2.3 Lysostilbene-4 selectively disrupts lysosomes and induces irreversible lysosomal damage
Lysostilbene-4 is really impressive compared to either parent compound. Have you thought about testing a CQ + DHS co-treatment, just to underline that the benefit comes specifically from conjugation
Caffeine rescued this early developmental defect in a dose-dependent manner (Figure 5E),
Very cool finding and clear recovery! Did the caffeine have any impact on the WT strain?
Alignment of DdADGF with human ADA2 yielded a root mean square deviation (RMSD) value of 1 Å, indicating strong structural similarity (Eidhammer et al., 2000; Koehl, 2001).
Very cool! Our in-house analysis comparing the predicted protein physiochemical properties corrected for phylogenetic distance indicates that DdADGF has essentially the same trait distance from human ADA2 as the Chimp ADA2! Great choice of a system to study ADA2!
Data: https://zoogle.arcadiascience.com/search?gene=ADA2-Q9NZK5
Paper: https://research.arcadiascience.com/pub/result-evolutionary-organismal-selection/release/4/
To evaluate the impact of these antioxidants, E. huxleyi algae were cultured both axenically and in co-culture with P. inhibens bacteria, and algal survival was monitored (Fig. 2).
Very cool and interesting findings! The selectivity of selenium protection is striking - it's fascinating that only selenium prevented algal death while ascorbate, DMSP, and α-tocopherol were all ineffective. I noticed there's quite a concentration disparity in the screen: selenium at 10 nM versus the others at 20 μM. This actually makes selenium's effectiveness even more remarkable, but do you think it would still be effective at 20 µM concentrations? or would there be diminishing returns and increased toxicity? It would be interesting to see dose-response curves for each antioxidant to confirm whether this represents true mechanistic selectivity versus concentration optimization differences. Also intriguing that selenium protects against ROS in axenic cultures too - suggesting it strengthens the algae's intrinsic antioxidant capacity independent of bacterial stress. Do you think there selenium would protect against chemically induced ROS? and given the naturally occuring ROS developed as the culture ages, do you think selenium supplementation would increase the lifespan of a culture of E. huxleyi before crashing out?
Very cool work and a really neat finding! Thanks for sharing!
Chlamydomonas cells were stained with the acidotropic dye Lysotracker DND-189 (Thermo Fisher), which was added to cells at 1 µM final concentration.
I can't find any lysotracker data in the paper and it looks like it didn't make it into the published article. Was the staining inconclusive?
Thanks!
Discussion
Thank you for developing what appears to be a really useful set of tools! I appreciate how you've made the techniques accessible and reproducible. The 96-well format and automated analysis should make it relatively straightforward for other researchers to adopt these methods.
I'm particularly grateful for your commitment to open science - making both your code and data freely available on GitHub is exactly what the research community needs more of. This transparency will help other labs build on your work and get the most value from your efforts.
Thank you for the thorough work and for sharing it so openly with the community!
45 media formulations (Table S2) had high absorbance (≥2.0) by SRB assay for all replicate wells at 48-72h of incubation (Fig S4), with four media leading to mean absorbance values ≥2.5 at 72 hours of incubation (Fig 1A).
It would be helpful to add a short line here like "..indicating more successful encystment"
ll FEM
I know you define this later in the results section, but it would be helpful to define it as encystment media here as well. As a reader with no direct experience with A. castellanii, the experimental design was confusing without the context.
Discussion
Really cool work — I appreciated the clear quantitative approach to comparing subcellular allocation in sperm of different sizes. One thing that stood out was the interpretation that increased mitochondrial number and area in C. macrosperma reflect elevated energetic demands. That makes intuitive sense, but I wonder if there’s an opportunity in future work to more directly test mitochondrial function and energy production.
For example, live imaging with fluorescent reporters could help quantify ATP levels or assess mitochondrial membrane potential. Coupling that with fluorescent or DIC imaging of active sperm crawling post-mating could reveal whether larger sperm are more motile, or adhere more strongly, in line with the energetic investment hypothesis.
I realize that TEM gives you high-resolution structure but not dynamics — so integrating these kinds of live-cell tools seems like a natural next step. Thanks for sharing this neat paper! These are cool worms!
ChatGPT was used to streamline thoughts
Conclusion
Congratulations on this exciting result of isolating multiple mutants with enhanced heterotrophic growth through random UV mutagenesis. I'm particularly curious about how these mutants would perform under mixotrophic conditions compared to the wild type. While your approach of developing chlorophyll-deficient Chlorella strains certainly streamlines the culture-to-food pipeline, it would be valuable to understand whether the growth advantages persist across different cultivation modes. If there's substantial growth reduction compared to wild type performance in mixotrophic or photoautotrophic settings, perhaps post-harvest photobleaching methods of a faster growing WT strain could offer complementary efficiency benefits. I'd be especially interested in future genetic characterization to identify which specific genes have been disrupted in these promising mutants.
Very exciting and thanks for sharing the work openly!
One of the major benefits of UV mutagenesis, in76comparison to chemical methods, is that UV mutagenesis is random (Trovão et al., 2022).77.CC-BY 4.0 International licenseavailable under awas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeThe copyright holder for this preprint (whichthis version posted April 30, 2025.;https://doi.org/10.1101/2025.04.28.651037doi:bioRxiv preprint
While it is true that UV mutagenesis is random, there are many methods of chemical mutagenesis that are also random. Chemical mutagens like EMS, MMS, and sodium azide all produce stochastic genetic alterations throughout the genome. The statement suggesting randomness as a distinctive benefit of UV over chemical methods is not entirely accurate, as both approaches fundamentally create unpredictable mutations.
Unlike other methods that introduce68external genetic material into the target cell, it does not produce genetically modified69organisms (Bleisch et al., 2022; Kanakdande et al., 2021; Trovão et al., 2022).
The statement that random mutagenesis 'does not produce genetically modified organisms' seems misleading. While certain regulatory frameworks may classify these differently than transgenic methods, random UV mutagenesis fundamentally alters genetic material. This regulatory distinction appears based more on historical precedent than biological reality. For scientific accuracy, perhaps acknowledging these as genetically modified organisms that fall under different regulatory classifications would be more appropriate.
In this study, we identified MpCAFA, a protein that functions in spermatozoid motility. Our results
Very cool work on MpCAFA's role in liverwort spermatazoid motility! This is a really neat and mysterious protein. It will be very interesting to see if both CAPS and FAP115 domains are both essential in contributing the ciliary rigidity. Nice work!
