Chytrid Fungi Construct Actin-rich Pseudopods, Implicating Actin Regulators Wasp and Scar in an Ancient Mode of Cell Motility

Various cells scattered throughout the eukaryotic tree crawl across surfaces or through three-dimensional environments. Evidence now indicates that cell crawling is not a single behavior, but rather a collection of processes, driven by different molecular mechanisms. Understanding these mechanisms and their evolutionary relationships first requires narrowly defining mechanical modes of locomotion, and then identifying phenotypic and molecular markers of each. Here, we focus on a widely dispersed form of cell crawling characterized by dynamic, actin-filled pseudopods and weak, nonspecific adhesion to external substrates, a mode we refer to as " α-motility. " Finding α-motility in cells ranging from free-living amoebae to immune cells hints at a single evolutionary origin of this complex process. By mining recently published genomic data, we identified a clear trend: only organisms with both WASP and SCAR/WAVE— two activators of branched actin assembly—make dynamic, three-dimensional pseudopods. While SCAR has been shown in some organisms to be an important driver of pseudopod formation, a role for WASP in this process is less clear. We hypothesize that these genes together represent a genetic signature of α-motility, and both proteins are used for pseudopod formation. We test our hypothesis by depleting WASP from human neutrophils, and confirm that both proteins are needed for explosive actin polymerization, pseudopod formation, and rapid cell migration. We also found that WASP and SCAR colocalize to the same dynamic signaling structures in living cells. Moreover, genomic retention of WASP together with SCAR also correctly predicts the presence of pseudopods in the disease-causing fungus Batrachochytrium dendrobatidis, making it the first fungus reported to undergo α-motility. By narrowing our focus to a single mode of cell migration while expanding our phylogenetic analysis to a variety of eukaryotes, we identified WASP together with SCAR as a conserved genetic marker of fast, low-adhesion cell crawling. Our cell-biology experiments argue that this conservation reflects their collaboration in the explosive actin assembly required to build dynamic pseudopods. These results represent the clearest evidence for a widely distributed mode of cell crawling with a single evolutionary origin.


Introduction
Eukaryotic cells move using several distinct modes of locomotion, including crawling and flagella-driven swimming. The stereotyped architecture of flagella and the conservation of their protein components render the evolutionary conservation of cell swimming relatively transparent. In contrast, "crawling motility" is a collection of processes whose functional and evolutionary relationships are not well understood (Lämmermann and Sixt, 2009;Paluch and Raz, 2013;Rodriguez et al., 2005). Some crawling cells require dedicated adhesion molecules to make specific, high-affinity contacts with their surroundings, while other cells rely on weaker, nonspecific interactions. Crawling cells also employ different mechanisms to advance their leading edge, either assembling polymerized actin networks to push the plasma membrane forward, or detaching the membrane from the underlying cytoskeleton to form a rapidly expanding bleb. Furthermore, some cell types have been shown to use contractile forces to generate forward movement (Bergert et al., 2012;Lämmermann et al., 2008;Liu et al., 2015). Different cells can also employ different sets of molecules to drive similar modes of crawling. In an extreme example, nematode sperm have evolved a method of crawling in which polymer assembly advances the leading-edge membrane but, in these cells, the force-generating polymer networks are composed of "major sperm protein" rather than actin. Given this variety of crawling behaviors, it is clear that one cannot simply assume that the underlying molecular mechanisms are the same.
The best understood mode of crawling is the slow (1-10 µm/hour) creeping of adherent animal cells, including fibroblasts and epithelial cells (Petrie and Yamada, 2015). These cells move by extending across a surface a sheet-like protrusion called a lamellipodium while gripping substrate molecules using integrins, often clustered into large focal adhesions. Although clinically and physiologically important, this form of adhesionbased crawling is unique to the animal lineage, and largely restricted to molecular "highways" formed by the extracellular matrix.
In contrast, many motile cells-including free-living amoebae and human immune cells-make three-dimensional, actin-filled pseudopods and navigate complex environments at speeds exceeding 20 µm/min (100-1000× faster than creeping fibroblasts) without forming specific molecular adhesions (Buenemann et al., 2010;Butler et al., 2010). Although this mode of fast cell crawling has been called "amoeboid motility," this term is also used to describe a range of behaviors, including cell motility that relies on membrane blebs rather than actin-filled pseudopods (Lämmermann and Sixt, 2009).
To narrow our mechanistic focus we use the term "α-motility" specifically to describe cell crawling that is characterized by: (i) highly dynamic three-dimensional pseudopods at the leading edge filled with branched-actin networks assembled by the Arp2/3 complex; (ii) fast migration, typically on the order of tens of µm/min; and (iii) the absence of specific, high-affinity adhesions to the extracellular environment. This independence from specific molecular adhesions separates α-motility from the adhesion-based motility of fibroblasts and epithelial cells. Furthermore, the use of pseudopods discriminates it ! 4 of 31 from the fast bleb-based motility adopted by fibroblasts in environments that preclude adhesion formation (Liu et al., 2015;Ruprecht et al., 2015). Some organisms using αmotility may also employ additional methods of generating forward movement, such as contractility, retrograde flow, and/or blebbing (Bergert et al., 2012;Lämmermann et al., 2008;Yoshida and Soldati, 2006), but here we focus on a single phenotype readily observable in diverse species, including non-model organisms.
Organisms with cells capable of α-motility appear throughout the eukaryotic tree, and we hypothesize that this form of locomotion reflects a single, discrete process that arose early in eukaryotic evolution and has since been conserved. If this hypothesis is correct, then elements of this ancient process-specific molecules and mechanisms-should be conserved and still associated with cell crawling in distantly related organisms that employ α-motility. Such molecular remnants would help to unravel the evolutionary history of cell locomotion, and might enable us to predict the existence of specific modes of motility in unexplored species. Identifying genes associated with a process such as α-motility is not trivial because the core machinery driving pseudopod formation (e.g. actin and the Arp2/3 complex) is shared with other cellular activities, including some types of endocytosis (Winter et al., 1997). We therefore turned our attention to upstream regulators of actin assembly. WASP and SCAR (also known as WAVE) are widely conserved "nucleation promoting factors" that stimulate branched actin network assembly by the Arp2/3 complex in response to various upstream cellular signals (Koronakis et al., 2011;Moreau et al., 2000;Rohatgi et al., 1999) and promote different levels of Arp2/3 activity (Zalevsky et al., 2001). The broad phylogenetic distributions of WASP and SCAR gene families suggest that both genes are ancient and likely to have been present in the eukaryotic ancestor (Kollmar et al., 2012;Veltman and Insall, 2010), making them appealing candidates for genetic markers of α-motility.
SCAR plays a major role in the formation of lamellipodia required for slow, adhesiondependent migration of fibroblasts (Miki et al., 1998;Steffen et al., 2004) as well as pseudopods at the leading edge of fast-moving amoebae and neutrophils (Veltman et al., 2012;Weiner et al., 2006). In contrast, the involvement of WASP genesparticularly the two mammalian homologs WASP and N-WASP-in cell crawling is less clear, perhaps due to confusion between adhesion-based cell migration and α-motility. N-WASP is ubiquitously expressed in mammals, and is dispensable for lamellipodia or filopodia formation by adherent fibroblasts (Lommel et al., 2001;Sarmiento et al., 2008;Snapper et al., 2001), which has lead many researchers to discount a role for any WASP protein in protrusions or motility (Small and Rottner, 2010). Mammalian WASP, on the other hand, is expressed only in blood cells, where it has been shown to be involved in migration and pseudopod formation (Badolato et al., 1998;Burns et al., 2001;Ishihara et al., 2012;G. E. Jones et al., 2002;R. A. Jones et al., 2013;Shi et al., 2009). Recent work has demonstrated that, despite having similar domain organization, the proteins must have distinct biological functions because N-WASP cannot compensate for the migration defect that results from loss of WASP expression in Tcells (Jain and Thanabalu, 2015).The use of WASP by blood cells, the only cells in the body known to build three-dimensional pseudopods, may therefore reflect unique requirements for α-motility. Further evidence of this has come from the handful of ! 5 of 31 papers studying WASP in non-mammallian cells that also point to a role of WASP in cell migration (Veltman et al., 2012;Zhu et al., 2016). (See also Table S1 for an annotated bibliography of 29 papers on WASP and N-WASP relating to cell migration, summarized above.) To understand the regulation of the actin cytoskeleton during pseudopod formation, we exploited the diversity of organisms that use α-motility: by comparing the genomes of many eukaryotes, we found that organisms with genes encoding both WASP and SCAR make pseudopods, and organisms that do not build pseudopods have lost either or both Arp2/3 activator. We validated this molecular signature using a negative test (depleting the protein disrupts pseudopod formation in well-studied cells), as well as a positive test (a new prediction of α-motility in a little-studied organism). Differentiating α-motility from slow/adhesive cell migration helps clarify the confusion over WASP's importance in cell motility, and shifts the major question from whether WASP or SCAR is required for motility in a given single cell type, to how WASP and SCAR work together to construct and maintain pseudopods in many species. The retention of WASP and SCAR by organisms that form pseudopods represents the first molecular support, to our knowledge, for a single origin of this widespread form of cell motility in an ancestor of extant eukaryotes (see summary Figure 6).

