Cytoskeletal polarization and cytokinetic signaling drives polar lobe formation in spiralian embryos.

In many spiralians, asymmetry in the first two cleavages is achieved through the formation of a polar lobe (PL), which transiently constricts to sequester vegetal cytoplasm into the CD and D blastomeres. While microtubules and actin filaments are required for polar lobe formation, little else is known regarding the structural and functional similarities with the contractile ring, or how the PL constriction is able to form perpendicular to the cleavage plane. Examination of scallop embryos revealed that while activated myosin II could be detected in both the cleavage furrow and early PL constriction, astral or central spindle microtubules were not observed associated with the PL neck until the constriction was nearly complete. Further, inhibition of Aurora B had no effect on polar lobe initiation, but blocked both contractile ring ingression and PL constriction beyond phase II. The cortex destined for PL sequestration was marked by enrichment of the Arp2/3 complex, which was first detected during meiosis and remained enriched at the vegetal pole through the first two cleavages. Inhibition of Arp2/3 affected PL formation and partitioning of cytoplasm into the two daughter cells, suggesting that Arp2/3 plays a functional role in defining the zone of cortex to be sequestered into the polar lobe. Together, these data offer for the first time a mechanism by which a cytoskeletal specialization defines the polar lobe in this atypical form of asymmetric cell division.

Cdc42 controls primary mesenchyme cell morphogenesis in the sea urchin embryo.

In the sea urchin embryo, gastrulation is characterized by the ingression and directed cell migration of primary mesenchyme cells (PMCs), as well as the primary invagination and convergent extension of the endomesoderm. Like all cell shape changes, individual and collective cell motility is orchestrated by Rho family GTPases and their modulation of the actomyosin cytoskeleton. And while endomesoderm specification has been intensively studied in echinoids, much less is known about the proximate regulators driving cell motility. Toward these ends, we employed anti-sense morpholinos, mutant alleles and pharmacological inhibitors to assess the role of Cdc42 during sea urchin gastrulation. While inhibition of Cdc42 expression or activity had only mild effects on PMC ingression, PMC migration, alignment and skeletogenesis were disrupted in the absence of Cdc42, as well as elongation of the archenteron. PMC migration and patterning of the larval skeleton relies on the extension of filopodia, and Cdc42 was required for filopodia in vivo as well as in cultured PMCs. Lastly, filopodial extension required both Arp2/3 and formin actin-nucleating factors, supporting models of filopodial nucleation observed in other systems. Together, these results suggest that Cdc42 plays essential roles during PMC cell motility and organogenesis.

The nanoscale organization of the Wnt signaling integrator Dishevelled in the vegetal cortex domain of an egg and early embryo.

Canonical Wnt/ß-catenin (cWnt) signaling is a crucial regulator of development and Dishevelled (Dsh/Dvl) functions as an integral part of this pathway by linking Wnt binding to the Frizzled:LRP5/6 receptor complex with ß-catenin-stimulated gene expression. In many cell types Dsh has been localized to ill-defined cytoplasmic puncta, however in sea urchin eggs and embryos confocal fluorescence microscopy has shown that Dsh is localized to puncta present in a novel and development-essential vegetal cortex domain (VCD). In the present study, we used super-resolution light microscopy and platinum replica transmission electron microscopy (TEM) to provide the first views of the ultrastructural organization of Dsh within the sea urchin VCD. 3D structured illumination microscopy (SIM) imaging of isolated egg cortices demonstrated the graded distribution of Dsh in the VCD, whereas higher resolution stimulated emission depletion (STED) imaging revealed that some individual Dsh puncta consisted of more than one fluorescent source. Platinum replica immuno-TEM localization showed that Dsh puncta on the cytoplasmic face of the plasma membrane consisted of aggregates of pedestal-like structures each individually labeled with the C-terminus specific Dsh antibody. These aggregates were resistant to detergent extraction and treatment with drugs that disrupt actin filaments or inhibit myosin II contraction, and coexisted with the first cleavage actomyosin contractile ring. These results confirm and extend previous studies and reveal, for the first time in any cell type, the nanoscale organization of plasma membrane tethered Dsh. Our current working hypothesis is that these Dsh pedestals represent a prepositioned scaffold organization that is important for the localized activation of the cWnt pathway at the sea urchin vegetal pole. These observations in sea urchins may also be relevant to the submembranous Dsh puncta present in other eggs and embryos.

Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network.

The cytokinetic contractile ring (CR) was first described some 50 years ago, however our understanding of the assembly and structure of the animal cell CR remains incomplete. We recently reported that mature CRs in sea urchin embryos contain myosin II mini-filaments organized into aligned concatenated arrays, and that in early CRs myosin II formed discrete clusters that transformed into the linearized structure over time. The present study extends our previous work by addressing the hypothesis that these myosin II clusters also contain the crucial scaffolding proteins anillin and septin, known to help link actin, myosin II, RhoA, and the membrane during cytokinesis. Super-resolution imaging of cortices from dividing embryos indicates that within each cluster, anillin and septin2 occupy a centralized position relative to the myosin II mini-filaments. As CR formation progresses, the myosin II, septin and anillin containing clusters enlarge and coalesce into patchy and faintly linear patterns. Our super-resolution images provide the initial visualization of anillin and septin nanostructure within an animal cell CR, including evidence of a septin filament-like network. Furthermore, Latrunculin-treated embryos indicated that the localization of septin or anillin to the myosin II clusters in the early CR was not dependent on actin filaments. These results highlight the structural progression of the CR in sea urchin embryos from an array of clusters to a linearized purse string, the association of anillin and septin with this process, and provide the visualization of an apparent septin filament network with the CR structure of an animal cell.

Echinoderm immunity.

A survey for immune genes in the genome for the purple sea urchin has shown that the immune system is complex and sophisticated. By inference, immune responses of all echinoderms maybe similar. The immune system is mediated by several types of coelomocytes that are also useful as sensors of environmental stresses. There are a number of large gene families in the purple sea urchin genome that function in immunity and of which at least one appears to employ novel approaches for sequence diversification. Echinoderms have a simpler complement system, a large set of lectin genes and a number of antimicrobial peptides. Profiling the immune genes expressed by coelomocytes and the proteins in the coelomic fluid provide detailed information about immune functions in the sea urchin. The importance of echinoderms in maintaining marine ecosystem stability and the disastrous effects of their removal due to disease will require future collaborations between ecologists and immunologists working towards understanding and preserving marine habitats.

Structure and dynamics of an Arp2/3 complex-independent component of the lamellipodial actin network.

Sea urchin coelomocytes contain an unusually broad lamellipodial region and have served as a useful model experimental system for studying the process of actin-based retrograde/centripetal flow. In the current study the small molecule drug 2,3-butanedione monoxime (BDM) was employed as a means of delocalizing the Arp2/3 complex from the cell edge in an effort to investigate the Arp2/3 complex-independent aspects of retrograde flow. Digitally-enhanced phase contrast, fluorescence and polarization light microscopy, along with rotary shadow transmission electron microscopy methods demonstrated that BDM treatment resulted in the centripetal displacement of the Arp2/3 complex and the associated dendritic lamellipodial (LP) actin network from the cell edge. In its wake there remained an array of elongate actin filaments organized into concave arcs that displayed retrograde flow at approximately one quarter the normal rate. Actin polymerization inhibitor experiments indicated that these arcs were generated by polymerization at the cell edge, while active myosin-based contraction in BDM treated cells was demonstrated by localization with antiphospho-myosin regulatory light chain (MRLC) antibody, the retraction of the cytoskeleton in the presence of BDM, and the response of the BDM arcs to laser-based severing. The results suggest that BDM treatment reveals an Arp2/3 complex-independent actin structure in coelomocytes consisting of elongate filaments integrated into the LP network and that these filaments represent a potential connection between the LP network and the central cytoskeleton.

Rac and Arp2/3-Nucleated Actin Networks Antagonize Rho During Mitotic and Meiotic Cleavages.

In motile cells, the activities of the different Rho family GTPases are spatially segregated within the cell, and during cytokinesis there is evidence that this may also be the case. But while Rho's role as the central organizer for contractile ring assembly is well established, the role of Rac and the branched actin networks it promotes is less well understood. To characterize the contributions of these proteins during cytokinesis, we manipulated Rac and Arp2/3 activity during mitosis and meiosis in sea urchin embryos and sea star oocytes. While neither Rac nor Arp2/3 were essential for early embryonic divisions, loss of either Rac or Arp2/3 activity resulted in polar body defects. Expression of activated Rac resulted in cytokinesis failure as early as the first division, and in oocytes, activated Rac suppressed both the Rho wave that traverses the oocyte prior to polar body extrusion as well as polar body formation itself. However, the inhibitory effect of Rac on cytokinesis, polar body formation and the Rho wave could be suppressed by effector-binding mutations or direct inhibition of Arp2/3. Together, these results suggest that Rac- and Arp2/3 mediated actin networks may directly antagonize Rho signaling, thus providing a potential mechanism to explain why Arp2/3-nucleated branched actin networks must be suppressed at the cell equator for successful cytokinesis.