While the swimmingdirection of most wild-type spermatozoids was predominantly straight, Mpcafage spermatozoids frequentlyexhibited circular trajectories or increased swing width (Fig. 7a). We examined two aspects of spermatozoidswimming behavior: swimming speed and linearity (see Materials and Methods).
Wow! The motility phenotype is very striking! It appears the WT and mutant spermatozoids have a similar number of cells in the field of view but the m-Citrine recovery strain has a lot more cells in the background. Were the sample densities normalized before imaging? You mentioned they perform chemotaxis so I'm curious if the recovery could be influenced by cell density. Regardless, it is a very clear and convincing recovery
Morphologicallynormal spermatozoids were released from all male Mpcafage plants upon the application of water to theirreceptacles (Fig. 6ab), and the cilia of spermatozoids from Mpcafage retained the typical 9+2 arrangement (Fig.6cd), indicating that MpCAFA is not essential for normal spermatozoid development.
I appreciate how you've shown that the mutant spermatozoids maintain normal morphology and axonemal structure despite their motility defects. I'm curious about the sample size that informed this conclusion - approximately how many spermatozoids did you analyze by phase-contrast microscopy, and how many axonemal cross-sections were examined by TEM? This information would help contextualize the robustness of the structural assessment, particularly since subtle ultrastructural differences might potentially contribute to the observed swimming phenotype.
This is a neat paper with impressive work! I'm curious about the dimerization claims though. Throughout the paper, you discuss "SPIN90 dimers," but all experiments use the SPIN90-C truncation (residues 274-722). Have you determined whether full-length SPIN90 also dimerizes?
The N-terminal domains could potentially prevent dimerization in vivo- perhaps by preventing it until binding partners are present, or through intramolecular interactions that affect the dimerization interface.
Your bidirectional actin nucleation model is very compelling, but some experiments with the full-length protein would significantly strengthen the biological implications of this interesting discovery. Although, I'm not sure if full-length SPIN90 purification is feasible.
Thanks again for sharing this great work!!!
Treatment of M. corti larvae with 10 μM colchicine for six hours did not result in any discernible effects on tegumental microtubules, and the microtubules of the distal tegument only disappeared when larvae were treated with 100 μM colchicine (Fig 4A).
Are the microtriches impacted by these drugs?
Exposure to ABZ or ABZSO resulted in the complete loss of microtubules in the distal tegument (Fig 4A,B), while no effect was observed in solvent controls with dimethyl sulfoxide (DMSO).
In the representative images (Fig 4A), it looks like the drug treatments reduce the length of the distal tegument; however, in 4D, the ABZ treatment seems to have increased the size.
I know y'all mentioned that distal tegument thickness can range from 5-11 µm across the body, but I think it'd be helpful to have some quantification to indicate if the MT structure is necessary for dt structure/size. *
this article presents an optimized electroporation method for high efficiency transformation of P. tricornutum, an emerging diatom chassis for algal synthetic biology (Supplementary Fig. S7)
Thank you for assembling this clear guide to an efficient and easier way to do Phaeodactylum transformations! We're so excited to see that the alcalase-induced protoplast method is reproducible and being used in a really important way! Super excited to try out these methods in our lab! Thank you for sharing!
Following electroporation, half of the total reaction was plated on ¼-salt L1 plates supplemented with 100 µg/ml nourseothricin. (d)
It seems the 1/2 salt L1 plates were previously used to allow for bacterial conjugation, but it is less clear why reduced salt (1/2 or 1/4) plates were used in these electroporation experiments?
Is it because of the protoplast's sensitivity to osmotic stress? or do you used reduced salt in the solid media plates to prevent the crashing out/precipitation that frequently occurs when making L1 agar plates?
I'm essentially wondering if the low salt is addressing a physiological concern for the cells or a technical issue in the lab.
Microaga
Spelling Error in the title
. For dark autotrophic cells of G. sulphuraria, an almost twofold smooth decrease in the rate of O2 uptake on the 4-th day of cultivation is evident due to depletion of respiratory substrates in the absence of photosynthesis (Fig. 5).
ok, so the increased oxygen uptake in day two with ethanol is not because of increased cell growth since the ethanol didn't impact cell density in the dark, right? very interesting!
This is very neat work! Thank you for sharing it
G
This is a very convincing figure and consistent with the increased cell density at day 4 when treated with ethanol! Very cool!
Thus, the non-usage of ethanol by G. sulphuraria cells in the dark and the consumption of ethyl alcohol from the culture medium in the light correlate with the data on additional light-induced cell growth
did y'all do any statistics on this ethanol loss dataset?
. Microscopic control showed that the cells in both dark cultures remained viable, i.e. in a state of survival, throughout the duration of the experiment.
Where there any notable morphology phenotypes observed? Also, I didn't see any mention of microscopy in the methods. What sort of imaging did you do?
rowth
minor spelling error
disoder
minor spelling error
No statistically significant changes in growth were also observed in the dark in the presence of ethanol, as opposed to light
So there was no statistical difference in the growth rate of normal cultures in light vs ethanol-treated cultures in the dark? The abstract/intro seemed to say that ethanol had no impact on dark cultures, correct? So there is no statistical difference in the growth rate of G. sulphuraria in normal media in the light vs normal media in the dark?
Is the legend saying Curve 3 represents dark cultures in normal media AND dark cultures with ethanol? If not, why is there no curve for the dark growth WITHOUT ethanol?
However, when amoebas were familiarized to Variovorax boronicumulans (Vb), they preferred all the novel bacteria over the familiar bacterium (Vb) (Estimated marginal means = -0.18, 95% CI (-0.32, -0.04), p = 0.01).
This is very interesting! Especially given their high growth rate when fed on Vb in Fig 2! Very cool paper and really neat work! Thank you for sharing it!
o account for day-specific effects (Figure S1), we scaled all amoeba movement estimated on a given day by dividing it by net amoeba movement towards Klebsiella pneumoniae (averaged across 3 replicates) on that day.
Wow! The fourth day of experiments had wildly different effects! Do you have any idea why things were so different on this day?
(2 g glucose (Fisher Scientific), 2 g BactoPeptone (Oxoid), 2 g yeast extract (Oxoid), 0.2 g MgSO4 (Fisher Scientific), 1.9 g KH2PO4 (Sigma-Aldrich), 1 g K2HPO4 (Fisher Scientific), 15g agar (Sigma-Aldrich))
I'm guessing these measurements are per Liter of medium, correct?