Evolutionary retention of both WASP and SCAR correlates with pseudopod formation
To trace the evolutionary history of α-motility, we first determined which sequenced eukaryotic organisms might employ α-motility, combing the literature for references to organisms with cells that form pseudopods. Eukaryotic phyla fall into at least six large clades, and species with sequenced genomes and that form pseudopods can be found in most (Table 1 and summary Figure 6).
On to this map of the phylogenetic distribution of pseudopods, we overlayed the conservation of WASP and SCAR/WAVE genes, using a recently published, manually curated database of nucleation promoting factors from genomes spanning eukaryotic diversity (Kollmar et al., 2012). Multiple analyses indicate that both WASP and SCAR were present in the last common ancestor of eukaryotes (Kollmar et al., 2012;Veltman and Insall, 2010), and therefore argue that a lack of either gene reflects loss during evolution.
To understand whether these gene loss events reveal a significant pattern, we compared the conservation of individual nucleation promoting factors across large evolutionary distances with the ability to assemble pseudopods. We observed a nearly perfect correlation between the conservation of WASP and SCAR and pseudopod formation (Table 1 and summary Figure 6); the only exceptions from this correlation were two little-studied species of chytrid fungi. For example, no plant cells build pseudopods and no sequenced plant genomes contain a WASP ortholog. Similarly, multicellular fungi-the dikarya-lack SCAR and are also not known to build pseudopods. Conversely, almost all sequenced genomes of Amoebozoan species (including dictyostelids) encode orthologs of WASP and SCAR, and almost all move with the help dynamic, actin-rich pseudopods. A interesting example is the amoeba Entamoeba histolytica, which forms Arp2/3-dependent phagocytic "food cups" to engulf bacteria (Babuta et al., 2015), but moves using blebs (Maugis et al., 2010), and also lacks genes encoding both WASP and SCAR. We were unable to find a single example of an organism lacking either WASP or SCAR that is capable of constructing motile, actin-rich pseudopods. We did, however, find two littlestudied species of chytrid fungi that had retained both nucleation promoting factors, but are not known for α-motility, Allomyces macrogynus and Batrachochytrium dendrobatidis.
We took a two-pronged approach to testing our hypothesis that retention of WASP together with SCAR serves as a molecular signature of pseudopod formation. First, we took the more traditional approach and confirmed that both genes are involved in pseudopod formation in mammalian cells. We followed this with an evolution-based approach by verifying the ability of this molecular signature to predict the capacity for pseudopod formation in chytrid fungi.

