to identify the specific phenotypic consequences of osmotic stress with respect to the cytoskeletal rearrangement.
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(Extended data Fig. 10)
I think this should say Extended data Fig. 9
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- Oct 2022
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www.biorxiv.org www.biorxiv.org
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It looks like each iteration involves permeabilization. How necessary is this step in each iteration rather than simply rounds of primary and secondary antibodies with washes in between?
Also we’ve found detection of actin with fluorescent phalloidin to require improved signal and reduced background, particularly in photosynthetic cells with a great deal of chlorophyll autofluorescence. This requires 1) low concentrations, 2) reduced incubation time, and 3) brighter fluorophore such as Atto compared to Alexa dyes. For your antibodies, you mention low concentrations also result in low signal, so this is suboptimal so I’m still wondering if we could improve phalloidin staining for difficult samples with iteration. I know you don’t find similar improvements in phalloidin staining with iteration but I’m wondering if your phalloidin staining protocol had been optimized as much as you’ve optimized the antibody concentrations for your 1X designations to minimize signal to noise before performing phalloidin iterations to increase signal. Further, it could be that the absolute target abundance matters for the changes in signal to noise ratio with iteration. So by comparing iteration for low abundance antibody targets with iteration for high abundance non-antibody labeling, you may not see comparable improvements for a non-antibody label. If you were to use this protocol on cells where the f-actin signal is harder to detect than in these cells, might you see improvements with iterative labeling? Or alternatively if you compared antibody iteration with a non-antibody label against a target of lower abundance in the same cells, perhaps the signal to noise difference would change more with iteration? It would be of general interest if iterative staining could also improve signal to noise for low signal or low abundance non-antibody labels.
Thanks for publishing this interesting study!
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- Sep 2022
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www.biorxiv.org www.biorxiv.org
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Competition between actin binding proteins for stabilizing different actin populations is a super interesting phenomenon. I always imagined this being due to some actin binding proteins acting as a sink for monomers dropping local actin concentration available for other ABPs or sequestering monomers to other spatial domains (something we’ve observed in other systems). However, I found it really interesting that an actin bundler forming presumably linear actin tunneling nanotubes that seems to be antagonistic to branched actin formation by ARP2/3 directly binds components of the ARP2/3 complex. Do you imagine that the higher branching ARP2/3 complex isoforms are also sequestered from the rest of the complex where lamellipodia would form? Are you able to localize those Eps8-interacting ARP2/3 components to nanotubes?
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