Methods for collection, handling, and analysis of sea urchin coelomocytes.

Sea urchin coelomocytes can be collected in large numbers from adult sea urchins of the species, Strongylocentrotus purpuratus, which typically has 12-40mL of coelomic fluid. Coelomocytes are used for analysis of immune reactions and immune gene expression in addition to basic functions of cells, in particular for understanding structure and modifications of the cytoskeleton in phagocytes. The methods described here include coelomocyte isolation, blocking the clotting reaction, establishing and maintaining primary cultures, separation of different types of coelomocytes into fractions, processing live coelomocytes for light microscopy, fixation and staining for light and electron microscopy, analysis of coelomocyte populations by flow cytometry, and sorting single cells for more detailed follow-up analyses including transcriptomics or genomic characteristics. These methods are provided to make working with coelomocytes accessible to researchers who are unfamiliar with these cells and perhaps to aid others who have worked extensively with invertebrate cells.

Wound closure in the lamellipodia of single cells: mediation by actin polymerization in the absence of an actomyosin purse string.

The actomyosin purse string is an evolutionarily conserved contractile structure that is involved in cytokinesis, morphogenesis, and wound healing. Recent studies suggested that an actomyosin purse string is crucial for the closure of wounds in single cells. In the present study, morphological and pharmacological methods were used to investigate the role of this structure in the closure of wounds in the peripheral cytoplasm of sea urchin coelomocytes. These discoidal shaped cells underwent a dramatic form of actin-based centripetal/retrograde flow and occasionally opened and closed spontaneous wounds in their lamellipodia. Fluorescent phalloidin staining indicated that a well defined fringe of actin filaments assembles from the margin of these holes, and drug studies with cytochalasin D and latrunculin A indicated that actin polymerization is required for wound closure. Additional evidence that actin polymerization is involved in wound closure was provided by the localization of components of the Arp2/3 complex to the wound margin. Significantly, myosin II immunolocalization demonstrated that it is not associated with wound margins despite being present in the perinuclear region. Pharmacological evidence for the lack of myosin II involvement in wound closure comes from experiments in which a microneedle was used to produce wounds in cells in which actomyosin contraction was inhibited by treatment with kinase inhibitors. Wounds produced in kinase inhibitor-treated cells closed in a manner similar to that seen with control cells. Taken together, our results suggest that an actomyosin purse string mechanism is not responsible for the closure of lamellar wounds in coelomocytes. We hypothesize that the wounds heal by means of a combination of the force produced by actin polymerization alone and centripetal flow. Interestingly, these cells did assemble an actomyosin structure around the margin of phagosome-like membrane invaginations, indicating that myosin is not simply excluded from the periphery by some general mechanism. The results indicate that the actomyosin purse string is not the only mechanism that can mediate wound closure in single cells.

The ultrastructural organization of actin and myosin II filaments in the contractile ring: new support for an old model of cytokinesis.

Despite recent advances in our understanding of the components and spatial regulation of the contractile ring (CR), the precise ultrastructure of actin and myosin II within the animal cell CR remains an unanswered question. We used superresolution light microscopy and platinum replica transmission electron microscopy (TEM) to determine the structural organization of actin and myosin II in isolated cortical cytoskeletons prepared from dividing sea urchin embryos. Three-dimensional structured illumination microscopy indicated that within the CR, actin and myosin II filaments were organized into tightly packed linear arrays oriented along the axis of constriction and restricted to a narrow zone within the furrow. In contrast, myosin II filaments in earlier stages of cytokinesis were organized into small, discrete, and regularly spaced clusters. TEM showed that actin within the CR formed a dense and anisotropic array of elongate, antiparallel filaments, whereas myosin II was organized into laterally associated, head-to-head filament chains highly reminiscent of mammalian cell stress fibers. Together these results not only support the canonical "purse-string" model for contractile ring constriction, but also suggest that the CR may be derived from foci of myosin II filaments in a manner similar to what has been demonstrated in fission yeast.