WASP and SCAR localize to the same dynamic arcs within pseudopods of human neutrophils
Our evolutionary evidence indicates that WASP and SCAR may both be required to build pseudopods. To test this hypothesis directly, we turned to human cell lines capable of forming pseudopods. HL-60 cells are derived from an acute myeloid leukemia (Collins et al., 1977) and retain many features of hematopoietic cells, including expression of hematopoietic WASP and the capacity to differentiate into fast-migrating neutrophils with dynamic pseudopods (Collins et al., 1978).
To follow the dynamics of WASP localization in live cells, we created an HL-60 line stably expressing full-length WASP fused at the N-terminus to the red florescent protein TagRFP-T. By confocal fluorescence microscopy, TagRFP-WASP concentrates in two distinct locations within migrating HL-60 cells: punctate foci distributed throughout the cell and a broad zone near the leading edge ( Figure S1A).
Others have shown that the SCAR regulatory complex localizes to fast-moving, anterograde "waves" that break against the leading edge of actively migrating HL-60 cells (Weiner et al., 2007). This localization pattern is most easily observed using total internal reflection fluorescence (TIRF) microscopy, which illuminates a ~100 nm thick region of the cell near the ventral surface (Axelrod, 1981). Using TIRF microscopy on rapidly migrating HL-60 cells, we observed that TagRFP-WASP concentrates near the leading edge in linear arcs that move in an anterograde direction, similar to previously observed patterns of the SCAR regulatory complex (Weiner et al., 2007).

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To see whether WASP and the SCAR travel together in the same waves, we introduced TagRFP-WASP into cells expressing YFP-Hem1, a core component of the SCAR regulatory complex (Weiner et al., 2007). TIRF microscopy of these cells revealed that WASP and the regulatory SCAR complex move together in the same dynamic, linear arcs ( Figure 1A-B, Figure S1B, and Video 1). Interestingly, however, the localization patterns of the two are not identical, an observation confirmed by quantifying WASP and SCAR localization across the leading edge ( Figure S1C). Spinning disk confocal microscopy indicates that WASP and SCAR colocalize throughout the growing pseudopods, not only at the ventral surface ( Figure 1C). Within the resolution limits of our imaging, the localization patterns move together, with neither protein consistently leading the other ( Figure S1B and Video 1). This dynamic localization pattern suggests that both WASP and SCAR activate the Arp2/3 complex in leading-edge pseudopods, promoting assembly of the branched actin networks required for membrane protrusion.

WASP participates in pseudopod assembly in neutrophils
To investigate whether WASP is involved in pseudopod assembly, we generated anti-WASP small hairpin RNAs (shRNAs), expression of which results in a >90% reduction of WASP protein by HL-60 cells (Figure 2A). We next examined whether WASPdepleted (WASP-KD) cells can form pseudopods. In a gradient of chemoattractant (the peptide fMet-Leu-Phe), wildtype HL-60 cells become strongly polarized with broad, actin-rich pseudopods used to rapidly move toward the source of chemoattractant ( Figure 2B and Video 2). Compared to control, 50% fewer WASP-KD cells formed pseudopods ( Figure 2B-C). Despite numerous attempts, we never succeeded in developing WASP-KD cell lines in which this phenotype was 100% penetrant. Although this might reflect the function of residual WASP protein, it is also consistent with the fact that WASP knockout mice show only a partial defect in neutrophil migration in vivo (Snapper et al., 2005).
In addition to the defect in pseudopod formation, approximately 20% of WASP-KD cells form large protrusions that taper to a point, reminiscent of a rhinoceros horn ( Figure 2B, D, Figure S2, and Video 2 WASP-KD cells 11, 32, 33, 39 and 42, for example). To verify its specificity, we rescued this "rhino" phenotype by expressing a functional WASP containing three silent mutations in the sequence targeted by to the shRNA ( Figure 2E). Additionally, a second shRNA that targets a separate region of the WASP gene results in a significantly smaller effect on both WASP expression and the number of cells with the rhino phenotype (not shown). Immunofluorescence combined with phalloidin staining of polymerized actin revealed that the aberrant rhino protrusions contain actin filaments but lack microtubules ( Figure 2D). The expression of a probe specific for polymerized actin (mCherry fused to calponin homology domain of Utrophin, Utr261 (Burkel et al., 2007)) revealed a highly dynamic and, surprisingly, hollow actin filament network inside the protrusions ( Figure S2). This distribution-enriched near the membrane but depleted from the core of the protrusion-is more reminiscent of cortical actin networks than of filopodia, which are packed tight with actin bundles (Tilney et al., 1973).

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Because some WASP family proteins contribute to endocytosis (Benesch et al., 2005;Merrifield et al., 2004;Naqvi et al., 1998), we investigated whether the defects in WASP-KD cells were caused by reduction of endocytosis. In undifferentiated HL-60s, we observed no difference in transferrin receptor endocytosis and recycling between WASP-KD and control cells ( Figure S3E). After differentiation into cells capable of making pseudopods, WASP-KD HL-60s actually showed increased internalization, recycling, and surface receptor densities compared to control cells ( Figure S3B-D). Therefore, we cannot attribute the WASP-KD phenotypes simply to a curtailment of endocytosis activity.

WASP-depleted neutrophils polymerize less actin in response to chemoattractant
Addition of chemoattractant to non-polarized (quiescent) HL-60 cells induces a burst of actin polymerization that drives polarization and pseudopod formation, nearly doubling the cell's polymerized actin content within 30 seconds of stimulation. This response is already known to depend on the activity of the SCAR regulatory complex (Weiner et al., 2006). To determine what role WASP might play in this explosive actin assembly, we synchronized pseudopod formation by stimulating populations of quiescent HL-60s with fMLP, fixed and stained the cells with phalloidin at different time points, and analyzed total polymerized actin content in each cell by confocal microscopy and fluorescenceactivated cell sorting (FACS) (Figure 2F-G). In the absence of chemoattractant, the amount of polymerized actin in quiescent WASP-KD cells is roughly equal to that in control cells. However, as reported for SCAR-depleted cells (Weiner et al., 2006), WASP-KD cells have greatly reduced actin polymerization at both short (30 seconds) and long times (3 min) following stimulation. This reduced actin polymerization indicates that WASP, like SCAR, is central to the explosive actin polymerization required for cell polarization and subsequent pseudopod formation.