For some bacterial species where the front was hard to see, we collected amoebas from the entire plate after ∼ 36 - 40 hours. We suspended the amoebas in KK2 buffer and centrifuged them at 300g, 10 °C for 3 minutes. We washed the amoeba pellet three more times to remove any bacteria.
How did you confirm axenic cultures at this point? Did they go through filtration as well as the centrifugation?
We used ImageJ version 1.53 (Schneider et al. 2012) to estimate number of amoebas that moved out of the amoeba spot towards each of the two potential attractants.
The depiction in Figure 1 is extremely helpful in understanding the assay! As a reader with no experience, it would also be helpful to see an example raw image. Reading the paper i was wondering if the ameoba crawl up or down as frequently as they crawl towards the KK2 or if the KK2 has its own chemo-attraction. Seeing the raw image could clear this up a bit
We found no significant relationship between these two measures suggesting that these patterns are not driven by chemokinesis (Figure S4: MCMCglmm, Fixed effect posterior mean = 0.04, 95% CI (-0.28, 0.10), pMCMC = 0.24).
This calculation was taking all data points into account, yes? But its possible that they are performing some chemokinesis in certain environments (specifically Ar), right? Just as they seem be chemostatic in some environments (Aa, Cr, Mm, Ba, Nc, Ra). It would be interesting to the breakdown by species
The bacteria for the experiments were grown on LB (Fisher Scientific) or SM/5 plates (2 g glucose (Fisher Scientific), 2 g BactoPeptone (Oxoid), 2 g yeast extract (Oxoid), 0.2 g MgSO4 (Fisher Scientific), 1.9 g KH2PO4 (Sigma-Aldrich), 1 g K2HPO4 (Fisher Scientific), 15g agar (Sigma-Aldrich)), and resuspended to a concentration of OD600 5 in KK2 buffer.
It would be helpful to include a column in Table 1 that shows the media each bacteria was grown on to see if the chemicals present in the growth media correlate with the chemotaxis observed
Summary and Discussion
This is some really neat work. This is a wonderful resource for the community! I really appreciated the logic and decision making highlighted throughout the paper leading to an optimal protocol for 4- or 5- color imaging in dicty! Very cool work and beautiful images!!!
We constructed strains expressing Achilles or GFP(S65T) under the control of a prestalk-specific ecmAO promoter
Why did y'all shift from mNeonGreen comparisons to GFP?
These result suggests that despite the low temperature (22 °C) of D. discoideum culture, maturation of Achilles was fast and comparable to that demonstrated in mice
Very neat & exciting finding!!!
22 h was required for a similar percentage of Dox-GFP(S65T)
Is there any data comparing Dox-GFP with a Dox-mNeonGreen FP? If so, are the two probes comparable in their response times?
Using the same microscopy setup as above, in vegetative cells, PHAkt-Achilles and PHAkt-mNeonGreen fluorescence appeared localized in the pinocytic cups (Fig. 2A),
Was the cytoplasmic background also brighter here (not just the localized signal)? I'm just naively wondering if the achilles tag is causing some stress that is increasing background autofluorescence in the cell (given the onset of uniform cytoplasmic fluorescence in Fig 1).
In the slug stage, Achilles fluorescence remained bright in the majority of cells and appeared uniform in the cytosol (Fig. 1C and D), whereas very few cells showed mNeonGreen fluorescence (Fig. 1C–E).
Interesting that there is a significant difference in the distribution of mNeonGreen (anterior vs posterior) but not with Achilles. Do you have any ideas why this would be the case? Are there any observable physiological defects caused by either of these FPs?
Although various FP has been utilized in D. discoideum (Table S1
Thank you for compiling this list! It is a very useful resource!
Organisms
This is really neat work highlighting incredible "new" organisms! Now that y'all have established methods for maintenance, will you be (or have you already) deposited these strains in a culture collection center? Thanks for doing this important work!
Both organisms are presumed anaerobes and have documented eukaryotrophy on breviates.
Did y'all try to feed the organisms other prey? I'm curious if the same predation Step 1 methods would apply when other prey are encountered
As with SSF, predation appears to occur in two decoupled steps, although PG may return and devour its prey only seconds following initial contact.
Amazing!!! How long is the gap between SSF predation steps if this one is only seconds?
The breviates appear increasingly tattered and eventually round upon. Upon contact with a detached or partly rounded up cell, SSF (not necessarily the same cell as in the initial encounter) contacts it again and then phagocytoses it (Fig 1J, K)
Fascinating!!!! Did y'all ever see any tattered breviates that weren't contacted again? Does the initial contact kill them?
Swimming cells have a snakey, ‘squishy’ movement.
These are really amazing cells! I'd love to see some dynamic videos to better understand this "snakey, 'squishy'" movement that y'all describe.
Immediately next to the flagellar insertion site is the nucleus (Fig 1D).
Is this cell stained? I don't see any mention of dyes/stains but there seems to be some blue & pink colors in the cytoplasm of this cell. Just curious if this is real pigment being picked up by the DS10 camera.
Conclusion
Thank you for sharing this really interesting paper! The bleaching methods will surely contribute to a lot of future work! Super cool system!!!
han A (Fig. 1 D).
Were there any benefits to the host M. viridis that you observed by having 1 strain of Chlorella instead of the other?
Quantification of symbiotic abilities of algae to M. viridis
Were you able to notice any differences in the appearance of Chlorella strains that were symbionts vs free-living? A recent pre-print showed extensive morphological changes to Chlorella species when they were established as endosymbionts of Paramecium bursaria (https://www.biorxiv.org/content/10.1101/2023.12.22.572971v1)
In addition, the symbiotic abilities of two Chlorella strains are different, though these strains were isolated from the original M. viridis culture.
Interesting! Have y'all tried trying to re-establish the symbiosis with a co-culture of Strain A & Strain B? I'm just wondering if there could be a synergistic benefit of both strains (since strain A was naturally able to compete its way into the host). Further, do you think that a non-Chlorella species of microalgae could become an endosymbiont? I'm wondering if the non-photosynthetic microalgae C. paramecium that you feed them could be a symbiont that isn't recognized because its not easily visible in the cell?
Cryptomonas paramecium (NIES-715) was used as feed for M. viridis.