Retention of structural and functional polarity in cultured skate hepatocytes undergoing in vitro morphogenesis.

The present study characterized a primary culture model of hepatocytes isolated from the little skate, Leucoraja erinacea, that maintain remarkable structural and functional polarity over 7 days in culture. Skate hepatocytes were isolated as clusters of 3-20 hepatocytes surrounding a bile canaliculus, rather than as single cells. Trypan blue and propidium iodide exclusion was found to be >98%, and the cells maintained high intracellular concentrations of K+, ATP, and reduced glutathione (GSH), and high ratios of ATP/ADP and GSH/GSSG. Glutathione S-transferase activity remained constant, whereas cytochrome P450 activity declined to 16% of initial levels after 7 days. Quantitative RT-PCR analysis revealed that the mRNA levels of several genes remained constant over the 7-day period, whereas Bsep, the canalicular bile salt export pump, levels declined slowly to 30% of initial values. In the presence of dexamethasone, the cells underwent a morphogenesis in which the clusters reannealed into a three-dimensional network of chords. During this morphogenesis, skate hepatocytes clusters maintained a polarized distribution of actin filaments and microtubules, as well as apical and basolateral membrane domains. Polarity of membrane transport systems was confirmed both morphologically, using antibodies raised against Bsep and Mrp2, the canalicular multispecific organic anion transporter, and functionally, by monitoring secretion of the fluorescent organic anions NBD-taurocholate, a Bsep substrate, and fluorescein-methotrexate, an Mrp2 substrate, into the bile canalicular spaces. Overall, the results indicate that in contrast with mammalian hepatocytes, isolated skate hepatocyte clusters retain polarity in culture, and provide an excellent system for investigating long-term effects of drugs and xenobiotics on hepatobiliary functions, and for studying in vitro morphogenesis.

Central Spindle Self-Organization and Cytokinesis in Artificially Activated Sea Urchin Eggs.

The ability of microtubules of the mitotic apparatus to control the positioning and initiation of the cleavage furrow during cytokinesis was first established from studies on early echinoderm embryos. However, the identity of the microtubule population that imparts cytokinetic signaling is unclear. The two main--and not necessarily mutually exclusive--candidates are the central spindle and the astral rays. In the present study, we examined cytokinesis in ammonia-activated sea urchin eggs, which lack paternally derived centrosomes and undergo mitosis mediated by unusual anastral, bipolar mini-spindles. Live cell imaging and immunolabeling for microtubules and the centralspindlin constituent and kinesin-related protein, MKLP1, demonstrated that furrowing in ammonia-activated eggs was associated with aligned arrays of centralspindlin-linked, opposed bundles of antiparallel microtubules. These autonomous, zipper-like arrays were not associated with a mitotic apparatus, but did possess characteristics similar to the central spindle region of control, fertilized embryos. Our results highlight the self-organizing nature of the central spindle region and its ability to induce cytokinesis-like furrowing, even in the absence of a complete mitotic apparatus.

Arp2/3 complex inhibition radically alters lamellipodial actin architecture, suspended cell shape, and the cell spreading process.

Recent studies have investigated the dendritic actin cytoskeleton of the cell edge's lamellipodial (LP) region by experimentally decreasing the activity of the actin filament nucleator and branch former, the Arp2/3 complex. Here we extend these studies via pharmacological inhibition of the Arp2/3 complex in sea urchin coelomocytes, cells that possess an unusually broad LP region and display correspondingly exaggerated centripetal flow. Using light and electron microscopy, we demonstrate that Arp2/3 complex inhibition via the drug CK666 dramatically altered LP actin architecture, slowed centripetal flow, drove a lamellipodial-to-filopodial shape change in suspended cells, and induced a novel actin structural organization during cell spreading. A general feature of the CK666 phenotype in coelomocytes was transverse actin arcs, and arc generation was arrested by a formin inhibitor. We also demonstrate that CK666 treatment produces actin arcs in other cells with broad LP regions, namely fish keratocytes and Drosophila S2 cells. We hypothesize that the actin arcs made visible by Arp2/3 complex inhibition in coelomocytes may represent an exaggerated manifestation of the elongate mother filaments that could possibly serve as the scaffold for the production of the dendritic actin network.