WASP depletion impairs neutrophil motility
To determine the effect of WASP depletion on cell locomotion, we imaged HL-60 cells migrating through a chemoattractant gradient in a 5 µm tall glass chamber (Millius and Weiner, 2010). Tracking individual cells revealed a severe migration defect in WASP-KD cells (reported mean ± standard deviation of three biological replicates): while control cells move at 12 ± 0.8 µm/min, WASP-KD cells average 5.5 ± 1.5 µm/min, and cells with rhino protrusions are almost completely immotile moving at 1.7 ± 0.4 µm/min ( Figure 3A-B and Video 2). We did not observe an effect of WASP depletion on directional persistence ( Figure 3C).
Because the chamber we used to measure directional migration is not pre-coated with fibronectin (or any other specific molecule), we doubt this migration defect is due to an integrin-mediated adhesion defect. We confirmed this by directly testing adhesion to fibronectin-coated surfaces found no significant difference between WASP-KD and control cells ( Figure S3A). We conclude that HL-60 cells use WASP, along with SCAR (Weiner et al., 2006), for normal pseudopod formation and efficient α-motility.

WASP and SCAR genes predict pseudopod formation by chytrid fungi
The only potential exceptions to the tight correlation between actin-rich pseudopods and the genomic retention of WASP and SCAR were two deeply branching species of fungi, the chytrids Allomyces macrogynus and Batrachochytrium dendrobatidis (Bd). These chytrid species contain genes encoding both WASP and SCAR, but have not been reported in the literature to migrate using pseudopods. We were, however, able to find references to pseudopod formation by unsequenced infectious species related to A. macrogynus (Catenaria anguillulae), which may employ these structures for motility across the surface of its target host (Deacon, 1997;Gleason and Lilje, 2009). However, because chytrid fungi are not a monophyletic group, but rather comprise multiple deeply branching clades that are estimated to have diverged around 800 million years ago (Stajich et al., 2009) (James et al., 2006), one cannot assume that distantly related species share this capacity. Therefore, we used Bd as a predictive test of our hypothesis that WASP and SCAR genes represent a marker for α-motility.
Like other species of chytrid fungi, the lifecycle of Bd has two stages: a large (10-40 µm) reproductive zoosporangium, which releases a host of small (3-5 µm), motile, flagellated zoospores (Berger et al., 2005;Longcore et al., 1999). These infectious zoospores can form cysts beneath the skin of an amphibian host that develop into new zoosporangia to complete the life cycle (Berger et al., 2005). We searched for α-motility in Bd zoospores because, unlike the sessile cyst and zoosporangium, these freeswimming flagellates lack a cell wall and have been reported to assume non-uniform shapes with dense "cytoplasmic extensions" (Longcore et al., 1999).
To restrict the fast-swimming Bd zoospores to the imaging plane, we adhered zoospores to Concalavin-A coated glass. In initial experiments, we observed only a small fraction (<1%) of zoospores forming pseudopod-like protrusions. The rarity of pseudopod-forming cells suggested that α-motility might only occur during a short phase of the life cycle. We therefore enriched for cells of the same age by washing zoosporangia to remove previously released zoospores and collecting flagellates released during the subsequent two hours.
During the first 6 hours after release from the zoosporangium, ~40% of zoospores create dynamic pseudopod-like protrusions ( Figure 4A-B, Figure S4A, and Video 3) that extend from the cell body at a rate of 25 ± 9 µm/min standard deviation ( Figure 4C), consistent with speeds expected for pseudopods (Chodniewicz and Zhelev, 2003;Zhelev et al., 2004). Unlike blebs, these cellular protrusions are not spherical but irregularly shaped and amorphous-similar to the actin-rich pseudopods of amoebae and neutrophils.
To ensure that these crawling cells were not contaminating organisms, we obtained independently isolated Bd cultures from three different laboratories, and observed similar pseudopods in each.
To better investigate the morphology of these tiny pseudopods, we performed scanning electron microscopy on fixed cells (Figure 4D and S4B), and observed a similar proportion of flagellated zoospores with one or more thick protrusions. Each protrusion ! 10 of 31 was about 1 µm long and 1 µm wide, and many appeared to be composed of multiple discrete terraces (Figure 4D and S4B).
Chytrid pseudopods contain actin and require Arp2/3 activity Using our assay to image chytrid zoospores, we next investigated whether extension of Bd pseudopods is driven by assembly of branched actin networks, as in other cells crawling using α-motility. We first fixed the cells to preserve the actin cytoskeleton and then stained them with fluorescent phalloidin to reveal a thin shell of cortical actin surrounding the cell body and a dense network of filamentous actin filling the pseudopod ( Figure 4E). To determine whether assembly of the pseudopodial actin network requires the nucleation and branching activity of the Arp2/3 complex, we incubated zoospores with CK-666, a small molecule that inhibits actin nucleation by mammalian and fungal Arp2/3 complexes (Nolen et al., 2009). Addition of 10 µM of CK-666 reduced the number of cells with active protrusions by nearly 100%, an effect reversed by washing out the drug (Figure 5A-B). These experiments reveal that protrusion of Bd pseudopods requires Arp2/3-dependent actin assembly.

Chytrid zoospores use pseudopods for α-motility
Although pseudopod-forming Bd cells adhere tightly to glass surfaces coated with Concalavin-A, they were not able to move or swim away from the site of initial attachment, and other coatings did not promote any form of attachment (including collagen, fibronectin, and human keratin, not shown). Several types of animal cells are known to migrate without specific molecular adhesions in confined environments (Lämmermann et al., 2008;Liu et al., 2015;Ruprecht et al., 2015). To test whether Bd zoospores might also be capable of migration in confined environments, we inserted cells between uncoated glass coverslips, held apart by 1 µm diameter glass microspheres, and observed rapidly migrating cells (Figure 5C-D, Video 4). Obviously migrating cells had an average instantaneous speed of 19 ± 9 µm/min standard deviation, with individual cells averaging speeds over 30 µm/min (Figure 5E), consistent with the rates of pseudopod extension described above ( Figure 4C). The trajectories of these cells appeared fairly straight ( Figure 5D), with an average directional persistence of 0.61 ± 0.25 standard deviation ( Figure 5F).
Some pseudopod-forming zoospores retained flagella, while other cells had clearly lost or resorbed their flagella and strongly resembled free-living amoebae ( Figure 5C and Videos 3-4). We also observed cells switching from crawling to flagellar motility and vice versa, as well as cells rapidly retracting their flagellar axonemes into the cell body (Video 5).

