Search

Understanding the origin of eukaryotic cells is one of the most difficult problems in all of biology. A key challenge relevant to the question of eukaryogenesis is reconstructing the gene repertoire of the last eukaryotic common ancestor (LECA). As data sets grow, sketching an accurate genomics-informed picture of early eukaryotic cellular complexity requires provision of analytical resources and a commitment to data sharing. Here, we summarise progress towards understanding the biology of LECA and outline a community approach to inferring its wider gene repertoire. Once assembled, a robust LECA gene set will be a useful tool for evaluating alternative hypotheses about the origin of eukaryotes and understanding the evolution of traits in all descendant lineages, with relevance in diverse fields such as cell biology, microbial ecology, biotechnology, agriculture, and medicine. In this Consensus View, we put forth the status quo and an agreed path forward to reconstruct LECA's gene content., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Richards et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Eukaryotic cells have been evolving for billions of years, giving rise to wildly diverse cell forms and functions. Despite their variability, all eukaryotic cells share key hallmarks, including membrane-bound organelles, heavily regulated cytoskeletal networks and complex signaling cascades. Because the actin cytoskeleton interfaces with each of these features, understanding how it evolved and diversified across eukaryotic phyla is essential to understanding the evolution and diversification of eukaryotic cells themselves. Here, we discuss what we know about the origin and diversity of actin networks in terms of their compositions, structures and regulation, and how actin evolution contributes to the diversity of eukaryotic form and function., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The global panzootic lineage (GPL) of the pathogenic fungus Batrachochytrium dendrobatidis (Bd) has caused severe amphibian population declines, yet the drivers underlying the high frequency of GPL in regions of amphibian decline are unclear. Using publicly available Bd genome sequences, we identified multiple non-GPL Bd isolates that contain a circular Rep-encoding single-stranded (CRESS)-like DNA virus, which we named Bd DNA virus 1 (BdDV-1). We further sequenced and constructed genome assemblies with long read sequences to find that the virus is integrated into the nuclear genome in some strains. Attempts to cure virus-positive isolates were unsuccessful; however, phenotypic differences between naturally virus-positive and virus-negative Bd isolates suggested that BdDV-1 decreases the growth of its host in vitro but increases the virulence of its host in vivo. BdDV-1 is the first-described CRESS DNA mycovirus of zoosporic true fungi, with a distribution inversely associated with the emergence of the panzootic lineage., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Batrachochytrium dendrobatidis ( Bd ), a causative agent of chytridiomycosis, is decimating amphibian populations around the world. Bd belongs to the chytrid lineage, a group of early-diverging fungi that are widely used to study fungal evolution. Like all chytrids, Bd develops from a motile form into a sessile, growth form, a transition that involves drastic changes in its cytoskeletal architecture. Efforts to study Bd cell biology, development, and pathogenicity have been limited by the lack of genetic tools with which to test hypotheses about underlying molecular mechanisms. Here, we report the development of a transient genetic transformation system for Bd . We used electroporation to deliver exogenous DNA into Bd cells and detected transgene expression for up to three generations under both heterologous and native promoters. We also adapted the transformation protocol for selection using an antibiotic resistance marker. Finally, we used this system to express fluorescent protein fusions and, as a proof of concept, expressed a genetically encoded probe for the actin cytoskeleton. Using live-cell imaging, we visualized the distribution and dynamics of polymerized actin at each stage of the Bd life cycle, as well as during key developmental transitions. This transformation system enables direct testing of key hypotheses regarding mechanisms of Bd pathogenesis. This technology also paves the way for answering fundamental questions of chytrid cell, developmental, and evolutionary biology., Competing Interests: Competing interests statement:The authors declare no competing interest.