Are the symbiotic-state cells able to survive without this external food (using Chlorella photosystems for energy)?
We found that the number of Chlorella cells ingested by M. viridis was significantly higher in strains B than A (Fig. 1 D).
If I read correctly, these measurements were taken after 1 hour co-incubation, correct? Were the re-established symbionts present after longer time periods? Is it possible these were ingested as a food source and weren't fully digested yet?
Two Chlorella strains (strain A and B) were isolated from M. viridis culture and grown in 20 mL
Were these free-living in the culture medium? or did you need to rupture the host cell to isolate these?
On the other hand, no chlorophyll fluorescence was observed in M. viridis cells treated with ACN for one week (Fig. 1 A).
Is the ACN actually killing the endosymbiont? or does it cause them to be flushed from the host?
Although eukaryotic species possessing photosymbionts have been found sporadically within phylogenetically distant taxa, such as Opisthokonta and Alveolata, and a limited number of model species have been studied.
Consider removing "Although" since the sentence doesn't continue the thought
Finally, we screened candidate formulations for operational toxicity to UC-1 cultures (i.e., live protists in a concentrated formulation should remain viable and actively moving)
Is this data available anywhere? I'd be interested to see the Colpoda behavior in each treatment.
5.3 Agrochemical Selection
It looks like the Spinosad is ~200x the concentration of chlorantraniliprole. Is this just the recommended concentration for each chemical?
. Truffles may become a model system for the investigation of complex biotic and chemical communications in the near future.
Thank you for sharing this work!!!!
The control group of C. vulgaris was cultivated with dry residue of methanol added, as a solvent of truffle extract. Truffle extracts were not added to the samples.
It could strengthen your argument to include a Yeast Extract control to see if your effects are truffle specific. There have been reports of Yeast extract increasing Chlorella growth rates, but it would be nice to see it in comparison to your data. (J. Algal Biomass Utln. 2018, 9(1): 72-85 ) / (https://www.nature.com/articles/1831404a0)
That being said, its nice to see how variable these effects can be depending on not only the source of the fungal extract, but the year & location of the sources isolation.
The experiment was conducted in three stages. In the first stage, the experimental group included the following extracts: Tuber sp. LPB2020-16, Tuber sp. LPB2020-17, Tuber sp. LPB2021-22, Tuber sp. LPB2021-23, Tuber sp. LPB2021-24, Tuber sp. LPB2021-26, Tuber sp. LPB2021-28, Tuber sp. LPB2021-29, and Tuber sp. LPB2021-27. In the second stage, the experimental group included the following extracts: Tuber sp. LPB2022-58, Tuber sp. LPB2022-59, Tuber sp. LPB2022-61, Tuber sp. LPB2022-67, and Tuber sp. LPB2022-69. In the third stage, the experimental group included the following extracts: Tuber sp. LPB2021-30, Tuber sp. LPB2022-39, Tuber sp. LPB2022-42, and Tuber sp. LPB2022-47. Each stage had its own control group of C. vulgaris.
You've listed 18 samples here and in table 1, but Figure 1 only shows 15 being used, including LPB2022-47, which could be very interesting to look at given its reported unpleasant scent. I see this data is listed in Table 2 though. Thank you.
To perform the extraction, 0.5 grams of each truffle sample were defrosted and homogenized using a mortar and pestle with methanol added in a ratio of 1:10.
How variable were the truffles in mass? I could image an entire 0.5 g truffle would be more nutrient-rich than a 0.5 g fragment of a 10 g truffle. It would be nice to have the full fruit mass listed in Table 1 to see if there are any relationships here.
To assess the dynamics of the algae growth, we measured the culture optical density on days 2, 4 and 6 of the experiment.
Was this also at 530 nm? Did the cultures look clean? I'm wondering if there were any spores/contaminants that were inside of the tuber that survived the 70% ethanol wash, freezing, and methanol treatment. The low wavelength OD would pick up on bacteria and fungal growth.
Also, Table 1 shows the dynamics of growth of C. vulgaris algae (expressed in % related to control group).
It looks like this should be referring to Table 2 instead
To test this, we performed a laboratory experiment using a representative Kordia strain (K. algicida OT1) and an isolate of the Rhodobacteraceae family (Sulfitobacter sp. N5S).
I was just reading another pre-print that showed co-culturing Phaeodactylum with a wild Sulfitobacter sp. isolate increases Phaeodactylum growth rate!
Not sure if the same mechanism would occur with the strain y'all used but just a fun parallel!
Very neat work! Thank you for sharing this! I'm always looking for ways to speed up my Phaeo growth!
The CCAP lists strain 1055/1 as 'contains bacteria'. In our lab it always looks clean on F/2 but when we grow it on more rich medium (F/2 + Peptone) tons of contaminants are present. Did you all isolate the axenic strain through antibiotics, streaking to single colonies, or some other means?
Further, did the axenic control include the addition of Peptone + Yeast Extract Seawater? or was it a "nothing added" type control?
Were you also able to get a cell count for the bacteria when co-cultured with Phaeo in F/2? I'm wondering if a strain like NFXS29 had better growth and thats why the effect is so much stronger.
Again, very neat work and thank you so much for sharing it!
Results and Discussion
I don't know anything about M. conductrix, but does it typically have flagella, an eyespot, and a cell wall (appears to be present in the EM images)?
I'm wondering if these features exist & are lost when they become endosymbionts, like Tetraselmis convoluta when they become symbionts of the acoel Convoluta roscoffenis (10.1111/j.1529-8817.1966.tb04603.x)
). The average number of algal symbionts further dropped at 10 μM (average: 0.5 symbionts / individual; Fig. 3 D), with a significant decrease compared to 5 μM (P = 0.01) and to all the previous concentrations (all P < 0.001; Fig. 3 E).
Do you think the high concentration of BPA is impacting Tetraselmis swimming, preventing them being taken up by the acoels? or that the Acoels are impacted, preventing them from eating?
Further, since they're obligate, do you think the acoel swimming defects and miRNA expression could be impacted by the reduced number of tetraselmis cells in the host body?
Futher, do acoels maintained in high conc. of BPA remain viable through adulthood?
This is a very neat paper taking advantage of a really cool system! Thanks for sharing!!!!
. Moreover, thanks to a newly optimized protocol for whole mount in situ hybridization, we reported for the first time the expression of the pan-neural miRNA, miR-124, in acoels and demonstrated that BPA disrupts neural development in S. roscoffensis. Similar effects have been already documented in other marine invertebrates.
