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

Thibaut Brunet, Marvin Albert, William Roman, Danielle C. Spitzer, Nicole King

Preprint posted on June 28, 2020 https://www.biorxiv.org/content/10.1101/2020.06.26.171736v1

Choanoflagellates’ divaricate morphological behavior.

Selected by Ankita Jha

Context:

There is an increasing understanding of the plasticity in eukaryotic cells where cells modulate and switch between different modes of migration. Only recently have we begun to understand how cells attain this plasticity. It has been depicted that the protozoan ancestors of eukaryotes have genetic programs that are required for these different modalities and the evolution of differentiated forms of crawling cells.

Major findings:

Authors report through fortuitous discovery that choanoflagellate Salpingoeca rosetta trans-differentiates from flagellate state to amoeboid state similar to what has been reported in a wide variety of eukaryotic cells. Like many reports in eukaryotic cells, S. rosetta when confined below 3 microns elicited protrusions and increasing confinement lead to switching to amoeboid phenotype. Inhibiting actin polymerization and myosin II prevented the formation of protrusions under confinement. Similar to animal cells undergoing bleb-based migration, the protrusions formed by S.rosetta under confinement were devoid of actin while expanding and were filled with actin during retraction, suggesting these protrusions to be blebs.

Authors suggest that this amoeboid switch might be essential for choanoflagellates as an escape mechanism. In a heterogeneous environment of confined and less confined spaces, cells would bleb and once the protrusions come in contact with non-confined spaces, cells attain an elongated shape and manage to escape. The amoeboid switch behavior is conserved across many branches of choanoflagellate suggesting the ancestral role of this behavior. How this is conserved and evolved across many different species remains elusive.

Reversible phenotypic transitions with and without confinement. It is made available under a CC-BY-NC 4.0 International license.

What I like about this preprint:

Along with my personal interest towards understanding how cells attain different modes of migration and morphology with a changing micro-environment that surrounds the cells, this preprint highlights that this morphological plasticity is conserved across many different relatives of animals. We have just begun to understand how cells attain this plasticity. Another important feature that is highlighted in this study is the use of amoeboid or bleb-like protrusions for the escape mechanism. Interestingly, a similar mechanism is used by cancer cells to move in a complex microenvironment. Cancer cells with higher contractility can utilize the formation of blebs to move in a dense matrix or confined environments to metastasize. It would be interesting to see if similar mechano-chemical mechanisms are conserved in choanoflagellate versus other types of animal cells undergoing bleb based migration. 

My questions to the authors:

  • Authors show that microtubule depolymerization induces blebbing in S. rosetta, even in the absence of confinement, this is attributed to the microtubule distribution under the plasma membrane wherein amoeboid cells, microtubules detach from the plasma membrane providing free zone to experience higher contractility and undergo blebbing. I was wondering if microtubule depolymerization might be concomitant with an increase in the overall contractility of the cells as it is seen in the animal cells with the release of GEFs?
  • Authors observe a variety of morphologies when various choanoflagellates are subjected to confinement, ranging from large blebs, lobopodia, or no morphological change. Could this also be attributed to the intrinsic contractility of these choanoflagellates?
  • One of the interesting observations made in this study was the maintenance of apical-basal polarity after the loss of flagella under confinement. When the confinement is removed, rosetta develops the flagella at the same location. Do authors have any notion about how cells maintain the memory of apical-basal polarity under confinement and are this reflected in the formation and localization of blebs?
  • One of the open questions raised by the authors is the involvement of the mechano-transduction pathway that might be involved in the detection of confinement and responses to it. I wonder if authors believe this is purely mechanical or might involve activation of certain pathways that control the contractility in these cells.

References-

Lomakin, A. J. et al. The nucleus acts as a ruler tailoring cell responses to spatial constraints. bioRxiv (2019). doi:10.1101/863514

Kopf, A. et al. Microtubules control cellular shape and coherence in amoeboid migrating  cells. J. Cell Biol. 219, e201907154 (2020).

Ruprecht V, Wieser S, Callan-Jones A, et al. Cortical contractility triggers a stochastic switch to fast amoeboid cell motility. Cell. 2015;160(4):673-685. doi:10.1016/j.cell.2015.01.008

Liu YJ, Le Berre M, Lautenschlaeger F, et al. Confinement and low adhesion induce fast amoeboid migration of slow mesenchymal cells. Cell. 2015;160(4):659-672. doi:10.1016/j.cell.2015.01.007

 

Tags: #amoeboid, confinement

Posted on: 30th July 2020 , updated on: 31st July 2020

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  • Author's response

    Thibaut Brunet shared

    Hi Ankita,

    Thanks for highlighting our paper and for your excellent questions! Here are some brief answers:

    1. Authors show that microtubule depolymerization induces blebbing in S. rosetta, even in the absence of confinement, this is attributed to the microtubule distribution under the plasma membrane wherein amoeboid cells, microtubules detach from the plasma membrane providing free zone to experience higher contractility and undergo blebbing. I was wondering if microtubule depolymerization might be concomitant with an increase in the overall contractility of the cells as it is seen in the animal cells with the release of GEFs?

    Absolutely. We did this experiment in Figure 2 – figure supplement 2 (panels L to P) and depolymerizing microtubules (with the compound carbendazim) did induce spectacular and unmistakable blebbing. See also Supplementary Movie 8 which shows a time lapse video of this phenomenon.

    media-9

    2. Authors observe a variety of morphologies when various choanoflagellates are subjected to confinement, ranging from large blebs, lobopodia, or no morphological change. Could this also be attributed to the intrinsic contractility of these choanoflagellates?

    This is definitely an interesting question for the future. Intrinsic contractility might play a role, as could the osmolarity of the extracellular medium – some of these choanoflagellate species live in freshwater and other in the ocean, so that might play a role.

    3. One of the interesting observations made in this study was the maintenance of apical-basal polarity after the loss of flagella under confinement. When the confinement is removed, S.rosetta develops the flagella at the same location. Do authors have any notion about how cells maintain the memory of apical-basal polarity under confinement and are this reflected in the formation and localization of blebs?

    We think the microtubule-organizing center is probably maintained in its original position during the entire amoeboid switch. One sign that this is the case is that the microtubule cytoskeleton (which normally forms a tight cage underlying the plasma membrane and converging underneath the base of the flagellum) is still present and recognizable in amoeboid cells – just detached from the plasma membrane. We think this means the MTOC persists, ready to regenerate a flagellum where it previously was, during the entire amoeboid switch.

    4. One of the open questions raised by the authors is the involvement of the mechano-transduction pathway that might be involved in the detection of confinement and responses to it. I wonder if authors believe this is purely mechanical or might involve activation of certain pathways that control the contractility in these cells.

    We do not know. The one observation we made is that calcium signaling does not seem to be necessary, as depleting both intracellular and extracellular calcium left the response unchanged – which is a difference from several other systems in which calcium seems involved in the activation of contractility under confinement (in mammalian cell culture, zebrafish embryonic cells and in slime molds). So it could be purely mechanical (similar to a crawling response recently analyzed in Euglena), but a (calcium-independent) transduction pathway (responsive to forces or to cell deformation) might be involved as well. Some active biochemical changes are likely needed at least to explain flagellar retraction. We are excited to explore this question in the future.

     

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