The actin cytoskeleton is a protein polymer system that underlies a wide variety of eukaryotic phenotypes. A new study reports that diversity in a key actin regulator, the Arp2/3 complex, drives species-specific sperm development within the Drosophila lineage., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Many eukaryotic cells, including animal cells and unicellular amoebae, use dynamic-actin networks to crawl across solid surfaces. Recent discoveries of actin-dependent crawling in additional lineages have sparked interest in understanding how and when this type of motility evolved. Tracing the evolution of cell crawling requires understanding the molecular mechanisms underlying motility. Here we outline what is known about the diversity and evolution of the molecular mechanisms that drive cell motility, with a focus on actin-dependent crawling. Classic studies and recent work have revealed a surprising number of distinct mechanical modes of actin-dependent crawling used by different cell types and species to navigate different environments. The overlap in actin network regulators driving multiple types of actin-dependent crawling, along with cortical-actin networks that support the plasma membrane in these cells, suggest that actin motility and cortical actin networks might have a common evolutionary origin. The rapid development of additional evolutionarily diverse model systems, advanced imaging technologies, and CRISPR-based genetic tools, is opening the door to testing these and other new ideas about the evolution of actin-dependent cell crawling.

Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments, such as freshwater, must constantly battle water influx to avoid swelling until they burst. Many eukaryotic cells use contractile vacuoles to collect excess water from the cytosol and pump it out of the cell. Although contractile vacuoles are essential to many species, including important pathogens, the mechanisms that control their dynamics remain unclear. To identify the basic principles governing contractile vacuole function, we investigate here the molecular mechanisms of two species with distinct vacuolar morphologies from different eukaryotic lineages: the discoban Naegleria gruberi and the amoebozoan slime mold Dictyostelium discoideum. Using quantitative cell biology, we find that although these species respond differently to osmotic challenges, they both use vacuolar-type proton pumps for filling contractile vacuoles and actin for osmoregulation, but not to power water expulsion. We also use analytical modeling to show that cytoplasmic pressure is sufficient to drive water out of contractile vacuoles in these species, similar to findings from the alveolate Paramecium multimicronucleatum. These analyses show that cytoplasmic pressure is sufficient to drive contractile vacuole emptying for a wide range of cellular pressures and vacuolar geometries. Because vacuolar-type proton-pump-dependent contractile vacuole filling and pressure-dependent emptying have now been validated in three eukaryotic lineages that diverged well over a billion years ago, we propose that this represents an ancient eukaryotic mechanism of osmoregulation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Chytrid fungi play key ecological roles in aquatic ecosystems, and some species cause a devastating skin disease in frogs and salamanders. Additionally, chytrids occupy a unique phylogenetic position- sister to the well-studied Dikarya (the group including yeasts, sac fungi, and mushrooms) and related to animals- making chytrids useful for answering important evolutionary questions. Despite their importance, little is known about the basic cell biology of chytrids. A major barrier to understanding chytrid biology has been a lack of genetic tools with which to test molecular hypotheses. Medina and colleagues recently developed a protocol for Agrobacterium -mediated transformation of Spizellomyces punctatus . In this manuscript, we describe the general procedure including planning steps and expected results. We also provide in-depth, step-by-step protocols and video guides for performing the entirety of this transformation procedure on protocols.io (dx.doi.org/10.17504/protocols.io.x54v9dd1pg3e/v1)., Competing Interests: The authors declare that there are no conflicts of interest., (© 2023 The Authors.)

The fungal kingdom represents an extraordinary diversity of organisms with profound impacts across animal, plant, and ecosystem health. Fungi simultaneously support life, by forming beneficial symbioses with plants and producing life-saving medicines, and bring death, by causing devastating diseases in humans, plants, and animals. With climate change, increased antimicrobial resistance, global trade, environmental degradation, and novel viruses altering the impact of fungi on health and disease, developing new approaches is now more crucial than ever to combat the threats posed by fungi and to harness their extraordinary potential for applications in human health, food supply, and environmental remediation. To address this aim, the Canadian Institute for Advanced Research (CIFAR) and the Burroughs Wellcome Fund convened a workshop to unite leading experts on fungal biology from academia and industry to strategize innovative solutions to global challenges and fungal threats. This report provides recommendations to accelerate fungal research and highlights the major research advances and ideas discussed at the meeting pertaining to 5 major topics: (1) Connections between fungi and climate change and ways to avert climate catastrophe; (2) Fungal threats to humans and ways to mitigate them; (3) Fungal threats to agriculture and food security and approaches to ensure a robust global food supply; (4) Fungal threats to animals and approaches to avoid species collapse and extinction; and (5) Opportunities presented by the fungal kingdom, including novel medicines and enzymes., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

