Figure 2, last row:

This experiment is very helpful and strengthens your interpretation. It would help the reader if the method used (SDS Lysis / Flow Cytometry) was briefly mentioned in the text here or in the figure legend, rather than solely in the methods section.

Figure 2, middle row:

The mean Cy5 intensity per ciliate is an interesting proxy for algal load, but it's hard to fully interpret without knowing the impact high light intensity has on the algae by themselves. Culturing the algal symbionts without the host under the same high and low light conditions and measuring their Cy5 signal would be a helpful control. If high light reduces chlorophyll autofluorescence in the algae independently of the host, that would confound the symbiont load interpretation, and you could be measuring photobleaching or photoinhibition rather than actual cell loss. A complementary approach would be z-stack imaging to directly count fluorescent puncta per host cell over time, which would give you a discrete count rather than an intensity proxy; however, this could be difficult if the symbionts are too compact together, making it difficult to segment out individual cells. The SDS lysis / flow cytometry validation at 96h mostly addresses this concern, but if high light reduces chlorophyll fluorescence intensity in individual algal cells below the flow cytometry gating threshold, stressed or damaged algae could fall outside the gate and be undercounted — leaving the same confound unresolved.

The E-modulus measurements and texture profile analysis in Figure 7 are a nice addition to the paper — it's genuinely useful to know that soy scaffolds fall within a stiffness range reported to support MSC growth. A natural extension of this characterization would be to apply the same mechanical measurements to the pea and lentil scaffolds, which would help interpret the cell performance differences observed across scaffold types in Figure 9.

At the moment it's not clear whether the superior attachment and biomass accumulation on soy scaffolds reflects something intrinsic to soy protein chemistry, or whether the three scaffold types are producing mechanically distinct structures under identical fabrication conditions. These are commercially sourced food-grade protein powders — pea, lentil, and soy — that likely differ in gelation behavior, water holding capacity, and network-forming properties during curing, meaning identical fabrication protocols could plausibly yield scaffolds with different stiffness, porosity, or surface roughness. If lentil scaffolds turn out to have a substantially different E-modulus or pore structure under these conditions, that would be a really informative finding — it would give the field a mechanistic handle on why material choice matters and when the soy preference might or might not generalize to other cell types or fabrication contexts.

The paper also notes that a stiffness range of 35-60 kPa supports MSC culture, but this is currently only established for soy. Knowing whether pea and lentil fall within or outside this range would help readers understand whether the cell performance differences are likely to be mechanistically meaningful or more context-dependent. Given that the fabrication protocol and equipment are already in place, this feels like a characterization that would add a lot of interpretive value to an already interesting dataset.

The time series is described as starting from "day of culture medium change," but the medium renewal process itself isn't described beyond "renewed every 4 to 5 weeks," leaving some important questions about how to interpret the temporal dynamics presented in Figure 4.

What does medium renewal involve practically? If it involves centrifugation, the differential sedimentation properties of chain-forming Biddulphia versus small spindle-shaped L. merlionensis cells would substantially reshape the population ratio at day 0, and zoospores — being small and motile — would likely be lost in the supernatant. If it's a simple partial medium exchange with minimal disturbance, the population structure carries over largely intact. These two scenarios lead to very different interpretations of the day 4 zoospore observations. Similarly, since the ectoplasmic net is an extracellular structure, the degree of physical disturbance during renewal matters — gentle exchange leaves existing networks intact and ongoing, whereas vigorous pipetting or centrifugation could fragment networks and synchronize cells artificially, making the "establishment phase" at day 4 look like a natural infection initiation when it may actually reflect synchronized recovery from mechanical disruption.

It's also not clear from the methods whether all experiments were performed on the original continuously passaged field isolate, or whether the co-culture was reconstituted using the separately maintained diatom isolate described later in the methods. If the former, both organisms have been co-passaged since 2020 and may have adapted to each other under lab conditions in ways that don't reflect natural infection dynamics. If the latter, at what ratio were the partners combined, and what was the physiological state of each at day 0? The methods also note that L. merlionensis was never successfully isolated in pure culture, which means there's no defined inoculum — but having a separately maintained naive diatom culture raises the question of whether a controlled infection experiment was attempted, which would have provided a cleaner starting point for the temporal framework and directly strengthened the parasitism interpretation in the conclusion.

I'm curious whether you have any insight into where in the infection cycle the bacterial protection is actually acting? Since aphelids are obligate intracellular parasites that have to physically attach to and penetrate the algal cell wall, there are a few pretty distinct points where bacteria could be intervening, disrupting zoospore motility or chemotaxis, blocking cell wall attachment, or producing antifungal compounds that inhibit development post-penetration. These would have pretty different implications for how stable and generalizable the protection is.