This is very cool! But since you also show a lack of uptake of Tetraselmis, do you think there could be an issue with the 5 µM BPA treated acoels not taking up the miRNA? I think it would be useful to add a control miRNA that shows expression of an uneffected pathway is detectable in the 5 µM BPA treated animals.
Merging evolutionary and cell biology will be critical to uncover the evolutionary origins of cellular processes, link the molecular evolution to evolution of cellular phenotypes, and clarify the role of adaptation versus random genetic drift that drives cell level evolution [66].
This is a really beautiful work that answers a lot of important questions about the evolutionary diversity of an essential process, and leads to even more exciting question! Thank you for sharing this work!
These data suggest that the membrane invagination length at the time of scission is different in each species: ∼100 nm in S. cerevisiae, ∼150 nm in S. pombe, and ∼50 nm in U. maydis.
Are there any physiological conditions that correlate with this finding?
Given the similar shape & size of U. maydis and S. pombe, I would have naively guessed that they'd have similar invagination lengths. Also, the short lifetime of endocytic proteins in U. maydis is consistent with shorter invaginations, but it looks like S. cerevisiae patch lifetimes are longer than S. pombe, but this doesn't correlate w/ invagination length.
Does the invagination depth scale with the turgor pressure? Or maybe the growth rate?
S.cerevisiae
minor error: missing space
. However, lifetimes of the orthologs differed across species (Figure 3,
This is a beautiful visualization of the protein dynamics across species! It hammers home how the different modules of the machinery have such different effects across species! Breathtaking!
All the proteins in the three species localized to small puncta at the plasma membrane consistent with a function in endocytosis (Figure 2A).
Beautiful images!
It looks like U. maydis consistently has a lot of cytoplasmic fluorescence, compared to the other yeasts.
Is their cytoplasm auto-fluorescent in untagged cells? If so, is it equally bright when grown in their preferred medium or could the synthetic medium be stressing them out?
or is it just a lot of tagged-protein that isn't localized to endocytic sites.
All strains were cultured at 24°C unless otherwise specified
Was the growth rate of each species similar on the synthetic medium compared to the preferred medium?
(SD)
Is this synthetic medium also supposed to be EMM? Table S2 does not have a recipe for SD medium but indicates S. cerevisiae grows on EMM.
Besides that, autumn-DOM exhibited a higher promotion of cell growth (Fig. 2c, d). Considering the projected warming scenario in the future, we propose a hypothesis that the outburst of HAB event in early autumn is likely to happen in ECS coastal waters, which may contribute to higher frequency of HABs shown in the model simulation (Xiao et al., 2018; Xiao et al., 2019; Zhou et al., 2022).
Thank you for sharing this important work. It seems that there are innumerable variables involved in the frequency of HABs and climate change will undoubtedly impact each of these. It seems these experiments were run at ambient temperature in the lab, correct? I assume the temperature in the estuary is wildly different in the spring & autumn (and throughout the day) which will impact the nutrients available in the DOM sample drastically. The NCMA collection center indicates this species can be maintained from 14-22 C. Have you tried maintaining the strains in the DOM while mimicking the temperatures of the seasonal samples?
In this study, mariculture DOM served as an effective nutrient source promoting the growth of bloom forming species P. donghaiense, the growth rate was elevated 87.2% in comparison with the control group supplied with inorganic nutrients (Fig. 2)
I didn't see this in the text, but are HAB in the Changjiang Esturay more common in the spring or autumn?
Fig. 5
Sorry, the text in this figure (and to a lesser extent fig 6) is very hard to read. The text and image is very pixelated.
4.1 Promoted cell growth by mariculture DOM
Just out of curiousity, how does the growth in DOM compare to growth in standard culture mediums like L1 or F/2?
s.
Will the newly isolated strains be deposited in any collection centers? These are very cool cells!!!
Chlorolobion obtusum Korshikov.
Figure 14 & Fig 15 are swapped
Fig. 6.
It would be nice to have the scale bars on all of the images, especially since there are multiple strains & species
Fig. 1.
The scale bars aren't defined for many of your figures. The first definition comes in Figure 5, despite the earlier figures having scale bars. Very beautiful images though!
Our results show, in accordance to previous works38,51, that the Bathypelagic Ocean is not an isolated environment but it is in continue connection with surface waters and with the land. In this regard the fungus G. minor shows an enormous potential to be an important active part of the global carbon cycle, pointing at the same time to the importance and necessity of more studies both on the Bathypelagic Ocean and on its fungal diversity.
Very fascinating work! I'd never heard about these mysterious fungi before so it was fun to learn about them! Its amazing how these microbes thrive in wildly different environments! I'd be very interested to see if y'all are able to replicate the yeast-like morphology in lab settings by mimicking the marine environment. Dissecting the Atlantic vs Pacific impact further would be very cool for future studies! Thanks for sharing this neat work!
TSA-FISH analysis targeting G. minor was performed on the deepest sample (between 2600 and 4000 m) of 34 stations.
Do you think the morphological distribution would be different when taken from different ocean depths? I imagine the pressure differences could lead to morphological changes/adaptations.
Also, are the yeast forms ever found in apples? or are those organisms most always elongated hyphae?
The elongated morphotypes, present only in Pacific waters, are probably hyphae, sometimes found in chains
I'm a bit confused. A few sentences above you said the yeast-like form never occurs in culture and now that the hyphae form is only in pacific waters. Does this mean neither morphologies are found in cultured cells?
The cultured cells in Fig 1 appear to be elongated hyphae as well? Sorry if I'm misreading things.
Edit: Oh! I see! Are you stating that, when in marine environments, the hyphae are only observed in the Pacific Ocean? In Fig 4 you show that there are no elongated morphotypes in the Atlantic Ocean and they are mostly observed in the Pacific Ocean; however, you do show their presence in the Indian Ocean as well, correct? I think this could be reworded for clarity
and fixed with 100 µL of 1X PHEM 2% PFA at 4°C.
Amazing images in this paper! How long was this fixation step at 4C? Does the cold temp prevent contraction?
Conclusion and Perspectives
Thank you for sharing this impressive piece of work! It is amazing how the entire function and morphology of a cell can change based on its environment! The use of multiple high-resolution techniques all converging on a consistent conclusion is very convincing!