The frog-killing chytrid fungus Batrachochytrium dendrobatidis (Bd) is decimating amphibian populations around the world. 1-4 Bd has a biphasic life cycle, alternating between motile zoospores that disperse within aquatic environments and sessile sporangia that grow within the mucus-coated skin of amphibians. 5 , 6 Zoospores lack cell walls and swim rapidly through aquatic environments using a posterior flagellum and crawl across solid surfaces using actin structures similar to those of human cells. 7 , 8 Bd transitions from this motile dispersal form to its reproductive form by absorbing its flagellum, rearranging its actin cytoskeleton, and rapidly building a chitin-based cell wall-a process called "encystation." 5-7 The resulting sporangium increases in volume by two or three orders of magnitude while undergoing rounds of mitosis without cytokinesis to form a large ceonocyte. The sporangium then cellurizes by dividing its cytoplasm into dozens of new zoospores. After exiting the sporangium through a discharge tube onto the amphibian skin, daughter zoospores can then reinfect the same individual or find a new host. 5 Although encystation is critical to Bd growth, whether and how this developmental transition is triggered by external signals was previously unknown. We discovered that exposure to amphibian mucus triggers rapid and reproducible encystation within minutes. This response can be recapitulated with purified mucin, the bulk component of mucus, but not by similarly viscous methylcellulose or simple sugars. Mucin-induced encystation does not require gene expression but does require surface adhesion, calcium signaling, and modulation of the actin cytoskeleton. Mucus-induced encystation may represent a key mechanism for synchronizing Bd development with the arrival at the host., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Naegleria gruberi is a unicellular eukaryote whose evolutionary distance from animals and fungi has made it useful for developing hypotheses about the last common eukaryotic ancestor. Naegleria amoebae lack a cytoplasmic microtubule cytoskeleton and assemble microtubules only during mitosis and thus represent a unique system for studying the evolution and functional specificity of mitotic tubulins and the spindles they assemble. Previous studies show that Naegleria amoebae express a divergent α-tubulin during mitosis, and we now show that Naegleria amoebae express a second mitotic α- and two mitotic β-tubulins. The mitotic tubulins are evolutionarily divergent relative to typical α- and β-tubulins and contain residues that suggest distinct microtubule properties. These distinct residues are conserved in mitotic tubulin homologs of the "brain-eating amoeba" Naegleria fowleri, making them potential drug targets. Using quantitative light microscopy, we find that Naegleria's mitotic spindle is a distinctive barrel-like structure built from a ring of microtubule bundles. Similar to those of other species, Naegleria's spindle is twisted, and its length increases during mitosis, suggesting that these aspects of mitosis are ancestral features. Because bundle numbers change during metaphase, we hypothesize that the initial bundles represent kinetochore fibers and secondary bundles function as bridging fibers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The chytrid fungus Batrachochytrium dendrobatidis (Bd) is a causative agent of chytridiomycosis, a skin disease associated with amphibian population declines around the world. Despite the major impact Bd is having on global ecosystems, much of Bd's basic biology remains unstudied. In addition to revealing mechanisms driving the spread of chytridiomycosis, studying Bd can shed light on the evolution of key fungal traits because chytrid fungi, including Bd, diverged before the radiation of the Dikaryotic fungi (multicellular fungi and yeast). Studying Bd in the laboratory is, therefore, of growing interest to a wide range of scientists, ranging from herpetologists and disease ecologists to molecular, cell, and evolutionary biologists. This protocol describes how to maintain developmentally synchronized liquid cultures of Bd for use in the laboratory, how to grow Bd on solid media, as well as cryopreservation and revival of frozen stocks. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Reviving cryopreserved Bd cultures Basic Protocol 2: Establishing synchronized liquid cultures of Bd Basic Protocol 3: Regular maintenance of synchronous Bd in liquid culture Alternate Protocol 1: Regular maintenance of asynchronous Bd in liquid culture Basic Protocol 4: Regular maintenance of synchronous Bd on solid medium Alternate Protocol 2: Starting a culture on solid medium from a liquid culture Basic Protocol 5: Cryopreservation of Bd., (© 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.)

