Mechanical stretch regulates macropinocytosis in Hydra vulgaris

  1. Taylor D. Skokan
  2. Bert Hobmayer
  3. Kara L. McKinley
  4. Ronald D. Vale

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Abstract

Cells rely on a diverse array of engulfment processes to sense, exploit, and adapt to their environments. Among these, macropinocytosis enables indiscriminate and rapid uptake of large volumes of fluid and membrane, rendering it a highly versatile engulfment strategy. Much of the molecular machinery required for macropinocytosis has been well established, yet how this process is regulated in the context of organs and organisms remains poorly understood. Here, we report the discovery of extensive macropinocytosis in the outer epithelium of the cnidarian Hydra vulgaris. Exploiting Hydra’s relatively simple body plan, we developed approaches to visualize macropinocytosis over extended periods of time, revealing constitutive engulfment across the entire body axis. We show that the direct application of planar stretch leads to calcium influx and the inhibition of macropinocytosis. Finally, we establish a role for stretch-activated channels in inhibiting this process. Together, our approaches provide a platform for the mechanistic dissection of constitutive macropinocytosis in physiological contexts and highlight a potential role for macropinocytosis in responding to cell surface tension.

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  1. Version published to 10.1091/mbc.e22-02-0065
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    Reply to the reviewers

    The authors do not wish to provide a response at this time.

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    Referee #3

    Evidence, reproducibility and clarity

    The article by Skokan and coworkers studies the regulation of macropinocytosis in the Hydra. They design a clever assay to image the formation of macropinosomes in the ectodermal cells of the Hydra body, by amputating the head and the foot of the animal and then helving it onto a thin glass rod, allowing them to study the dynamics of actin rings formation, associated with uptake of external fluid phase. They also observe the cyclic formation of macropinosomes during the oscillatory contractions of spheroids formed from amputated animals during regeneration. By using agonist and antagonist drugs targeting mechano-sensitive calcium channels, they show that the formation of macropinosomes correlates with the reduction of cell tension. Overall, the article is succint, but clear and convincing. However, in my opinion, two major points should be clarified, if not solved before considering publication.

    Major points:

    1. the function of macropinocytosis in the Hydra is not known. The author postulates that it could be linked to a regulation of membrane area during animal contractions. However, one may wonder if the membrane cell surface really changes during contractions. I wonder if another explanation is possible: most of the organisms leaving in fresh water require an efficient mechanism to remove excess water that comes in the cells through osmosis. The hydra regular contractile movement are part of this, and I am wondering the macropinocytosis could be linked to this mechanism. Would the author be able to apply osmotic shocks, in particular hypertonic shocks, and see how it changes the formation rate and the dynamics of macropinosomes? On the reverse, in paralyzed animal, I am wondering if macropinosomes are still formed? Results from these experiments may give a clue about the function of macropinocytosis in the Hydra.
    2. Because of the role of Piezo and other mechano-sensitive calcium channels, the author conclude that the factor that limits macropinocytosis is membrane tension. However, unless I am mistaken, actin cytoskeleton has also been involved in mechano-sensing channels, it could be that cortical tension, rather than membrane tension is playing a regulatory role. A direct proof of membrane tension (by measuring it) changes would be required to conclude as the authors do. The role of membrane tension versus macropinocytosis could be directly assessed using membrane tension probes such as FliptR or flipper probes. Otherwise, a less clearly defined term, that combines both cortical tension and membrane tension, such as cell surface tension or cell tension would be preferable.
    3. Number of macropinocytic cups(actin rings) per cell is used as a readout for rate of macropinocytosis. Yet in addition to the number of cups parameters the diameter increases in certain conditions such as GdCi3. It would ideally be interesting to show the changes in diameter of cups and how this varies per in different conditions. For example, in videos of Jedi1 treated body columns the cups seem bigger in size. Supporting experiments of monitoring macropinosomes via dextran uptake assays needs to be performed for quantifications a rate of change in macropinocytosis is proposed. Alternatively, dextran beads of different molecular sizes with different fluorophores could also be used to assess the differences in rate and volume of uptake via macropinocytosis in various conditions of this study.
    4. If membrane tension is altered upon dissecting Hydra fragments, would it make sense to study potential changes in macropinocytosis within the regenerating body column? Such as differences in actin ring formation in cells close to wound edges versus equatorial regions of regenerating body columns and spheroids?
    5. Reasoning for selection of Piezo as molecular target over other stretch activated channels has not been provided. Piezo activators have been used, on the contrary depletion of Piezo via RNAi could be performed in intact animals to assess increased macropinocytosis. Furthermore, rate of macropinocytosis could be assessed in body columns generated from Piezo depleted animals. This would further support the direct role of Piezo in the process.