Cell volume was 5.6-fold higher than the free-living stage (43.55 ± 12.97 µm3 vs 7.79 ± 1.94 µm3).
This is very striking and interesting! We noticed a similar phenotype in the species Chlamydomonas smithii when we grew them in nutrient rich marine broth (https://doi.org/10.57844/arcadia-35f0-3e16). The cells became much larger compared to when grown in standard low-nutrient TAP media. Do you think this could have anything to do with increased nutrient availability within the host cell?
liquid medium
Is this the same bacterized volvic natural mineral water supplemented w/ protozoan pellets that the P. bursaria are grown in? or liquid HSM? or different media? just wondering if there are any external nutrients contributing to any of the differences.
Released symbiotic microalgae were recovered after filtration through a 40 µm cell strainer and 10 µm filter that removed host debris. The filtrate was centrifuged (2 min at 2 500 g) and plated on modified solid High Salt Medium (HSM) (Gorman and Levine 1965; Sueoka 1960). Microalgae were maintained at 20°C with a 12:12 h light/dark cycle - light intensity of 40 μE m−2 s−1 - and re-streaked on plates every week.
Interesting! Since the average volume of the symbiotic algae was ~43 um, do you think you're selecting for smaller cells from the start?
For the symbiotic algae in the study, were the isolated M. conductrix cultures re-introduced into vacant P. bursaria cells to correct for any inadvertent size selection?
The most important differences involved the energy-producing organelles, with the volume of the chloroplast and mitochondrion increasing 6.4 and 7.3-fold in symbiotic microalgae, respectivel
In your FIB-SEM, did you notice any differences in the cell wall of the free-living vs symbiotic algae? Has the 5.6-fold larger cell lost the wall, which might not be as necessary anymore since it has a protective host?
Pak1 activity promotes successful internalization of endocytic vesicles
Very cool! Did you see Pak1 at endocytic sites along the side of the cell? If not, were the success rates similar at the side & at the tip of the cell? It would be neat if the Myo1 in the tip patches are phosphorylated and the Myo1 in the side patches aren't. I know there are a lot of other factors that could contribute to differences in side & tip patch internalization (membrane curvature/hardness, crowdedness, etc) but could be interesting to see!
The data from each cell was then analyzed to find the correlation coefficient between fluorescent signals of both ends within the cell
Can you expand on how this analysis is done?
Was there a consistently sized ROI used in each cell? The LatA treated cells seemed to favor CRIB localization on the side of the cell instead of the proper tip so I'm curious what fraction of the image goes into the analysis.
Similar to CK-666 treated cells, myo1Δ mutants showed a decrease in Scd1-mNG levels at the cell ends (Fig.5C, D)
Are these images maximum intensity projections or just single z-planes? If its a max intensity, it looks like scd1 might go to endocytic sites in the absence of Myo1?!
Moreover, in keeping with previous reports, Scd1-mNG levels at the cell ends increases in pak1-ts mutant at permissive temperature (Fig.4A, B)
Oh wow! The nuclear localization of Scd1 in CK-666 goes away in the pak1-ts mutant!!!
In CK-666 treated cells, in addition to decrease in levels of Scd1-mNG, we also observe bipolar localization in only 37% of cells. This suggests that in the absence of branched actin, Scd1 localization is restricted possibly due to the disruption of its oscillation.
Very cool! It also looks like there is more localization of Scd1 in the nucleus in CK-666 treated cells! Is this true across the dataset or just in the representative images?
To this end, Cdc42 oscillations were analyzed in four conditions: DMSO, Lat-A, for3Δ, and CK-666. DMSO treatment served as a control (Fig.1A, B and S1A, B).
I found the S1 figure VERY helpful in understanding these claims. You've got such great signal that it is hard to see the differences the in polar signal intensities through the fluorescent images alone. I think it could be a lot easier to interpret with the temporal quantification from S1 shown in the main figure
Red arrow heads mark the site of Cdc42 activation
Can you provide more context on how you know this is the site of Cdc42 activation when there is dense CRIB-3xGFP localization on both ends of the cell? and in many places theres no visible fluctuation from the earlier cells to the marked cells
solated THATCH dimers displayed a diffusive localization throughout the fission yeast cell, indicating a low affinity to F-actin, similarly to THATCH homologues (Fig. 2b)(44, 45). In contrast, overexpression of THATCH-ΔUSH dimers induced the formation of thick F-actin bundles and large puncta (Fig. 2c)(34)
Very Interesting! Did you ever notice THATCH dimers associated at cables when expressed at endogenous levels? Would this indicate something in the THATCH domain is contributing to patch localization? Do the (1-856) and THATCH fragments co-localize? If so, could the 2 fragments be re-associating (infrequently)?
Abstract
Really nice & convincing work! I really enjoyed reading this paper. Its fascinating how these mutations can have such a broad impact on different parts of the endocytic machinery but with no measurable impact on the accumulation of actin in patches or the internalization of vesicles! Thank you for sharing it on here!
he analysis of Pan1 motility showed that membrane invagination and vesicleformation are not affected by the deletion of Bsp1 (Fig. 3C).
Did you compare the intensity of tagged Pan1? it looks like it might be brighter in the bsp1 mutant compared to WT.
Next we wanted to study if Bsp1 interacts directly with CP.
Its probably worth noting that in Drees 2001 they state "Ypr171w also interacted with Cap1, the α subunit of the yeast actin filament capping protein", but the evidence you provide is definitely much more convincing.
one of our mutants showed complete mislocalization,indicating that the proline-rich N-terminal part of the protein also has a role in the localizationof Bsp1.
Very cool! Did you try to make the 408-470 peptide in vivo as well? Since the inclusion of the WH2-like domain enhances patch localization, it'd be neat to see if the domain is sufficient to recruit the peptide to the patches. Meaning it could be recruited through both the proline-rich region and the WH2-like domain. Or alternatively if the WH2-dependent enhancement is because of a feedback loop of sorts.
Theputative WH2 and CPI sequences together with the localization of Bsp1 actin structuressuggest a role in actin regulation
It could be useful to sight its reported interactions with Bzz1, CAP1, Las17, Lsb3, RSV161, RSV167, Sla1, and YSC84 (several sources logged at yeastgenome.org)
Bsp1 localizes to endocytic actin patches and cytokinetic actin rings (Drees et al., 2001;Wright et al., 2008; Tonikian et al., 2009).