Correlating the location of subcellular structures with dynamic cellular behaviors is difficult when working with organisms that lack the molecular genetic tools needed for expressing fluorescent protein fusions. Here, we describe a protocol for fixing, permeabilizing, and staining cells in a single step while imaging on a microscope. In contrast to traditional, multi-step fixing and staining protocols that take hours, the protocol outlined here achieves satisfactory staining within minutes. This approach takes advantage of well-characterized small molecules that stain specific subcellular structures, including nuclei, mitochondria, and actin networks. Direct visualization of the entire process allows for rapid optimization of cell fixation and staining, as well as straightforward identification of fixation artifacts. Moreover, live imaging prior to fixation reveals the dynamic history of cellular features, making it particularly useful for model systems without the capacity for expressing fluorescent protein fusions. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Fixing, permeabilizing, and staining mammalian cells in one step on the microscope., (© 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.)

Cell motility is critical for animal biology, but its evolutionary history is unclear. A new study reports blebbing motility - a form of cell crawling - in the closest living relative of animals, suggesting that the unicellular ancestors of animals could crawl., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cells from across the eukaryotic tree use actin polymer networks for a wide variety of functions, including endocytosis, cytokinesis, and cell migration. Despite this functional conservation, the actin cytoskeleton has undergone significant diversification, highlighted by the differences in the actin networks of mammalian cells and yeast. Chytrid fungi diverged before the emergence of the Dikarya (multicellular fungi and yeast) and therefore provide a unique opportunity to study actin cytoskeletal evolution. Chytrids have two life stages: zoospore cells that can swim with a flagellum and sessile sporangial cells that, like multicellular fungi, are encased in a chitinous cell wall. Here, we show that zoospores of the amphibian-killing chytrid Batrachochytrium dendrobatidis (Bd) build dynamic actin structures resembling those of animal cells, including an actin cortex, pseudopods, and filopodia-like spikes. In contrast, Bd sporangia assemble perinuclear actin shells and actin patches similar to those of yeast. The use of specific small-molecule inhibitors indicate that nearly all of Bd's actin structures are dynamic and use distinct nucleators: although pseudopods and actin patches are Arp2/3 dependent, the actin cortex appears formin dependent and actin spikes require both nucleators. Our analysis of multiple chytrid genomes reveals actin regulators and myosin motors found in animals, but not dikaryotic fungi, as well as fungal-specific components. The presence of animal- and yeast-like actin cytoskeletal components in the genome combined with the intermediate actin phenotypes in Bd suggests that the simplicity of the yeast cytoskeleton may be due to evolutionary loss., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Much of our understanding of actin-driven phenotypes in eukaryotes has come from the "yeast-to-human" opisthokont lineage and the related amoebozoa. Outside of these groups lies the genus Naegleria, which shared a common ancestor with humans >1 billion years ago and includes the "brain-eating amoeba." Unlike nearly all other known eukaryotic cells, Naegleria amoebae lack interphase microtubules; this suggests that actin alone drives phenotypes like cell crawling and phagocytosis. Naegleria therefore represents a powerful system to probe actin-driven functions in the absence of microtubules, yet surprisingly little is known about its actin cytoskeleton. Using genomic analysis, microscopy, and molecular perturbations, we show that Naegleria encodes conserved actin nucleators and builds Arp2/3-dependent lamellar protrusions. These protrusions correlate with the capacity to migrate and eat bacteria. Because human cells also use Arp2/3-dependent lamellar protrusions for motility and phagocytosis, this work supports an evolutionarily ancient origin for these processes and establishes Naegleria as a natural model system for studying microtubule-independent cytoskeletal phenotypes., (© 2020 Velle and Fritz-Laylin.)

Global amphibian populations are being decimated by chytridiomycosis, a deadly skin infection caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal). Although ongoing efforts are attempting to limit the spread of these infections, targeted treatments are necessary to manage the disease. Currently, no tools for genetic manipulation are available to identify and test specific drug targets in these fungi. To facilitate the development of genetic tools in Bd and Bsal, we have tested five commonly used antibiotics with available resistance genes: Hygromycin, Blasticidin, Puromycin, Zeocin, and Neomycin. We have identified effective concentrations of each for selection in both liquid culture and on solid media. These concentrations are within the range of concentrations used for selecting genetically modified cells from a variety of other eukaryotic species., Competing Interests: The authors have declared that no competing interests exist.