Discussion
Our results reveal that, across eukaryotic phyla, cells capable of constructing actin-rich pseudopods and performing fast, low-adhesion crawling retain, in addition to the Arp2/3 complex, two distinct activators of its actin nucleation activity: WASP and SCAR/WAVE. Our data is well supported by a recent paper implicating both WASP and SCAR in C. elegans neuroblast cell migration (Zhu et al., 2016). In that system, the phenotype of SCAR mutants is enhanced by loss of WASP, and suggests that both WASP and SCAR is found at the leading edge of migrating neuroblasts in vivo. Our model is also consistent with requirements for building protrusions that mediate myoblast cell fusion events during muscle formation: like pseudopods, these protrusions are actin-filled force generating machines, and require WASP and SCAR .
Organisms without the capacity to crawl using pseudopods turn out to have lost one or both of these nucleation-promoting factors (Table 1 and Figure 6). The presence of genes encoding both WASP and SCAR, therefore, provides a molecular correlate for a suite of behaviors that we call "α-motility." The conservation and phylogeny of WASP and SCAR indicate that both were present in a common ancestor of living eukaryotes (Kollmar et al., 2012;Veltman and Insall, 2010). The power of WASP and SCAR as a genomic marker with the ability to identify cryptic pseudopod-forming organisms, together with cell-biology evidence that both WASP and SCAR are required for α-motility in well-studied organisms, argues that this widespread behavior arose from single, and ancient origin. Alternatively, it is possible that α-motility did not have a single evolutionary origin, but that scenario would require both WASP and SCAR to be co-opted together for pseudopod assembly multiple times during eukaryotic history. Because WASP and SCAR are only two of a large number of Arp2/3 activators , we have no reason to believe that motility would repeatedly converge on these two in particular.
Together with the metazoans and a handful of protists, fungi form a major clade known as the "opisthokonts" (Figure 6). Our identification of α-motility in a fungal species argues that the ancestor of all the opisthokonts was capable of fast, pseudopodassociated crawling and that multicellular fungi represent lineages that have lost αmotility (Fritz-Laylin et al., 2010). This loss appears to coincide with the disappearance from multicellular fungi of cell types lacking a cell wall. We suggest that the limitations on pseudopod formation imposed by a rigid cell wall obviated any possible selection pressure to preserve gene networks specific for fast crawling motility and the genes unique to this behavior were lost. The fungi would therefore represent a large eukaryotic lineage from which crawling motility has almost completely disappeared.
Images from earlier studies revealed individual Bd zoospores with irregular shapes and "cytoplasmic extensions" (Longcore et al., 1999). Actin-driven pseudopod formation and cell motility, however, was not previously described in Bd cells, in part because this species was discovered quite recently (Longcore et al., 1999) and relatively few studies have been devoted to its cell biology. In addition, Bd zoospores are quite small (<5 µm) and highly motile so that visualizing their tiny (<1 µm) pseudopods requires physical confinement and high-resolution microscopy. Finally, because crawling motility appears most frequently during a specific stage of the Bd life cycle-in zoospores recently released from zoosporangia-synchronization of cell cultures was crucial. These advances not only enabled us to observe α-motility, but also revealed Bd zoospores retracting their flagella by coiling the entire axoneme into the cell body in less than a ! 12 of 31 second (Video 5), a process that has been observed to take minutes in other chytrid species (Koch, 1968).
The α-motility of Bd fills an important gap in our understanding of the life cycle of this pathogen. As proposed for other chytrid species (Gleason and Lilje, 2009), Bd zoospores may use pseudopods during the initial stages of their interaction with a host: either to move across epithelia or crawl between epithelial cells and invade the underlying stroma. Alternatively, our observation that newly hatched zoospores make more pseudopods suggests that Bd may rely on α-motility to crawl along or within the epithelial surface to uninfected tissues, or to exit the host.
Imaging chytrid zoospores provided key evidence for the involvement of WASP and SCAR in a conserved mode of cell migration, but further exploration of WASP and SCAR function in Bd was hampered by several factors. First, the small size of Bd zoospores (<5 µm) poses challenges to live-cell imaging. Second, a lack of genetic tools for this species makes it difficult to fluorescently label or deplete proteins in the cells. Third, the lack of potent and specific chemical inhibitors of WASP and SCAR (Bompard et al., 2008;Guerriero and Weisz, 2007) means that it is currently impossible to disrupt their activity chemically.
Several protein families are known to stimulate nucleation activity of the Arp2/3 complex, including WASP, SCAR, JMY, and WHAMM. A conventional explanation for this multiplicity of nucleation promoting factors is that each one directs construction of branched actin networks with different functions at different locations. The conserved connection between WASP, SCAR, and pseudopod formation, however, suggests that this pair of nucleation promoting factors works together to drive the explosive actin polymerization required for pseudopod assembly during α-motility. It is possible that WASP and SCAR act together as a "coincidence detector" that integrates multiple signals and damps formation of spurious pseudopods (Stradal and Scita, 2006). This model is supported by studies of the upstream activators of WASP (Cdc42) and SCAR (Rac) (Koronakis et al., 2011;Lebensohn and Kirschner, 2009;Rohatgi et al., 1999;. Specifically, Srinivasan et al. suggest that Rac mediates a positive feedback loop required for leading-edge formation, but the stability of the resulting protrusion requires Cdc42 (Srinivasan et al., 2003).
This model fits the colocalization of WASP and the SCAR regulatory complex at the leading edge (Figure 1) and the observation that, while depleting the SCAR regulatory complex leads to a loss of pseudopods in nearly 100% of cells (Weiner et al., 2006), WASP depletion gives only a partially penetrant phenotype. Although it is possible that this is due to residual WASP protein, this seems an insufficient explanation as WASP knockout mice also show only a partial defect in the gross motility of neutrophils in vivo (Snapper et al., 2005). This implies a requirement for SCAR in mammalian cells, with WASP playing a supporting role. However, the two proteins may play opposite roles in Dictyostelium discoideum, the only non-metazoan amoeboid organism in which WASP and SCAR have been studied: Myers et al. (Myers et al., 2005) reported that high levels of WASP expression are required for cell polarization and chemotaxis, and Veltman et ! 13 of 31 al. (Veltman et al., 2012) found that WASP partially compensates for a deletion of SCAR, maintaining cell motility. This coincidence detection model may also explain the rhino cell morphology observed in WASP-depleted cells: if the formation of robust pseudopods requires activation of Arp2/3 by both WASP and SCAR, the resulting more sparsely branched actin networks generated by WASP-depleted cells might collapse and coalesce into the observed rhino horn structures.
Our results are also supported by papers showing that blood cells rely on WASP for efficient cell migration (Anderson et al., 2003;Binks et al., 1998;Blundell et al., 2008;Dovas et al., 2009;Kumar et al., 2012;Snapper et al., 2005;Worth et al., 2013;Zhang et al., 2006;Zicha et al., 1998), and others suggesting that WASP plays a direct role in protrusion formation, including pseudopods (Badolato et al., 1998;Burns et al., 2001;Ishihara et al., 2012;G. E. Jones et al., 2002;R. A. Jones et al., 2013;Shi et al., 2009). However, these data have been overshadowed by studies showing that fibroblasts do not require N-WASP for filopodia or sheet-like, surface-adhered lamellipodia (Lommel et al., 2001;Sarmiento et al., 2008;Snapper et al., 2001). Such papers have been cited as proof that all WASP family proteins are dispensable for protrusions in general (Small and Rottner, 2010). Such generalizations depend on two assumptions: that N-WASP and WASP have the same molecular function, and that the adherent motility of fibroblasts and α-motility use the same molecular pathways. However, recent molecular replacement studies have shown that WASP and the ubiquitously expressed N-WASP have different functions and cannot compensate for each other (Jain and Thanabalu, 2015). Furthermore, when one considers the large body of mammalian WASP literature in the light of distinct modes of motility, a simple pattern emerges: cell types that do not natively express WASP do not make pseudopods (although they may make surfacebound lamellipodia, linear filopodia, or adhesive structures called podosomes); WASP is only expressed in blood cells, and these cells use WASP for pseudopod-based migration. (See Table S1 for an annotated summary of WASP/N-WASP literature.) The predominant view that WASP is not involved in cell migration demonstrates the peril of assuming that insights based on adhesion-dependent cell motility apply to other modes of cell crawling.
In addition to α-motility, WASP family proteins have been shown to play roles in other cellular processes, including endocytosis (Benesch et al., 2005;Merrifield et al., 2004;Naqvi et al., 1998). The relationship between cell motility and endocytosis and is complex and not completely understood (Schiefermeier et al., 2011;Traynor and Kay, 2007). Rapid pseudopod extension requires not only a large quantity of actin polymerization (Weiner et al., 2006), but also increases membrane tension (Diz-Muñoz et al., 2016), both of which counteract efficient clathrin-and actin-mediated endocytosis (Boulant et al., 2011). Despite this apparent dichotomy between protrusion formation and endocytosis, both WASP and SCAR protein families have been shown to interact with endocytosis pathways. In adherent Drosophila cells, for example, membranebound clathrin recruits SCAR and promotes lamellipodia formation without additional endocytic machinery (Gautier et al., 2011), and T-cells lacking WASP exhibit impaired receptor internalization (Badour et al., 2007). Here, we find that WASP-deficient HL-60 cells maintain normal receptor internalization and recycling (Figure S3E), until pseudopod activity is activated by differentiation into neutrophils. After differentiation, in addition to being defective in building pseudopods, WASP-KD cells exhibit increased endocytosis and receptor recycling (Figure S3B-D). This is consistent with the idea that actin-mediated endocytosis is more efficient when cells are not making pseudopods.
Although a large number of eukaryotes make pseudopods (Table 1 and Figure 6), only two such lineages are currently molecularly tractable: animals and Dictyostelium. Experimental data in both of these lineages confirm that pseudopod formation involves both WASP and SCAR proteins. Taken together, this indicates that while the adhesiondependent migration used by some animal cell types may require a single Arp2/3 activator (SCAR), the explosive actin polymerization required to build three-dimensional pseudopods involves both SCAR and WASP, and both proteins are conserved together to facilitate this evolutionarily ancient mode of cell motility.
Generation of HL-60 cell lines: HL-60 lines were derived from ATCC #CCL-240, and were grown in medium RPMI 1640 supplemented with 15% FBS, 25 mM Hepes, and 2.0 g/L NaHCO3, and grown at 37C and 5% CO2. WASP-KD was achieved using Sigma's Mission Control shRNA vector (TRCN0000029819), with corresponding control vector expressing anti-GFP shRNA (Catalog# SHC005). Lentivirus was produced in HEK293T grown in 6-well plates and transfected with equal amounts of the lentiviral backbone vector (either protein expression vector derived from pHRSIN-CSGW (Demaison et al., 2002), or shRNA expression vectors described above), pCMV∆8.91 (encoding essential packaging genes) and pMD2.G (encoding VSV-G gene to pseudotype virus). After 48hr, the supernatant from each well was removed, centrifuged at 14,000 g for 5 min to remove debris and then incubated with ~1×10 6 HL-60 cells suspended in 1 mL complete RPMI for 5-12 hours. Fresh medium was then added and the cells were recovered for 3 days to allow for target protein or shRNA expression. TagRFPt-WASP fusion was cloned by first swapping out eGFP for TagRFP-T (Shaner et al., 2008) in the pHRSIN-CSGW by PCR amplifying TagRFP-T with 5'CCCGGGATCCACCGGTCGCCACCATGGTGTCTAAGGGCGAAGAGCTGATTAAG G3' and 5'GAGTCGCGGCCGCTTTAACTAGTCCCGCTGCCCTTGTACAGCTCGTCCATGCCA TTAAGTTTGTGCCCC3' primers, and cloning the resulting PCR product into pHRSIN-CSGW using NotI and BamHI to produce the pHR-TagRFP-T vector. Then, the WASP open reading frame was PCR amplified from cDNA (NCBI accession BC012738) using 5'GCACTAGTATGAGTGGGGGCCCAATGGGAGGAA3' and 5'AAGCGGCCGCTCAGTCATCCCATTCATCATCTTCATCTTCA3' primers, and cloned into the pHR-TAGRFP-T backbone using NotI and SpeI to result in a single open reading frame containing TagRFPt, a flexible linker (amino acids GSGTS), followed by full length WASP. The WASP shRNA rescue vector was cloned by inserting a P2A cleavage site (Kim et al., 2011) between the linker and a WASP open reading frame edited with site-directed mutagenesis to contain three silent mutations within the shRNA-targetting region (5'cgagacctctaaacttatcta3' was changed to 5'CGAaACCTCTAAgCTcATCTA3', with silent mutations in lower case). A corresponding control vector was designed to express TagRFPt with the flexible linker, but no portion of WASP. The Hem1-YFP line was previously described (Weiner et al., 2006). shRNA lines were selected by puromycin (1 µg/mL for at least 1 week), and florescent cell lines by FACS. HL-60 cells were differentiated by treatment with 1.3% DMSO for 5 days.
Cytometry: Fluorescence-activated cell sorting (FACS) analysis was performed on a FACSCalibur analyzer (Bd), Data were analyzed with FlowJo software (Tree Star) and dead cells were gated out using forward and side scatter for all analyses. A FACS Aria II was used for sorting. All FACS analysis was performed at the Laboratory for Cell Analysis (UCSF).
Imaging: EZ-TAXIScan (Effector Cell Institute, Tokyo) analysis of HL-60 cell migration between glass surfaces was conducted as previously described (Millius and Weiner, 2010), and cell migration analyzed using Chemotaxis and Migration Tool (Ibidi). Fixed HL-60 cells were imaged with a 100× 1.40 NA oil Plan Apo objective on a motorized inverted microscope (Nikon Ti-E) equipped with a spinning disk (Yokogawa CSU22) and EMCCD camera (Photometrics Evolve). Live TIRF images were acquired by plating HL-60 cells on coverglass cleaned by a 30 min incubation in 3M NaOH, followed by four washes with PBS, pH 7.2, coated for 30 min with 100 µg/mL bovine fibronectin (Sigma F4759) resuspended in PBS. TIRF microscopy images were acquired on a Nikon TE2000 inverted microscope equipped with a 1.45 NA oil 60× or 100× PlanApo TIRF objective and an EMCCD (Andor iXon+), using previously described imaging conditions (Weiner et al., 2007). Fixed chytrid cells were imaged using an inverted microscope (Nikon Ti-E, Tokyo, Japan) equipped with a spinning-disk confocal system with 33 µm pinholes and a 1.8× tube lens (Spectral Diskovery), a Nikon 60× 1.45 NA Apo TIRF objective, and a CMOS camera (Andor Zyla 4.2). DIC microscopy was performed on an inverted microscope (Nikon Ti-E) with a light-emitting diode illuminator (Sutter TLED) and a Nikon 100× 1.45 NA Apo TIRF objective; images were acquired on a CMOS camera (Andor Zyla 4.2). All microscopy hardware was controlled with Micro-Manager software (Edelstein et al., 2010). Image analysis was performed with the ImageJ bundle Fiji. Three-dimensional reconstructions done with the ClearVolume plugin for Fiji/ImageJ (Royer et al., 2015).