This was also shown in Wicky et al 2003 where they also showed actin patch defects when BSP1 was overexpressed. (https://doi.org/10.1016/S0014-5793(03)00067-X)
.
minor punctuation error
Thereafter, we decided to use the concentration of 20 µg/mL to further evaluate nanoalgosome effects on animal fitness45.
Thank you for running this experiment and reporting the concentration gradient! I was a bit confused why 20 ug/ml was selected for the worms in the last paragraph until reading this. Thank you for making it clear!
2 µg/mL
Is this the same concentration of nanoalgosomes that were used in the colocalization studies? I see in the materials & methods that 2 ug were added to cells in 12-well plates which can usually hold 1-2 mL, so I assume its the same concentration, but the actual concentration is not currently clear. Sorry if I missed it somewhere.
Both fluorescence and confocal images showed that intracellular LAMP-1- and calnexin-positive compartments (green) did not co-localize with internalized PKH26-labeled vesicles, suggesting that lysosomes and ER were not involved in their intracellular trafficking.
In panel A the algosome signal looks pretty ubiquitously dispersed. While the colocalization w/ CD63 is clear, I think the figure could benefit from showing the individual channels like in panel b. Its hard to tell if the algosome signal is excluded at sites of Lamp1 & Calnexin or if its just ubiquitous signal that isn't enriched at these sites.
We measured the growth rate of D. discoideum on 22 species of soil bacteria and the commonly used food bacterium K. pneumoniae.
Thank you for sharing this work as an open access preprint! It is a fun read and I learned a lot!
Each condition has 3 dots representing QS1, QS6, and QS9, correct? Are these strains maintained on K. pneumoniae in the lab, explaining their quickest doubling time on Kp?
Discussion
There was a recent paper that showed differences in growth of a heterotrophic algae when fed different strains of the same genus! It would be interesting to see the variability of Dicty growth when fed different strains of the same species!
D. discoideum do not experience switching costs if they undergo spore formation before the prey switch
While not statistically significant as a whole, it does look like the Fg-Sm & Pv-At effects observed in Figure 3 are conserved after undergoing spore formation. Do you think certain bacterium might induce these genetic changes?
10 OD600 bacterial suspension on starving agar
Were all 22 species of bacteria maintained in the same media? I could imagine different bacterial media components (even though low concentration) could impact Dicty's growth.
Similarly, do all 22 bacteria have similar doubling times? If Kp doubles 2x faster than Po, that could impact why amoeba double quicker on Kp than Po
Thus, it would be interesting to test if D. discoideum amoebas can avoid some concurrent costs by preferentially eating the most profitable prey bacteria first, but if they do, our data show that it is apparently not enough to fully overcome such cost
Interesting! Do you think this picky-eating theory could explain delayed growth? It takes longer to eat because they have to sort through the less-profitable prey to find their favorite meal?
Finally, our work is a proof of methodology that could support marine ecology research and modeling of carbon fluxes. With the development of automated sample preparation methods that separate large algae aggregates from single cells of different sizes, the SMR could be adapted for measurements of algae directly from the ocean. Such future approaches could provide high throughput sinking velocity measurements in situ.
Thank you for sharing this work as an open source preprint! I'm very intrigued by the nutrient-dependent changes in buoyancy and I'm excited to see the future use of this technique!
As the highly elongated diatom Phaeodactylum tricornutum was the only species where sinking velocities were influenced >5% by cell shape (Supplemental dataset S3), all sinking velocities reported in figures are assuming a spherical object.
I love the inclusion of a diverse panel of algal species. The P. tricornutum strain used in this study (CCMP632) was reported to be fusiform 95-100% of the time (DOI: 10.1111/j.1529-8817.2007.00384.x). It'd be interesting to see how buoyancy is impacted by morphology of the same species. In my hands, I always find triradiate cells to be harder to pellet.
Unialgal cultures were grown in filter-sterilized L1 or L1-Si medium, as appropriate for cell type (see table 1).
It would be really cool to see how the presence of silica is impacting the buoyancy. P. tricornutum (which looks like one of the slowest sinkers measured here) can survive without the presence of silica. It would be interesting to see if there are notable differences in P. tricornutum buoyancy in L1-Si compared to standard L1.
Phaeodactylum traceroute
I think this is a typo? According to wikipedia, the only member of the Phaeodactylum genus is P. tricornutum and P. traceroute isn't mentioned again in the text.
Working model of the mechanism for PbE3 to regulate plant immunity.
This is a very thorough study that uses clever techniques to describe the function of a protein from a Rhizarian species that doesn't have developed genetic tools. The data tells a convincing story and supports the claims made in this paper. Thank you for sharing this work as an open access preprint
esides, rd21a was more susceptible to B. cinerea than Col-0 (Fig. 6D)
Was S. sclerotiorum also tested like in the PbE3-2 overexpression plants?
PbE3-2 interacts with the plant cysteine protease RD21A
Figure 4 provides very convincing evidence that pbE3-2 interacts with RD21A in vivo. A minor point would be Fig4E could be improved by displaying the mCherry signal in magenta rather than red for color blind readers who can't distinguish the yellow-green differences in the merge. But very convincing figure!
Data were normalized to ACTIN2 expression in RT-qPCR analys
Do you know that PbE3-2 expression isn't impacting actin expression? If you normalized to 3 random "house-keeping" genes would the data look the same? The data makes sense and fits nicely with the other experiments but actin is highly involved in plant immunity so might not be the best control?
BAX-induced cell death.
It could be helpful to define BAX at some point. It can be difficult for non-plant scientists (myself included) to understand what it means that PbE3-2 confers BAX resistance without having this information readily available.
We found that the mutant strains had a higher frequency of cell death than the UVM4 strain (Figure 4A)
Very interesting and convincing phenotype. Do you think the lack of a cell wall contributes to the amount of cell death? If this experiment were done in cells with cell walls, would you miss out on this observation?
Also, why do you think ii-23 is so much more susceptible to heat than ii-9, since they're essentially the same strain, right?
A Crispr/Cas9 based targeted insertional mutagenesis approach (Picariello et al., 2020) was employed to knockout the CrMCA-II gene.
This was done on the the uvm4 strain, correct? Just confirming since the Picariello et al 2020 paper doesn't use uvm4. Have you found this Crispr/Cas9 protocol to be more effective on the uvm4 strain, compared to the strains used in Picariello et al 2020?