    Minor points:

    1. The authors report differences in macropinocytosis based on different parts of the animal (Fig.S1), upon treating intact animals with GdCI3 (Fig.2C) how does this vary? Do the differences still persist in spite of increased macropinocytosis?
    2. Hydra are animals with an elongated body column. Dissecting body columns of different lengths could give rise to spheroids of different volume, these could then be inflated to establish a comparative volume study with different volumes and macropinocytosis.
    3. For all graphical representation it would also be ideal to state the p-values for each significant comparison to better appreciate differences instead of stars.
    4. For better understanding of figures, highlight in graph legends and figure panels which tissue sample has been used i.e intact animal, body column or spheroid.
    5. A graphical representation could be given for the comparison of macropinocytic cups between intact hydra versus body column samples with statistical analysis. To appreciate the claims made by the authors regarding the trend of more cups being observed in body columns versus intact animals, as only the mean values are stated in the text.
    6. In Fig.1F, there exist streaks of dextran distinctly outlining apical membranes of cell sets in the hydra epithelia, what are these suggestive of?
    7. Fig.S1 No p-values

    Significance

    Overall, the work is of interest for several research communities. The significance could be increase by providing a few more experiments about the physiological role of macropinocytosis in the Hydra.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript, Skokan et al. develop a platform of cnidarian Hydra vulgaris, a powerful model for cellular self-assembly and organismal regeneration, to enable visualization of macropinocytosis in living tissue. Utilizing this system and small molecule perturbation, authors discover that macropinocytosis occurs constitutively at the ectoderm across the entire body axis of Hydra, and is constrained by membrane tension through stretch-activated channels and the downstream calcium influx.

    Major Comments:

    The manuscript is clearly written and logically organized, and the imaging results are properly quantified. With the logical interpretation, adequate biological repeats and statistical analysis, the method and data in this manuscript are clear and compelling. The major concern is the missing physiological significance of macropinocytosis induced by membrane relaxation in Hydra, if any.

    Suggested experiments:

    1. In Fig 2, the importance of SAC and Ca2+ for macropinocytosis are addressed. However, only one SAC inhibitor was used, whereas Ca2+ concentration in Ionomycin treated Hydro remained high even after 60 min when macropinocytic cup density had recovered (Fig 2E and G). As the authors mentioned in the DISCUSSION, other SAC transported cations may be involved in and thus need to be tested. Simply, the medium depleted of specific cation or water containing specific cation could be used to monitor the requirement of each cation on Jedi2 treated Hydra.
    2. In Fig 3, the authors demonstrate that increased membrane tension leads to higher Ca2+ concentration and less macropinocytic cups in Hydras. The SAC inhibitors and EDTA (or calcium free buffer) used in Fig2 should be applied in the inflated regenerative spheroids to confirm that membrane tension inhibits macropinocytosis via SAC and Ca2+.
    3. The authors observed an increase of macropinocytic cups in both amputated Hydra and regenerative spheroids than intact animal (0.186 and ~0.3 compared to 0.015 cups per cell, Fig S1, 2E, 3C). Would the inflation or inhibition of macropinocytosis perturb spheroid regeneration or polarization/sorting? Authors have discussed several potential biological functions of macropinocytosis in Hydra, including tension homeostasis and surface remodeling that are important during spheroid regeneration. It will be worthy to examine if mild membrane tension increase or SAC activation would delay the sorting process of regenerating Hydra tissues.

    Minor points:

    1. Fig 2C is the quantification results of 2B but include three sets of data (labeled as 1, 2, and 3) without explanation.
    2. Would amputation of one tentacle lead to local or global Ca2+ reduction and macropinocytosis in a Hydra?

    Significance

    Macropinocytosis is an evolutionary conserved, from amoeba to human, and versatile endocytic route critical for mammalian immune and cancer cells for antigen surveillance and nutrient uptake. Despite ample understanding of macropinocytosis in cultured cells has been made, the function and mechanism of macropinocytosis at the organ or organismal level remains poorly studied. Therefore, this work is intriguing and timely to support the physiological occurence of macropinocytosis from the tissue and evolutionary aspects.

    Macropinocytosis is critical process for membrane trafficking, cell signaling, immune surveliance and cancer cell growth, and Hydra vulgaris is a powerful model organism for regeneration biology, neuro biology and marine biology. Therefore, audiences from these fields will be interested and influenced by this report studying developing a new method for visualizing macropinocytosis in living Hydra.