Two species of parasitic fungi from the phylum Chytridiomycota (chytrids) are annihilating global amphibian populations. These chytrid species-Batrachochytrium dendrobatidis and B. salamandrivorans-have high rates of mortality and transmission. Upon establishing infection in amphibians, chytrids rapidly multiply within the skin and disrupt their hosts' vital homeostasis mechanisms. Current disease models suggest that chytrid fungi locate and infect their hosts during a motile, unicellular 'zoospore' life stage. Moreover, other chytrid species parasitize organisms from across the tree of life, making future epidemics in new hosts a likely possibility. Efforts to mitigate the damage and spread of chytrid disease have been stymied by the lack of knowledge about basic chytrid biology and tools with which to test molecular hypotheses about disease mechanisms. To overcome this bottleneck, we have developed high-efficiency delivery of molecular payloads into chytrid zoospores using electroporation. Our electroporation protocols result in payload delivery to between 75 and 97% of living cells of three species: B. dendrobatidis, B. salamandrivorans, and a non-pathogenic relative, Spizellomyces punctatus. This method lays the foundation for molecular genetic tools needed to establish ecological mitigation strategies and answer broader questions in evolutionary and cell biology.

P values and error bars help readers infer whether a reported difference would likely recur, with the sample size n used for statistical tests representing biological replicates, independent measurements of the population from separate experiments. We provide examples and practical tutorials for creating figures that communicate both the cell-level variability and the experimental reproducibility., (© 2020 Lord et al.)

Eukaryotic cells use a number of diverse mechanisms to swim through liquid or crawl across solid surfaces. The two most prevalent forms of eukaryotic cell motility are flagellar-dependent swimming and actin-dependent cell migration, both of which are used by animal cells and unicellular eukaryotes alike. Evolutionary cell biologists have used morphological and molecular phenotypes to trace the evolution of flagellar-based swimming. These efforts have resulted in a large body of evidence supporting a single evolutionary origin of the eukaryotic flagellum, an origin that dates back to before the diversification of modern eukaryotes. Actin-dependent crawling, in contrast, involves mutiple distinct molecular mechanisms, the evolution of which is just beginning to be explored., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Chytrids are early-diverging fungi that share features with animals that have been lost in most other fungi. They hold promise as a system to study fungal and animal evolution, but we lack genetic tools for hypothesis testing. Here, we generated transgenic lines of the chytrid Spizellomyces punctatus , and used fluorescence microscopy to explore chytrid cell biology and development during its life cycle. We show that the chytrid undergoes multiple rounds of synchronous nuclear division, followed by cellularization, to create and release many daughter 'zoospores'. The zoospores, akin to animal cells, crawl using actin-mediated cell migration. After forming a cell wall, polymerized actin reorganizes into fungal-like cortical patches and cables that extend into hyphal-like structures. Actin perinuclear shells form each cell cycle and polygonal territories emerge during cellularization. This work makes Spizellomyces a genetically tractable model for comparative cell biology and understanding the evolution of fungi and early eukaryotes., Competing Interests: EM, KR, KB, GI, CL, LF, NB No competing interests declared, (© 2020, Medina et al.)

The actin cytoskeleton governs a vast array of core eukaryotic phenotypes that include cell movement, endocytosis, vesicular trafficking, and cytokinesis. Although the basic principle underlying these processes is strikingly simple - actin monomers polymerize into filaments that can depolymerize back into monomers - eukaryotic cells have sophisticated and layered control systems to regulate actin dynamics. The evolutionary origin of these complex systems is an area of active research. Here, we review the regulation and diversity of actin networks to provide a conceptual framework for cell biologists interested in evolution and for evolutionary biologists interested in actin-dependent phenotypes., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

As outlined in their recent paper (A Tubulin Mutation Database: A Resource for the Cytoskeletal Community), Catherine Pham and Naomi Morrissette from the University of California, Irvine, scoured the literature and catalogued data for 489 point mutations for 𝛂-tubulin, 729 for β-tubulin, and 343 for 𝛄, ẟ, 𝛆, and 𝛇 tubulins to create the tubulin mutation database (http://tubulinmutations.bio.uci.edu). The database is a searchable catalog of missense mutations and phenotypes that is expected to grow with biannual updates. Data entries regarding the species and isoform, as well as links to available sequences and the original study which characterized the mutant are intuitively displayed and color coded (Pham & Morrissette, 2019). This database represents a unique opportunity for clinicians and cell biologists to rapidly connect sequence data to mutant phenotypes and gather primary literature which promises to facilitate discoveries on topics including microtubule dynamics, antimitotic drug use and resistance, and evolution. We expect that many researchers will find this tool of great use to their research. This article is protected by copyright. All rights reserved., (© 2019 Wiley Periodicals, Inc.)