If so, were the CrMCA genes effected by the UV mutagenesis used to generate the uvm4 strain?
PM localization of CrMCA-II-mVenus was confirmed using strain CC-4533, distinguished from UVM4 by the presence of both an intact cell wall and flagella (Supplemental Figure S12), suggesting that this localization is a strain-independent characteristic of Chlamydomonas.
Thank you for including this! Its nice to see such clear membrane localization in a more "normal" cell line with flagella! While this does have a cell wall, its still a cw15 mutant. I'm guessing this was necessary to get sufficient mVenus expression? I'd be super interested to see if this strain (and a fully intact cell wall strain) with MCA-II deletions are able to survive after HS
After transformation into UVM4 cells (Neupert et al., 2009), we obtained a strain overexpressing CrMCA-II with a C-terminal mVenus tag, that we named CrMCA-II-overexpressor 14-3 (OE14-3).
Are these over-expression cells more resistant to HS than WT cells?
n ank1Δ cells, Myo1-GFP was instead localized uniformly along the plasma membrane (PM) (Fig. 1C).
Such an exciting and striking effect!!!! This paper is a lot of fun! Thank you for sharing!
In contrast, in myo1-mNG fim1-mCherry cells overproducing Ank1, while Fim1-mCherry was at patches, almost all Myo1-mNG was cytoplasmic (Fig. 3A).
There are still some faint myo1-mNG puncta visible. Do you think this would go away with higher expression of Ank1? or do you expect there is another mechanism that allows some Myo1 to get to the membrane / evade Ank1?
This suggests that, in addition to inhibiting membrane binding, myosin-1 F-actin binding and/or motor activity may also be inhibited by Ank1/OSTF1.
This is super cool! I love the 3 species inclusion in this paper! I'd be interested to see if Ank1/OSTF1 are interchangeable between species (expressing OSTF1 in Ank1 delta pombe)! Given its small size it might be an easily purifiable myosin "inhibitor"!
In accord, Myo1Δ(1-772)-mNG localized to endocytic patches in wildtype cells but in ank1Δ cells, Myo1Δ(1-772)-mNG localized along the PM (Fig. 2C), consistent with the Myo1 C-terminal domains being dispensable for Ank1 interaction.
Was the Myo1 mutant localized to the contractile ring in the ank1 delta strain? One of the representative max intensity projections in Fig 2C looks like it has the beautiful elliptical ring!!!
Cooperative Motility, Force Generation and Mechanosensingin a Foraging Non-Photosynthetic Diatom
This is really fun work! Thank you for sharing all of the neat images/movies! Very exciting observations that will surely bring about a ton more questions!
he inset photo-graph shows Sargassum (yellow tag) at the Sentosa, Singapore collection site.(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprintthis version posted April 12, 2023.;https://doi.org/10.1101/2023.03.27.533254doi:bioRxiv preprint
Thank you for including this information!!! Being able to see the actual collection site / environment provides a lot of information that is often never publicly reported and gets lost with time!
DAPI
Did the DAPI staining not work? It isn't included in any of the figures.
For solid media, salt solutions I and II wereprepared as 4X stock solutions, while polysaccharides were prepared at 2Xconcentrations.
What are the components of these solutions? or if unknown, what is the source?
The relationship between runners and blazers leads tocooperative motility
Has this type of cooperative motility between clonal diatoms been shown before?
The graph shows thespeed of diatoms shown in (C). The bar shows the average with stand-ard deviation indicated. Related to Supplementary Movies 1, 2 and 3.(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprintthis version posted April 12, 2023.;https://doi.org/10.1101/2023.03.27.533254doi:bioRxiv preprint
Very interesting! Was the difference between agar & SW-air in the SW-overlay statistically significant?
Theprojections show diatom motility under the indicated conditions. Totaltime = 305 seconds. Scale bar = 100 μm.
Is this on the 1 or 2% agarose medium?
3: Environmental control of EPS and motility.
Just curious, do motile Nitzschia cells move on glass or inorganic substrates?
Related to Figure S5
Should this be referencing Figure S4? Also, is Fig S5 just showing more examples of this? If so, Awesome and thank you for providing these images!! If not, its unclear to me what additional information is shown in Figure S4.
) Lipiddroplet accumulation of N. sing1-1 and N. putrida on seawater agarosemedia in the presence (+) and absence (-) of glucose. The dotted linetraces the cell periphery. Scale bar = 10 μm. Related to Figure S5
Is this stained with BODIPY like cells in Figure S4?
nterestingly,diatoms are also observed gliding in a monolayer at theseawater-air interface, indicating that N. sing1 motility isnot strictly dependent on substratum attachment (
This is wild! How do you distinguish between gliding movement and simply drifting in the liquid?
Here, trails are not seen because the EPS is not at the airinterface.
Is the EPS trail visible after the seawater is removed / dried up? My limited understanding is that the EPS is a sort of mucusal "slip-n-slide" but if the entire surface is wet, there'd be no need for EPS trails, right?
Also, did you notice any changes in the directionality of these cells at the different substrates?
Did the cells at the SW-air interface also show a bump-induced cooperative motility mechanism?
F-actin staining of N. sing1-1. The arrows point to the approximate posi-tion of proximal raphe ends. A bright-field (BF) image of N. putrida isshown for comparison. Scale bar = 10 μm.(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprintthis version posted April 12, 2023.;https://doi.org/10.1101/2023.03.27.533254doi:bioRxiv preprint
Very cool! In the bright-field image, is the structure on the left an EPS trail? or a pipette tip like in Fig 6?
cytoplasmically connect cells
Will a TNT fuse to another cell's TNT or will it attach directly to the membrane?
. (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.
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.
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
(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?
Arpc5
Also ARPC3 disappeared, based on the STRING figure
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
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
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?
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.
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?
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).
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?
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
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?
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?
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.
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?
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?
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!
VLP
Very cool! It'd be neat to see if cdc42 is upregulated in covid patients to a similar degree!
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?
Discussion
Very cool work! I really enjoyed reading through this preprint! Thank you for sharing your work!
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?
Transcriptomic analysis of atm1-1 seedlings in response to sugars
Was there altered expression of other myosins? or cytoskeletal proteins?
(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.
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!
nt center: ***
The first image in Panel E has 2 asterisks instead of the 3. Not sure if intentional.
(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.
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?
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!
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)?
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!
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!
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!
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!!!
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?
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?