    I am a cell biologist studying the regulation of membrane remodeling and trafficking upon mechanical or biochemical stimuli. Due to my unfamiliar with Hydra as a model organism, the details of suggested experiments may need to be adjusted.

    Referees cross-commenting

    I agree with other reviewers and think their comments important and valid. This manuscript will be more clear and compelling after addressing these questions.

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    Referee #1

    Evidence, reproducibility and clarity

    In the manuscript by Skokan et al, the authors demonstrate a constitutive and robust program of macropinocytosis in the outer epithelial layer of the cnidarian Hydra vulgaris. While the model system is less tractable than others including mammalian cell types from a genetic stand-point, the authors have devised a neat approach to visualizing the planar epithelium in live organisms and provide clear evidence for macropinocytosis by a tissue monolayer in vivo. This model also supports the ancient conservation of macropinocytosis, supporting the studies in Dictyostelium, and may represent early modes of nutrient acquisition in complex fluid environments. Using probes for the cytoskeleton, fluid phase indicators, and mechanical and pharmacological interventions, the authors describe how stretch-activated calcium channels inhibit micropinocytosis. In general, while the manuscript is concisely written, and the available data are compelling, much more rigorous experimentation is required to make such a conclusion. In addition, the physiological importance for mechanical stretch in orchestrating the arrest of macropinocytosis remains unclear. Conceivably, this may be involved in the regulation of membrane tension since macropinocytosis (high membrane turnover) would demand that cells have a high rate of membrane recycling to compensate. Below, I have outlined some approaches that the authors could take to improve the study without demanding them to utilize additional model systems, which I think would be outside the scope of the work.

    Major comments:

    Most importantly, the role of Ca2+ entry via stretch-activated channels and how this would inhibit macropinocytosis remains unclear. In fact, the findings are somewhat counterintuitive since stretch applied to the monolayer would increase membrane tension while Ca2+ influx would support membrane delivery and exocytosis, thereby restoring tensional homeostasis.

    In Fig 3, the authors demonstrate that applied stretch to the epithelium increases cytosolic Ca2+ and decreases membrane tension as expected. But whether the Ca2+ influx is required for the loss of macropinocytosis is not clear. This can be tested by either chelating Ca2+ transients in the cytosol or depleting the cells of Ca2+ by inhibiting ER-resident Ca2+ pumps and removing Ca2+ from the medium. In fact, if the authors think that extracellular Ca2+ is the only issue to arresting macropinocytosis, substituting Ca2+ for another divalent cation (or removing all divalent cations from the medium, should the epithelium be amenable to it for short periods of time) could be employed.

    The connection between [Ca2+]cyto and macropinocytosis is established by Jedi and ionomycin. In the case of ionomycin, the large and sustained increase [Ca2+]cyto, well beyond what could be expected in physiological conditions, leads to the loss of plasma membrane PIP2, PIP3, and membrane associated F-actin. Jedi1/2 are certainly more targeted, but it is difficult to attribute their effects to Piezo in this system. More worryingly, the Ca2+ influx in response to Jedi2 and especially Jedi1 occurs maximally after 10 min of exposure. Yet, the authors show the complete loss of macropinocytic cups after 10 min (Fig 2E). It's difficult to reconcile that the Ca2+ is the issue.

    The authors do not quantify macropinocytosis beyond Figure 1. Instead, they use "macropinocytic cups" as their surrogate for bona fide, sealed macropinosomes. Macropinocytosis can occur at different scales and different rates, so the authors should instead use the 70 kDa dextran as the gold standard in Figure 2. And as part of gold standard approaches, the authors would appease the macropinocytosis field if they tested the requirement for PI3K and Na H+ exchangers in Figure 1.

    The appearance of the GCAMP6s in Figure 2F before given Jedi2 is interesting. Aside from the Ca2+ signal that appears where the Hydra has been severed, the Ca2+ through the epithelium appears very heterogeneous. Does this Ca2+ signal oscillate in the cells and/or across the epithelium? Since the authors are able to image the cytoskeleton and Ca2+ in this system, it would be interesting to determine any correlations in their kinetics.

    Minor comments:

    At this point, minor comments may be less useful to the authors since some of the more major suggestions are likely to impact the overall breadth of the work.

    Significance

    The work represents a technical advance and new system to consider macropinocytosis, albeit with limited mechanistic insights owed to some intrinsic challenges and remaining experimentation.

  7. Version published to 10.1101/2021.12.03.471193v1 on bioRxiv