The amount of polymerized actin within a cell can vary widely due to natural processes and/or experimentally induced perturbations. We routinely use this protocol to measure relative polymerized actin content between cell populations by staining cells in suspension with fluorescent phalloidin, then measuring total cell fluorescence using flow cytometry. Although developed for human cells, we have easily adapted this method for use with diverse eukaryotic cell types., Competing Interests: Competing interests We have no conflict of interest or competing interest to declare.

Leukocytes and other amoeboid cells change shape as they move, forming highly dynamic, actin-filled pseudopods. Although we understand much about the architecture and dynamics of thin lamellipodia made by slow-moving cells on flat surfaces, conventional light microscopy lacks the spatial and temporal resolution required to track complex pseudopods of cells moving in three dimensions. We therefore employed lattice light sheet microscopy to perform three-dimensional, time-lapse imaging of neutrophil-like HL-60 cells crawling through collagen matrices. To analyze three-dimensional pseudopods we: (i) developed fluorescent probe combinations that distinguish cortical actin from dynamic, pseudopod-forming actin networks, and (ii) adapted molecular visualization tools from structural biology to render and analyze complex cell surfaces. Surprisingly, three-dimensional pseudopods turn out to be composed of thin (<0.75 µm), flat sheets that sometimes interleave to form rosettes. Their laminar nature is not templated by an external surface, but likely reflects a linear arrangement of regulatory molecules. Although we find that Arp2/3-dependent pseudopods are dispensable for three-dimensional locomotion, their elimination dramatically decreases the frequency of cell turning, and pseudopod dynamics increase when cells change direction, highlighting the important role pseudopods play in pathfinding.

Diverse eukaryotic cells crawl through complex environments using distinct modes of migration. To understand the underlying mechanisms and their evolutionary relationships, we must define each mode and identify its phenotypic and molecular markers. In this study, we focus on a widely dispersed migration mode characterized by dynamic actin-filled pseudopods that we call "α-motility." Mining genomic data reveals a clear trend: only organisms with both WASP and SCAR/WAVE-activators of branched actin assembly-make actin-filled pseudopods. Although SCAR has been shown to drive pseudopod formation, WASP's role in this process is controversial. We hypothesize that these genes collectively represent a genetic signature of α-motility because both are used for pseudopod formation. WASP depletion from human neutrophils confirms that both proteins are involved in explosive actin polymerization, pseudopod formation, and cell migration. WASP and WAVE also colocalize to dynamic signaling structures. Moreover, retention of WASP together with SCAR correctly predicts α-motility in disease-causing chytrid fungi, which we show crawl at >30 µm/min with actin-filled pseudopods. By focusing on one migration mode in many eukaryotes, we identify a genetic marker of pseudopod formation, the morphological feature of α-motility, providing evidence for a widely distributed mode of cell crawling with a single evolutionary origin., (© 2017 Fritz-Laylin et al.)

The amoeboflagellate Naegleria was one of the first organisms in which de novo basal body/centriole assembly was documented. When in its flagellate form, this single-celled protist has two flagella that are templated by two basal bodies. Each of these basal bodies is structurally well conserved, with triplet microtubules and well-defined proximal cartwheel structures, similar to most other eukaryotic centrioles. The basal bodies are anchored to the nucleus by a single, long striated rootlet. The Naegleria genome encodes many conserved basal body genes whose expression is induced prior to basal body assembly. Because of the rapid and synchronous differentiation from centriole-less amoebae to temporary flagellates with basal bodies, Naegleria offers one of the most promising systems to study de novo basal body assembly, as well as the mechanisms regulating the number of centrioles assembled per cell.