Quantitation of actin polymerization:
HL-60 cells were depolarized in serum free medium supplemented with 2% low-endotoxin BSA (Sigma) for 1 hour at 37C and 5% CO2 before simulation with 20nM fMLP for the indicated time. Cells were immediately fixed with 4% paraformaldehyde in cytoskeleton buffer on ice for 20 min, stained with PBS supplemented with 2% BSA, 0.1% Triton X-100, and 66 nM Alexaflor-488 ! 16 of 31 conjugated phalloidin (Molecular Probes, catalog # A12379) for 20 min, and washed thrice with PBST before FACS analysis.
Cell adhesion assay: Differentiated control and WASP-KD HL-60 cells were each stained with either green or blue acetoxymethyl (AM) ester dyes!(CellTrace calcein green and blue, ThermoFisher)) and equal numbers mixed and allowed to attach to fibronectin-coated coverglass-bottomed 96 well plates for 30 min at 37C. One set of wells was gently washed three times with fresh media. 100 random locations within the well were immediately imaged, and the percent remaining cells calculated and normalized to control unwashed wells.! Transferrin uptake endocytosis assays: 5×10 6 differentiated HL-60 cells were washed twice with ice-cold serum-free growth medium (SF), transferred to 37C for 5 min (to clear surface-bound transferrin), and chilled on ice for 1 min. An equal volume of cold SF supplemented with 100ug/mL Alexa488 conjugated transferrin (McGraw and Subtil, 2001) (Molecular Probes T-13342) was added, and incubated on ice for 10 min. Cells were then washed twice with cold SF medium, transferred to 37C for the indicated time period, washed twice with ice-cold acid buffer (8.76 g NaCl, 9.74 g MES in 900 mL, pH to 4.0, water to 1 L), fixed in 4% paraformaldehyde in 1x PBS for 20 min, and washed twice more with ice-cold PBS before immediate FACS analysis.

