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Arp2/3 complex-mediated actin assembly drives microtubule-independent motility and phagocytosis in the evolutionarily divergent amoeba Naegleria

Katrina B Velle, Lillian K Fritz-Laylin

Preprint posted on May 15, 2020 https://www.biorxiv.org/content/10.1101/2020.05.12.091538v2

Actin vs tubulin: the Naegleria edition

Selected by Gautam Dey

Context 

We all know actin is important – but why study it in brain-eating amoebae? Velle and Fritz-Laylin provide more than a few compelling reasons to do so in their elegant new preprint. 

The beautiful textbook drawings of cells that many of us grew up with are based on decades of careful cell biology – research that has, for historical or pragmatic reasons, largely been limited to a very small number of animal and fungal model systems. Strange as it might seem, humans and yeast, both opisthokonts, are close cousins occupying a tiny fraction of the ever-expanding tree of life [1]. 

At the same time, the fundamental building blocks of eukaryotic cellular architecture – such as the actin, tubulin and ESCRTIII protein families – are over 2 billion years old, and were in fact directly inherited by the first eukaryotes from their archaeal ancestors [2]. Despite being finely tuned machines that exhibit an impressive degree of sequence conservation, these key proteins have had (literal) aeons to tweak interactions and modifications, acquire new binding partners, and to duplicate and diversify many times over [3]. It is becoming increasingly clear that our understanding of cytoskeletal function in complex processes such as cell migration and phagocytosis, and of their evolutionary origins [4], will remain incomplete until we do a better job of studying the cell biology of divergent branches of the species tree (Figure 1). 

 

Figure 1. Reproduced with Katrina Velle’s permission. Images by: Qiong Nan & Michelle Facette (Zea mays); Evan Craig & Prachee Avasthi (Chlamydomonas reinhardtii); Samantha Dundon & Thomas Pollard (Schizosaccharomyces pombe); Sarah Prostak & Lillian Fritz-Laylin (Batrachochytrium dendrobatidis); Alison Wirshing & Bruce Goode (Saccharomyces cerevisiae); Alexander Paredez (Giardia lamblia); Aoife Heaslip (Toxoplasma gondii); Katrina Velle (Naegleria gruberi, Listeria monocytogenes); Michelle Mei Kheng Yee, Ross Douglas, & Friedrich Frischknecht (Plasmodium berghei); Sarah Guest (Filoreta ramosa); Sabine Mueller (Arabidopsis thaliana); Erin Goley (Caulobacter crescentus); Ryan McQuillen & Jie Xiao (Escherichia coli); Arthur Molines (Physcomitrella patens); and Alexandre Bisson (Haloferax volcanii). Assembled by: Katrina Velle.

 

Enter the heterolobosean Naegleria gruberi and it’s brain-eating cousin Naegleria fowleri. Devoid of interphase cytoplasmic microtubules in their crawling amoeboid state, and possessed of a diverse complement of actins and actin-associated regulators, these little-studied cells provide excellent systems for the investigation of actin-driven motility and phagocytosis. 

Key take-home 

Velle and Fritz-Laylin combine advanced light and electron microscopy with an extensive toolkit of probes and small molecules used to label and perturb actin (it helps that Naegleria actin is 85% identical to mammalian actin) for their experiments. 

They show that Naegleria assembles Arp2/3-dependent actin structures, including lamellae (Figure 2); if Arp2/3 activity is compromised, the cells can no longer migrate or phagocytose effectively; without Arp2/3, cellular actin is reorganised into filopodia. These actin network properties are shared with other amoeboid cell types across the tree, supporting the notion of a deeply conserved, ancient origin for actin-powered crawling motility. 

Collectively, the authors’ systematic experiments establish Naegleria as a great new system for studying the emergent cellular behaviour of a complex actin cytoskeleton – without the confounding presence of microtubules.  

 

Figure 2: Taken from Figure 3 of Velle and Fritz-Laylin 2020 under a CC-BY-NC 4.0 International License. Structured illumination microscopy (SIM) of amoebae reveals several morphologically distinct, actin-rich structures. NEGM amoebae were fixed and stained with Phalloidin to detect actin (green) and DAPI to label DNA (magenta), prior to imaging using SIM to image actin and widefield fluorescence to image DNA. A single Z slice (top) and maximum intensity projections (bottom) are shown for representative control cells (treated with 0.1% DMSO). Actin structures defined in the text are indicated.