Great work! The core finding that environmental bacterial consortia can meaningfully delay pond crashes is really promising for large, outdoor cultivation. One thing I kept wondering about as I read through: have you looked at whether the bacterial co-culture (or the aphelid infection itself) affects lipid accumulation in the algae? Some of the Rhizobiaceae you're seeing enriched have been shown to boost fatty acid production, but high-stress conditions like infection are also known to push microalgae toward lipid accumulation, so bacterialized vs unbacterialized cultures might look similar by biomass but be pretty different in terms of actual fuel yield. A quick Nile Red screen across your conditions might be really informative. Have you thought about following up on this?

probably good to link (Figure 1K) here

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Using endosome size as a fission surrogate is reasonable and well-supported by the Figure 3 data. One question, could larger endosomes alternatively reflect increased fusion or altered EEA1 recruitment rather than impaired fission? Also, in the representative images the endosomes are often very closely clustered, and it's not clear how Imaris is distinguishing individual endosomes from clumps. How were individual endosomes distinguished from multi-endosome clumps during segmentation? It could be helpful to include a representative segmentation output image in the supplemental to let the reader evaluate this.

And was endosome count per cell considered as a complementary readout?

SMIFH2 has been reported to inhibit myosin family members in addition to formins, were any controls considered to rule out myosin-dependent effects on the observed phenotypes? https://pmc.ncbi.nlm.nih.gov/articles/PMC8121067/

Very cool work! In Figure 3A, both algae from Clade A and Clade B appear to have flagella. Is it known whether symbiont motility/flagellar beating impacts host development? For instance, does it affect gastrocoel roof plate function during left-right asymmetry establishment? This would be interesting to explore, especially given the potential for both clades to colonize eggs in the same populations.

This is a really cool and thorough study and enjoyable to read detailing a system that’s rarely explored. Thanks for sharing!

Fascinating screen showing how ineffective many common actin inhibitors can be in a non-traditional system, and how essential it is to have a diverse toolkit of compounds with different chemistries, binding affinities, and environmental stabilities. It really highlights that inhibitors developed for eukaryotic actin behave very differently in Lokiactin-based cells, and that relying on a single ‘standard’ drug would have missed most of the biology.

Related to the poly-L-lysine experiments in Fig. S2, did you ever see protrusions actually breaking off or being left behind on the surface at higher poly-L-lysine concentrations, or was the main effect just a slowdown in protrusion dynamics and cell migration? And did stronger adhesion noticeably change protrusion length distributions—for example, by increasing retraction/catastrophe events or biasing protrusions toward shorter lengths?

Does the "Round" phenotype include Bulbs and Complex Arms? or just Bulbs?

This is an exceptionally cool and ambitious piece of work. It’s impressive to see such a deep level of structural and dynamic characterization developed from an organism that was essentially uncharacterized and self-isolated by the authors. The combination of live imaging, high-resolution cytoskeletal staining, and careful morphometric analysis provides a remarkably detailed view of Filoreta’s network architecture across different states. This represents a substantial contribution both technically and conceptually, and it’s exciting to see such a comprehensive foundation being built for a new model system. Amazing work!

Incredibly cool analysis of this amazing phenotype!

Do you see evidence of these actin-dense patches staying at the base of newly formed branchlets? I was curious whether you have observations that help distinguish patches functioning as nucleation sites for new protrusions versus representing increased endocytic activity in the nutrient-enriched conditions. Also, do you think these puncta might correspond to Arp2/3-mediated branched actin networks, or is that still unclear?

I was curious whether you ever see a change in feeding behavior when the medium is enriched with YET. Since the network responds so quickly to added nutrients, do think cells would reduce their phagocytosis of bacteria under these conditions, or do you think feeding branchlets would stay just as active?

Just curious if you ever see the "loops" close overtime or if "feeding" branchlets might extend into the loop incase a food source is captured within, or if you believe these are fully functioning as support systems for the reticulated network?

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

Very cool finding and clear recovery! Did the caffeine have any impact on the WT strain?

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/

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!

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!

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!

It would be helpful to add a short line here like "..indicating more successful encystment"

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.

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

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!

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.

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.

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!

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

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!!!

Are the microtriches impacted by these drugs?

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. *

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!

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.

Spelling Error in the title

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

This is a very convincing figure and consistent with the increased cell density at day 4 when treated with ethanol! Very cool!

did y'all do any statistics on this ethanol loss dataset?

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?

minor spelling error

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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?

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!

Wow! The fourth day of experiments had wildly different effects! Do you have any idea why things were so different on this day?

I'm guessing these measurements are per Liter of medium, correct?