Motility of Chytrid zoospores:
Batrachochytrium dendrobatidis strain JEL423 was obtained from the laboratory of Joyce Longcore (University of Maine), and grown in 1% tryptone broth or on agar plates (1% tryptone, 2% agar) at 25C. Before imaging, cultures were synchronized by either three washes in 1% tryptone, and zoospores harvested 2 hours later (for liquid cultures), or flooding agar plates with ~2 mL water (for agar plates), passed through a 40 µm filter (Falcon), and collected by centrifuging at 1200 g for 5 min, and resuspended in Bonner's Salts (BONNER, 1947). Cell motility was imaged by sandwiching cells between a #1.5 glass coverslip and glass slide (cleaned by sonicating in pure water) separated using 1 µm glass microspheres (Bangs Laboratories). Coverslip and glass slide were sonicated in deionized water, and dried. Cells were treated with 10 µM CK-666 (Sigma) while cells were adhered to Concalavin-A coated glass. For visualization of polymerized actin: 400 µL fixation buffer (50 mM cacodylate buffer, pH 7.2) supplemented with 4% gluteraldehyde, was added to 100 µL cells attached to a Concalavin-A coated coverslip, and incubated for 20 min at 4C. Samples were quenched with tetraborohydride, permeabilized with 0.1% Triton X-100, incubated for 20 min with Alexa488-labeled phalloidin (Invitrogen), rinsed 4 times, and imaged as above. Samples for scanning electron microscopy were fixed as above, stained with osmium tetroxide, dehydrated, critical point dried, and Au/Pd sputter coated according to standard protocols and imaged using a Hitachi S-5000 scanning electron microscope in the University of California, Berkeley Electron Microscopy Laboratory.!    (B) Depletion of WASP protein leads to reduced cell speed. The average instantaneous speed for each cell in (A) is plotted as a dot, color-coded by biological replicate to highlight the consistency from experiment to experiment. The average of the three replicates is displayed as a horizontal line; p values from two-tailed paired t-test. (C) Reduction of WASP protein leads to no significant change in directional persistence (the ratio of the Euclidean distance to the accumulated distance) of cells tracked in (A).   Diagram showing the relationships of extant eukaryotes (based on (Fritz-Laylin et al., 2010)), with presence or absence of SCAR (blue) and WASP (green) genes from complete genome sequences as described (Kollmar et al., 2012). The representative organism whose genome was used for the analysis is listed to the right. For groups with similar morphological and sequence patterns, a single species is used. For example, there is no known plant species that forms pseudopods or retains the WASP gene, so only a single species is shown (Arabidopsis thaliana); similarly, Aspergillus nidulans represents all dikarya. See (Kollmar et al., 2012) for additional sequence information. An amoeba glyph indicates organisms that build pseudopods. Outlined rectangles indicate a lack of identifiable gene. See Table 1 for citations and full species names. *Although we were not able to find a reference to pseudopod formation in A. macrogynus, a relative (Catenaria anguillulae) does assemble pseudopods used for motility (Gleason and Lilje, 2009). Because of this, and the conservation of both WASP and SCAR in B. dendrobatidis, we predict this species is also capable of pseudopod formation. ++These species form pseudopods for feeding, rather than motility. "?" Indicates uncertainty regarding the structure of the protrusions for phagocytosis in Entamoeba histolytica (see text). The time of divergence of extant eukaryotic groups has been estimated to be 1.1-2.3 billion years ago (Chernikova et al., 2011) (Knoll, 2014;Parfrey et al., 2011) and has been predicted to have possessed both WASP and SCAR gene families (Kollmar et al., 2012) and therefore may have built pseudopods.