Some questions for the authors 

An obvious one: would the authors be willing to speculate on the possible nature of the hollow actin spheres? 

Given that the regulatory networks involved in both phagocytosis and motility intersect with various signaling and trafficking pathways, it has been difficult to infer the composition of a “minimal” phagocytic or motility system, and equally challenging to reconstruct the likely actin machinery of early eukaryotes [4]. This in turn has major implications for models of eukaryogenesis. Could you comment on how your findings might influence the status quo? 

It appears that Naegleria also presents a fantastic system for investigating – and perturbing – the control of nuclear division and spindle dynamics in mitosis without the confounding effects of disrupting interphase microtubules! Any thoughts? 

 

Further reading

Don’t miss the beautiful movies and images in the first author’s tweetorial

[1] Burki, F., Roger, A. J., Brown, M. W. & Simpson, A. G. B. The New Tree of Eukaryotes. Trends in Ecology and Evolution 35, 43–55 (2020).

[2] Eme, L., Spang, A., Lombard, J., Stairs, C. W. & Ettema, T. J. G. Archaea and the origin of eukaryotes. Nature Reviews Microbiology 15, 711–723 (2017).

[3] Stoddard, P. R., Williams, T. A., Garner, E. & Baum, B. Evolution of polymer formation within the actin superfamily. Molecular Biology of the Cell 28, 2461–2469 (2017).

[4] Burns, J. A., Pittis, A. A. & Kim, E. Gene-based predictive models of trophic modes suggest Asgard archaea are not phagocytotic. Nat. Ecol. Evol. 2, 697–704 (2018).

 

Posted on: 18th May 2020

doi: https://doi.org/10.1242/prelights.20789

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  • The authors respond to questions raised in the post

    Katrina Velle and Lillian Fritz-Laylin shared

    An obvious one: would the authors be willing to speculate on the possible nature of the hollow actin spheres? 

    We think they are probably contractile vacuoles–organelles freshwater protists use to pump water out of the cell. We can see these vacuoles pumping in living cells (https://vimeo.com/358286936), and they’re about the right size. Once we can visualize actin in live cells, we hope to confirm this.

    Given that the regulatory networks involved in both phagocytosis and motility intersect with various signaling and trafficking pathways, it has been difficult to infer the composition of a “minimal” phagocytic or motility system, and equally challenging to reconstruct the likely actin machinery of early eukaryotes [4]. This in turn has major implications for models of eukaryogenesis. Could you comment on how your findings might influence the status quo? 

    We agree completely that it is hard to untangle the origin of phagocytosis, since it is not as simple as one gene controlling one phenotype. Instead, as we discuss in our recent review (https://doi.org/10.1016/j.gde.2019.07.016), extensive and interlaced networks of actin regulators control phagocytosis in addition to dozens of other cell processes. As Burns et al. find, the broad conservation of core sets of proteins in cells known to use phagocytosis supports the hypothesis that phagocytosis arose very early in eukaryogenesis. However, it has been assumed that because organisms from both opisthokonts and amoebozoa use parallel actin-based pathways, other organisms that phagocytose use the same mechanism. While the importance of actin has been shown for some lineages, our work is the first study (that we are aware of) showing that Arp2/3 derived, thin lamellar ruffles are important for motility and phagocytosis outside of opisthokonts and amoebozoa.

    It appears that Naegleria also presents a fantastic system for investigating – and perturbing – the control of nuclear division and spindle dynamics in mitosis without the confounding effects of disrupting interphase microtubules! Any thoughts? 

    Yes, in fact this is the topic of our next paper! Naegleria mitosis looks pretty different from the “textbook” examples. Like yeast, mitosis occurs without nuclear envelope breakdown, and like some oocytes, Naegleria spindles lack centrioles. Naegleria also use distinct sets of tubulins for different tasks; amoebae use highly divergent tubulins specifically for mitosis, and conventional, “vanilla” tubulins for the flagellate cytoskeleton. We have recently been collaborating with Pat Wadsworth and Iva Tolić to characterize Naegleria spindles throughout mitosis, and 3D reconstructions using ChimeraX show they have a hollow, barrel shape (https://twitter.com/KatrinaVelle/status/1258704856103124992?s=20). We are excited to use Nageleria as a model system for these types of microtubule-centric questions, in addition to studying actin in interphase amoebae.

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