An internally controlled system to study microtubule network diversification links tubulin evolution to the use of distinct microtubule regulators.

Diverse eukaryotic cells assemble microtubule networks that vary in structure and composition. While we understand how cells build microtubule networks with specialized functions, we do not know how microtubule networks diversify across deep evolutionary timescales. This problem has remained unresolved because most organisms use shared pools of tubulins for multiple networks, making it impossible to trace the evolution of any single network. In contrast, the amoeboflagellate Naegleria uses distinct tubulin genes to build distinct microtubule networks: while Naegleria builds flagella from conserved tubulins during differentiation, it uses divergent tubulins to build its mitotic spindle. This genetic separation makes for an internally controlled system to study independent microtubule networks in a single organismal and genomic context. To explore the evolution of these microtubule networks, we identified conserved microtubule binding proteins and used transcriptional profiling of mitosis and differentiation to determine which are upregulated during the assembly of each network. Surprisingly, most microtubule binding proteins are upregulated during only one process, suggesting that Naegleria uses distinct component pools to specialize its microtubule networks. Furthermore, the divergent residues of mitotic tubulins tend to fall within the binding sites of differentiation-specific microtubule regulators, suggesting that interactions between microtubules and their binding proteins constrain tubulin sequence diversification. We therefore propose a model for cytoskeletal evolution in which pools of microtubule network components constrain and guide the diversification of the entire network, so that the evolution of tubulin is inextricably linked to that of its binding partners.

Evolutionary cell biology: New roles for Arp2/3 complex evolution in eukaryotic diversification.

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.

A conserved pressure-driven mechanism for regulating cytosolic osmolarity.

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.

A conserved pressure-driven mechanism for regulating cytosolic osmolarity.

Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments like 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 basic principles governing contractile vacuole function, we here investigate the molecular mechanisms of two species with distinct vacuolar morphologies from different eukaryotic lineagesâ€"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 actin for osmoregulation, as well as vacuolar-type proton pumps for filling contractile vacuoles. 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 . Because these three lineages diverged well over a billion years ago, we propose that this represents an ancient eukaryotic mechanism of osmoregulation.

Naegleria's mitotic spindles are built from unique tubulins and highlight core spindle features.

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 ß-tubulins. The mitotic tubulins are evolutionarily divergent relative to typical α- and ß-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.

A OneStep Solution to Fix and Stain Cells for Correlative Live and Fixed Microscopy.

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.

Evolutionary cell biology: Closest unicellular relatives of animals crawl when squeezed.

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.

Conserved actin machinery drives microtubule-independent motility and phagocytosis in Naegleria.

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.

SuperPlots: Communicating reproducibility and variability in 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.

Diversity and evolution of actin-dependent phenotypes.

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.

Enteropathogenic E. coli relies on collaboration between the formin mDia1 and the Arp2/3 complex for actin pedestal biogenesis and maintenance.

Enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC) are closely related extracellular pathogens that reorganize host cell actin into "pedestals" beneath the tightly adherent bacteria. This pedestal-forming activity is both a critical step in pathogenesis, and it makes EPEC and EHEC useful models for studying the actin rearrangements that underlie membrane protrusions. To generate pedestals, EPEC relies on the tyrosine phosphorylated bacterial effector protein Tir to bind host adaptor proteins that recruit N-WASP, a nucleation-promoting factor that activates the Arp2/3 complex to drive actin polymerization. In contrast, EHEC depends on the effector EspFU to multimerize N-WASP and promote Arp2/3 activation. Although these core pathways of pedestal assembly are well-characterized, the contributions of additional actin nucleation factors are unknown. We investigated potential cooperation between the Arp2/3 complex and other classes of nucleators using chemical inhibitors, siRNAs, and knockout cell lines. We found that inhibition of formins impairs actin pedestal assembly, motility, and cellular colonization for bacteria using the EPEC, but not the EHEC, pathway of actin polymerization. We also identified mDia1 as the formin contributing to EPEC pedestal assembly, as its expression level positively correlates with the efficiency of pedestal formation, and it localizes to the base of pedestals both during their initiation and once they have reached steady state. Collectively, our data suggest that mDia1 enhances EPEC pedestal biogenesis and maintenance by generating seed filaments to be used by the N-WASP-Arp2/3-dependent actin nucleation machinery and by sustaining Src-mediated phosphorylation of Tir.

Relative Quantitation of Polymerized Actin in Suspension Cells by Flow Cytometry.

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.

Extracellular motility and cell-to-cell transmission of enterohemorrhagic E. coli is driven by EspFU-mediated actin assembly.

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are closely-related pathogens that attach tightly to intestinal epithelial cells, efface microvilli, and promote cytoskeletal rearrangements into protrusions called actin pedestals. To trigger pedestal formation, EPEC employs the tyrosine phosphorylated transmembrane receptor Tir, while EHEC relies on the multivalent scaffolding protein EspFU. The ability to generate these structures correlates with bacterial colonization in several animal models, but the precise function of pedestals in infection remains unclear. To address this uncertainty, we characterized the colonization properties of EPEC and EHEC during infection of polarized epithelial cells. We found that EPEC and EHEC both formed distinct bacterial communities, or "macrocolonies," that encompassed multiple host cells. Tir and EspFU, as well as the host Arp2/3 complex, were all critical for the expansion of macrocolonies over time. Unexpectedly, EspFU accelerated the formation of larger macrocolonies compared to EPEC Tir, as EspFU-mediated actin assembly drove faster bacterial motility to cell junctions, where bacteria formed a secondary pedestal on a neighboring cell and divided, allowing one of the daughters to disengage and infect the second cell. Collectively, these data reveal that EspFU enhances epithelial colonization by increasing actin-based motility and promoting an efficient method of cell-to-cell transmission.

Integration of linear and dendritic actin nucleation in Nck-induced actin comets.

The Nck adaptor protein recruits cytosolic effectors such as N-WASP that induce localized actin polymerization. Experimental aggregation of Nck SH3 domains at the membrane induces actin comet tails--dynamic, elongated filamentous actin structures similar to those that drive the movement of microbial pathogens such as vaccinia virus. Here we show that experimental manipulation of the balance between unbranched/branched nucleation altered the morphology and dynamics of Nck-induced actin comets. Inhibition of linear, formin-based nucleation with the small-molecule inhibitor SMIFH2 or overexpression of the formin FH1 domain resulted in formation of predominantly circular-shaped actin structures with low mobility (actin blobs). These results indicate that formin-based linear actin polymerization is critical for the formation and maintenance of Nck-dependent actin comet tails. Consistent with this, aggregation of an exclusively branched nucleation-promoting factor (the VCA domain of N-WASP), with density and turnover similar to those of N-WASP in Nck comets, did not reconstitute dynamic, elongated actin comets. Furthermore, enhancement of branched Arp2/3-mediated nucleation by N-WASP overexpression caused loss of the typical actin comet tail shape induced by Nck aggregation. Thus the ratio of linear to dendritic nucleation activity may serve to distinguish the properties of actin structures induced by various viral and bacterial pathogens.