Figure S3. WASP is not required for adhesion or endocytosis by HL-60 cells. (A)
WASP-KD does not significantly reduce the HL-60 cells' ability to adhere to fibronectin-coated surfaces. Bars represent averages from three biological replicates, normalized to control in each experiment; p value from two-tailed paired t-test. (B) Steady state endocytosis was measured for WASP-KD (green) and control cells (black) by incubating cells for 10 minutes at 37C with fluorescent transferrin, immediately washing with ice-cold acid buffer (to remove surface-bound transferrin). Cells were then fixed, and endocytosed tranferrin quantified by FACS analysis. Values were normalized to percent control within each experiment. (C) Quantitation of actin-mediated endocytosis and receptor recycling in differentiated (neutrophil-like) control (black circles) and WASP-KD HL-60 cells (green squares). Cells were incubated with florescent transferrin, placed at 37C for the indicated time, washed with ice-cold acid buffer (to remove surface-bound transferrin), and fixed for FACS analysis. Within each experiment, samples for each cell line were normalized to time zero. Averages and standard deviations for three independent experiments are shown. (D) Transferrin receptor density was measured by incubating cells at 37C in serum-free medium (to remove surface-bound transferrin), chilling cells and incubating on ice with florescent transferrin. Cells were then washed with PBS to remove unbound transferrin and fixed. Surface-bound transferrin was then quantitated by FACS analysis. Values were normalized to percent control within each experiment. (E) Quantitation of actin-mediated endocytosis and receptor recycling in undifferentiated control (black squares) and WASP-KD HL-60 cells (green triangles). Cells were incubated with florescent transferrin, placed at 37C for the indicated time, washed with ice-cold acid buffer (to remove surface-bound transferrin), and fixed for FACS analysis. Within each experiment, samples for each cell line were normalized to time zero. Averages and standard deviations for three independent experiments are shown.  Figure S1B.
Video 2. Migration of control differentiated HL-60 neutrophil cells and of differentiated WASP-KD HL-60 cells in 2D environment (EZ-TAXIScan assay). Cells expressing control shRNA migrating in a chemoattractant gradient (source is at top) between two glass surfaces with 5 µm spacing. All cells initially within the field of view were manually tracked, as shown. Images were acquired every 20 seconds and are displayed at 300X speed. Many WASP-KD cells exhibited the rhino phenotype and their motility was effectively abolished (e.g. cells 11, 32, 33, 39 and 42).     A m oe baz oa P l a n t s