How did you confirm axenic cultures at this point? Did they go through filtration as well as the centrifugation?

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

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

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

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!!!

Why did y'all shift from mNeonGreen comparisons to GFP?

Very neat & exciting finding!!!

Is there any data comparing Dox-GFP with a Dox-mNeonGreen FP? If so, are the two probes comparable in their response times?

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).

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?

Thank you for compiling this list! It is a very useful resource!

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!

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

Amazing!!! How long is the gap between SSF predation steps if this one is only seconds?

Fascinating!!!! Did y'all ever see any tattered breviates that weren't contacted again? Does the initial contact kill them?

These are really amazing cells! I'd love to see some dynamic videos to better understand this "snakey, 'squishy'" movement that y'all describe.

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.

Thank you for sharing this really interesting paper! The bleaching methods will surely contribute to a lot of future work! Super cool system!!!

Were there any benefits to the host M. viridis that you observed by having 1 strain of Chlorella instead of the other?

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)

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?

Are the symbiotic-state cells able to survive without this external food (using Chlorella photosystems for energy)?

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?

Were these free-living in the culture medium? or did you need to rupture the host cell to isolate these?

Is the ACN actually killing the endosymbiont? or does it cause them to be flushed from the host?

Consider removing "Although" since the sentence doesn't continue the thought

Is this data available anywhere? I'd be interested to see the Colpoda behavior in each treatment.

It looks like the Spinosad is ~200x the concentration of chlorantraniliprole. Is this just the recommended concentration for each chemical?

Thank you for sharing this work!!!!

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.

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.

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.

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.

It looks like this should be referring to Table 2 instead

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!

https://doi.org/10.1099/acmi.0.000864.v1

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!

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)

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!!!!

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.

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!

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?

minor error: missing space

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!

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.

Was the growth rate of each species similar on the synthetic medium compared to the preferred medium?

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.

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?

I didn't see this in the text, but are HAB in the Changjiang Esturay more common in the spring or autumn?

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.

Just out of curiousity, how does the growth in DOM compare to growth in standard culture mediums like L1 or F/2?

Will the newly isolated strains be deposited in any collection centers? These are very cool cells!!!

Figure 14 & Fig 15 are swapped

It would be nice to have the scale bars on all of the images, especially since there are multiple strains & species

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!

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!

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?

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

Amazing images in this paper! How long was this fixation step at 4C? Does the cold temp prevent contraction?

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!

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?

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.

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?

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?

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!

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.

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?!

Oh wow! The nuclear localization of Scd1 in CK-666 goes away in the pak1-ts mutant!!!

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?

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

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

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)?

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!

Did you compare the intensity of tagged Pan1? it looks like it might be brighter in the bsp1 mutant compared to WT.

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.

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.

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)

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

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!

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.

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.

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?

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!

https://doi.org/10.1080/00318884.2021.1941567

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?

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

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?

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!

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.

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.

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.

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

Was S. sclerotiorum also tested like in the PbE3-2 overexpression plants?

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!

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?

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.

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?

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?

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

Are these over-expression cells more resistant to HS than WT cells?

Such an exciting and striking effect!!!! This paper is a lot of fun! Thank you for sharing!

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 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"!

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!!!

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!

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!

Did the DAPI staining not work? It isn't included in any of the figures.

What are the components of these solutions? or if unknown, what is the source?

Has this type of cooperative motility between clonal diatoms been shown before?

Very interesting! Was the difference between agar & SW-air in the SW-overlay statistically significant?

Is this on the 1 or 2% agarose medium?

Just curious, do motile Nitzschia cells move on glass or inorganic substrates?

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.

Is this stained with BODIPY like cells in Figure S4?

This is wild! How do you distinguish between gliding movement and simply drifting in the liquid?

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?

Very cool! In the bright-field image, is the structure on the left an EPS trail? or a pipette tip like in Fig 6?

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

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.

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

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

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?

Also ARPC3 disappeared, based on the STRING figure

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 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

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?

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.

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?

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).

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?

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

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?

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?

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.

Did Cdc42 inhibition reduce the number of / length of filopodia, or just the viral 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?

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?

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?

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!

Very cool! It'd be neat to see if cdc42 is upregulated in covid patients to a similar degree!

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?

Very cool work! I really enjoyed reading through this preprint! Thank you for sharing your work!

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?

Was there altered expression of other myosins? or cytoskeletal proteins?

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?

Just a minor point. I think y'all meant fluorescence.

What is marked in red? is that propidium iodide like in Fig 2D?

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!

The first image in Panel E has 2 asterisks instead of the 3. Not sure if intentional.

Very cool process! Have you taken the MA cells through several passages an they continue to retain this phenotype?