A flagellate-to-amoeboid switch in the closest living relatives of animals

  1. Thibaut Brunet
  2. Marvin Albert
  3. William Roman
  4. Maxwell C Coyle
  5. Danielle C Spitzer
  6. Nicole King

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Abstract

Amoeboid cell types are fundamental to animal biology and broadly distributed across animal diversity, but their evolutionary origin is unclear. The closest living relatives of animals, the choanoflagellates, display a polarized cell architecture (with an apical flagellum encircled by microvilli) that resembles that of epithelial cells and suggests homology, but this architecture differs strikingly from the deformable phenotype of animal amoeboid cells, which instead evoke more distantly related eukaryotes, such as diverse amoebae. Here, we show that choanoflagellates subjected to confinement become amoeboid by retracting their flagella and activating myosin-based motility. This switch allows escape from confinement and is conserved across choanoflagellate diversity. The conservation of the amoeboid cell phenotype across animals and choanoflagellates, together with the conserved role of myosin, is consistent with homology of amoeboid motility in both lineages. We hypothesize that the differentiation between animal epithelial and crawling cells might have evolved from a stress-induced switch between flagellate and amoeboid forms in their single-celled ancestors.

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  1. Version published to 10.7554/elife.61037 on eLife
  2. eLife

    ###This manuscript is in revision at eLife

    The decision letter after peer review, sent to the authors on August 24 2020, follows.

    Summary

    In this paper, Brunet and coauthors show that various species of choanoflagellates have the capacity to switch from the typical flagellated stage to an amoeboid, non-flagellated stage. They show these amoeboid cells move using blebbing migration. The paper makes four major points:

    1. Several species of choanoflagellates make dynamic protrusions that appear similar to blebs in DIC when confined. This claim is well supported by nice quantitative analysis of blebbing using a diversity of choanoflagellate species.

    2. The blebs made by choanoflagellates, like those of animal cells and Dictyostelium, involve breakage and healing of the actomyosin cortex. This is the weakest part of the paper (see below further details).

    3. Choanoflagellate cells can use this blebbing motility to escape the confinement, a concept supported by movies of cells doing this. This is supported by movies of cells doing this.

    4. Amoeboid motility is homologous in choanoflagellates and animals. In particular, the authors postulate that epithelial and crawling cells in animals differentiated by exploiting the switch from the flagellated to amoeboid stages that existed in unicellular opisthokonts. Although this is an interesting hypothesis, we believe the data is not conclusive and both the implications and the conclusions need to be better explained in the general context of eukaryotic evolution.

    Below we define what we believe should be done to improve the manuscript.

    Essential Revisions

    1. The idea that amoeboid morphology and blebbing motility is older than animals is not particularly controversial: blebbing has been documented in various microbial lineages for some time, and blebbing motility uses a contractile actin cortex, which are also widely distributed. This, combined with a lack of engagement with alternative hypotheses weakens the conceptual significance of the paper. Fleshing out what the alternative hypotheses are, and/or providing context for how this work provides new insight into the evolution of blebbing would improve the paper. Furthermore, the introduction should include more background information to distinguish blebbing motility from actin pseudopod based motility. Also, the use of the term "actin-filled" to discuss bleb retraction is confusing; do the authors mean actin-encased? Also a clear definition of what a "retracted bleb" is should be provided. In general, some information in the results that could be better incorporated in the introduction as it explains the authors' motivation for the work.

    2. Similarly, the idea of homology between the amoeboid cells of choanoflagellates and animal amoeboid cells seems not well supported or at least no more than a potential homology of many eukaryotic amoeboid cells. This needs to be toned-down and/or discussed into the context of the potential ancestral eukaryotic feature. The same with the related idea that epithelial and crawling cells in animals differentiated by exploiting the switch from the flagellated to amoeboid stages that existed in unicellular opisthokonts.

    Finally, the authors say that the switch between flagellated and crawling cells in choanoflagellates is triggered by particular size-related stress. However, it is difficult to imagine that animals evolved under such a type of stress. It may be interesting to discuss whether the authors have tried to see if alternative sources of stress induce transition to amoeboid states or, alternatively, discuss particular hypotheses about which kinds of stress might trigger this response. When discussing this, it may be worth considering an alternative: that choanoflagellates might be a side phylogenetic group having evolved specific characteristics and virtually lost amoeboid stages except for extreme situations like the ones shown here. The ancestor of metazoans would then had simply retained an ancestral (eukaryotic) capability to transition from flagellated to amoeboid states during the opisthokont life cycle without this capability being in any way related to volumetric stress but rather to particular environmental clues.

    Overall, all these ideas should be discussed in the context of eukaryotic evolution and toning down the potential implications.

    1. The authors use the standard definition of blebbing: actomyosin cortex breakage and healing concomitant with production of round protrusions devoid of actin. The paper provides insufficient data to support the claim that choanoflagellate cells defined as "amoeboid" based on the lack of microvilli undergo this form of blebbing. Providing additional examples and/or quantitative analyses of the data would strengthen the paper. Specifically:

    a. Only a single cell with lifeact is shown (Fig. 2C, with what looks to be the same cell in Video 7). Additional examples would be welcome. However, this cell has high levels of septin overexpression: could this be interfering with the native phenotype? Fig. 3K looks very different from Fig. 2q. Is septin localizing to the membrane? The WT cells shown in P do not show cortical actin. It seems likely worthwhile repeating this experiment with lifeact but without septin overexpression. Additionally, linescans to quantitate the cortical actin levels before, during, and after cortical breakage would provide quantitative support, particularly with a membrane stain, or at the very least, the septin localization.

    b. The phalloidin staining in Figures 2 and Figure 2-figure supplement 1 is not particularly convincing in terms of the presence of cortical actin. Why do the cells in Figure 2P and 2W look different? The additional examples also show weak actin staining.

    c. Figure 2, Panels S and V show myosin staining which does not appear to be localized to the cortex. More than a single cell should be shown, along with quantitation by line scan analysis to support the claim of cortical localization. d. Definition of amoeboid seems problematic as many of the images show "amoeboid" cells with what look to be microvilli: 2C-K, 2U-W, 3A-F

    e. The text describing the phalloidin staining is a bit circular as it assumes blebbing to interpret the staining patterns, but then uses the staining pattern as confirmation of blebbing.

    1. The authors indicate that the actin in amoeboid choanoflagellates undergo retrograde flow. The authors show a single cell imaged with widefield fluorescence (Fig. 3 F-G, Video 9). Typically retrograde flow is visualized using TIRF microscopy to show the movement of individual actin filaments within a network. The data presented makes it difficult to evaluate whether this is the retraction of the entire cortical network, or flow of actin filaments within a network. To support a claim of retrograde flow, additional data and analysis should be provided. Moreover, the concept of retrograde flow in the context of blebbing motility needs to be explained more fully in the text.

    2. The microtubule inhibition experiments involve treating cells for 36 hours with a non-standard microtubule inhibitor. Due to the possibility of off-target effects, the authors should repeat this experiment with a second microtubule inhibitor to cross-validate the result. A second, orthogonal approach would be to stain cells and look for anti-correlation between microtubule density and bleb formation.

  4. Version published to 10.1101/2020.06.26.171736v1 on bioRxiv