Centrioles are eukaryotic organelles whose number and position are critical for cilia formation and mitosis. Many cell types assemble new centrioles next to existing ones ("templated" or mentored assembly). Under certain conditions, centrioles also form without pre-existing centrioles (de novo). The synchronous differentiation of Naegleria amoebae to flagellates represents a unique opportunity to study centriole assembly, as nearly 100% of the population transitions from having no centrioles to having two within minutes. Here, we find that Naegleria forms its first centriole de novo, immediately followed by mentored assembly of the second. We also find both de novo and mentored assembly distributed among all major eukaryote lineages. We therefore propose that both modes are ancestral and have been conserved because they serve complementary roles, with de novo assembly as the default when no pre-existing centriole is available, and mentored assembly allowing precise regulation of number, timing, and location of centriole assembly., (© 2016 Wiley Periodicals, Inc.)

Horizontal gene transfer allows organisms to rapidly acquire adaptive traits. Although documented instances of horizontal gene transfer from bacteria to eukaryotes remain rare, bacteria represent a rich source of new functions potentially available for co-option. One benefit that genes of bacterial origin could provide to eukaryotes is the capacity to produce antibacterials, which have evolved in prokaryotes as the result of eons of interbacterial competition. The type VI secretion amidase effector (Tae) proteins are potent bacteriocidal enzymes that degrade the cell wall when delivered into competing bacterial cells by the type VI secretion system. Here we show that tae genes have been transferred to eukaryotes on at least six occasions, and that the resulting domesticated amidase effector (dae) genes have been preserved for hundreds of millions of years through purifying selection. We show that the dae genes acquired eukaryotic secretion signals, are expressed within recipient organisms, and encode active antibacterial toxins that possess substrate specificity matching extant Tae proteins of the same lineage. Finally, we show that a dae gene in the deer tick Ixodes scapularis limits proliferation of Borrelia burgdorferi, the aetiologic agent of Lyme disease. Our work demonstrates that a family of horizontally acquired toxins honed to mediate interbacterial antagonism confers previously undescribed antibacterial capacity to eukaryotes. We speculate that the selective pressure imposed by competition between bacteria has produced a reservoir of genes encoding diverse antimicrobial functions that are tailored for co-option by eukaryotic innate immune systems.

Naegleria gruberi, a free-living protist, has long been treasured as a model for basal body and flagellar assembly due to its ability to differentiate from crawling amoebae into swimming flagellates. The full genome sequence of Naegleria gruberi has recently been used to estimate gene families ancestral to all eukaryotes and to identify novel aspects of Naegleria biology, including likely facultative anaerobic metabolism, extensive signaling cascades, and evidence for sexuality. Distinctive features of the Naegleria genome and nuclear biology provide unique perspectives for comparative cell biology, including cell division, RNA processing and nucleolar assembly. We highlight here exciting new and novel aspects of Naegleria biology identified through genomic analysis., (Copyright © 2011 Institut Pasteur. Published by Elsevier SAS. All rights reserved.)

Naegleria gruberi is a single-celled eukaryote best known for its remarkable ability to form an entire microtubule cytoskeleton de novo during its metamorphosis from an amoeba into a flagellate, including basal bodies (equivalent to centrioles), flagella and a cytoplasmic microtubule array. Our publicly available full-genome transcriptional analysis, performed at 20-minute intervals throughout Naegleria differentiation, reveals vast transcriptional changes, including the differential expression of genes involved in metabolism, signaling and the stress response. Cluster analysis of the transcriptional profiles of predicted cytoskeletal genes reveals a set of 55 genes enriched in centriole components (induced early) and a set of 82 genes enriched in flagella proteins (induced late). The early set includes genes encoding nearly every known conserved centriole component, as well as eight previously uncharacterized, highly conserved genes. The human orthologs of at least five genes localize to the centrosomes of human cells, one of which (here named Friggin) localizes specifically to mother centrioles.

Protists account for the bulk of eukaryotic diversity. Through studies of gene and especially genome sequences the molecular basis for this diversity can be determined. Evident from genome sequencing are examples of versatile metabolism that go far beyond the canonical pathways described for eukaryotes in textbooks. In the last 2-3 years, genome sequencing and transcript profiling has unveiled several examples of heterotrophic and phototrophic protists that are unexpectedly well-equipped for ATP production using a facultative anaerobic metabolism, including some protists that can (Chlamydomonas reinhardtii) or are predicted (Naegleria gruberi, Acanthamoeba castellanii, Amoebidium parasiticum) to produce H(2) in their metabolism. It is possible that some enzymes of anaerobic metabolism were acquired and distributed among eukaryotes by lateral transfer, but it is also likely that the common ancestor of eukaryotes already had far more metabolic versatility than was widely thought a few years ago. The discussion of core energy metabolism in unicellular eukaryotes is the subject of this review. Since genomic sequencing has so far only touched the surface of protist diversity, it is anticipated that sequences of additional protists may reveal an even wider range of metabolic capabilities, while simultaneously enriching our understanding of the early evolution of eukaryotes., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

The multicellular green alga Volvox carteri and its morphologically diverse close relatives (the volvocine algae) are well suited for the investigation of the evolution of multicellularity and development. We sequenced the 138-mega-base pair genome of V. carteri and compared its approximately 14,500 predicted proteins to those of its unicellular relative Chlamydomonas reinhardtii. Despite fundamental differences in organismal complexity and life history, the two species have similar protein-coding potentials and few species-specific protein-coding gene predictions. Volvox is enriched in volvocine-algal-specific proteins, including those associated with an expanded and highly compartmentalized extracellular matrix. Our analysis shows that increases in organismal complexity can be associated with modifications of lineage-specific proteins rather than large-scale invention of protein-coding capacity.

Centrioles and basal bodies are discrete structures composed of a cylinder of nine microtubule triplets and associated proteins. Metazoan centrioles can be found at mitotic spindle poles and are called basal bodies when used to organize microtubules to form the core structure of flagella. Naegleria gruberi, a unicellular eukaryote, grows as an amoeba that lacks a cytoplasmic microtubule cytoskeleton. When stressed, Naegleria rapidly (and synchronously) differentiates into a flagellate, forming a complete cytoplasmic cytoskeleton de novo, including two basal bodies and flagella. Here, we show that Naegleria has genes encoding conserved centriole proteins. Using novel antibodies, we describe the localization of three centrosomal protein homologs (SAS-6, gamma-tubulin, and centrin-1) during the assembly of the flagellate microtubule cytoskeleton. We also used these antibodies to show that Naegleria expresses the proteins in the same order as their incorporation into basal bodies, with SAS-6 localizing first, followed by centrin and finally gamma-tubulin. The similarities between basal body assembly in Naegleria and centriole assembly in animals indicate that mechanisms of assembly, as well as structure, have been conserved throughout eukaryotic evolution.

Genome sequences of diverse free-living protists are essential for understanding eukaryotic evolution and molecular and cell biology. The free-living amoeboflagellate Naegleria gruberi belongs to a varied and ubiquitous protist clade (Heterolobosea) that diverged from other eukaryotic lineages over a billion years ago. Analysis of the 15,727 protein-coding genes encoded by Naegleria's 41 Mb nuclear genome indicates a capacity for both aerobic respiration and anaerobic metabolism with concomitant hydrogen production, with fundamental implications for the evolution of organelle metabolism. The Naegleria genome facilitates substantially broader phylogenomic comparisons of free-living eukaryotes than previously possible, allowing us to identify thousands of genes likely present in the pan-eukaryotic ancestor, with 40% likely eukaryotic inventions. Moreover, we construct a comprehensive catalog of amoeboid-motility genes. The Naegleria genome, analyzed in the context of other protists, reveals a remarkably complex ancestral eukaryote with a rich repertoire of cytoskeletal, sexual, signaling, and metabolic modules., ((c) 2010 Elsevier Inc. All rights reserved.)

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the approximately 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

The tomato (Lycopersicon esculentum) Cf-9 resistance gene encodes the first characterized member of the plant receptor-like protein (RLP) family. Other RLPs such as CLAVATA2 and TOO MANY MOUTHS are known to regulate development. The domain structure of RLPs consists of extracellular leucine-rich repeats, a transmembrane helix, and a short cytoplasmic region. Here, we identify 90 RLPs in rice (Oryza sativa) and compare them with functionally characterized RLPs from different plant species and with 56 Arabidopsis (Arabidopsis thaliana) RLPs, including the downy mildew resistance protein RPP27. Many RLPs cluster into four distinct superclades, three of which include RLPs known to be involved in plant defense. Sequence comparisons reveal diagnostic amino acid residues that may specify different molecular functions in different RLP subtypes. This analysis of rice RLPs thus identified at least 73 candidate resistance genes and four genes potentially involved in development. Due to the synteny between rice and other Gramineae, this analysis should provide valuable tools for experimental studies in rice and other cereals.