Broadening the spectrum of actin-based protrusive activity mediated by Arp2/3 complex-facilitated polymerization: motility of cytoplasmic ridges and tubular projections.

Arp2/3 complex-facilitated actin polymerization plays an essential role in a variety of cellular functions including motility, adherence, endocytosis, and trafficking. In the present study, we employ the sea urchin coelomocyte experimental model system to test the hypotheses that Arp2/3 complex-nucleated actin assembly mediates the motility of two unusual cellular protrusions; the cytoplasmic ridges present during coelomocyte spreading, and inducible, tubular-shaped, and neurite-like projections. Our investigations couple pharmacological manipulation employing inhibitors of actin polymerization and the Arp2/3 complex with a wide array of imaging methods including digitally enhanced phase contrast, DIC, and polarization light microscopy of live cells; conventional, confocal and super-resolution light microscopy of fluorescently labeled cells; and scanning and transmission electron microscopy. Taken together, the results of this study indicate that Arp2/3 complex-facilitated actin polymerization underlies the motility of coelomocyte cytoplasmic ridges and tubular projections, that these processes are related to each other, and that they have been preliminarily identified in other cell types. The results also highlight the broad spectrum of actin-based protrusive activities dependent on the Arp2/3 complex and provide additional insights into the pervasive nature of this ubiquitous actin nucleator. Furthermore, we provide the first evidence of a possible mechanistic difference between the impacts of the small molecule drugs BDM and CK666 on the Arp2/3 complex.

Localization and diversity of 185/333 proteins from the purple sea urchin--unexpected protein-size range and protein expression in a new coelomocyte type.

The current paradigm proposes that the innate immune systems of invertebrates are much more complex than previously thought. The highly diverse 185/333 gene family in the purple sea urchin encodes a family of closely related proteins of varying length and sequence composition. Subsets of small phagocytes and polygonal cells express 185/333 proteins with localization on the surface of the small phagocytes and within perinuclear vesicles in both cell types. In short-term cultures, coelomocytes form small aggregates that progress to syncytia that are thought to be equivalent to encapsulation in vivo. These aggregates were found to be enriched for 185/333-positive (185/333(+)) small phagocytes. In response to lipopolysaccharide challenge, coelomocytes transiently increased, including frequencies of both 185/333(+) and 185/333-negative (185/333(-)) small phagocytes and 185/333(+) polygonal cells. The 185/333 proteins were present in a broad array of sizes, most of which were larger than that predicted from the cDNAs. Recombinant 185/333 proteins expressed in bacteria and insect cells were also larger than expected, suggesting that the proteins dimerize and multimerize. The diversity of the 185/333 proteins, their expression in response to immune challenge, and their cellular localization suggests this protein family and the small phagocytes have an important immunological role in the sea urchin.

Bipolar, anastral spindle development in artificially activated sea urchin eggs.

The mitotic apparatus of the early sea urchin embryo is the archetype example of a centrosome-dominated, large aster spindle organized by means of the centriole of the fertilizing sperm. In this study, we tested the hypothesis that artificially activated sea urchin eggs possess the capacity to assemble the anastral, bipolar spindles present in many acentrosomal systems. Control fertilized Lytechinus pictus embryos and ammonia-activated eggs were immunolabeled for tubulin, centrosomal material, the spindle pole structuring protein NuMA and the mitotic kinesins MKLP1/Kinesin-6, Eg5/Kinesin-5, and KinI/Kinesin-13. Confocal imaging showed that a subset of ammonia-activated eggs contained bipolar "mini-spindles" that were anastral; displayed metaphase and anaphase-like stages; labeled for centrosomal material, NuMA, and the three mitotic kinesins; and were observed in living eggs using polarization optics. These results suggest that spindle structural and motor proteins have the ability to organize bipolar, anastral spindles in sea urchin eggs activated in the absence of the paternal centriole.

Actin-based centripetal flow: phosphatase inhibition by calyculin-A alters flow pattern, actin organization, and actomyosin distribution.

Previous studies have suggested that the actin-based centripetal flow process in sea urchin coelomocytes is the result of a two-part mechanism, actin polymerization at the cell edge coupled with actomyosin contraction at the cell center. In the present study, we have extended the testing of this two-part model by attempting to stimulate actomyosin contraction via treatment of coelomocytes with the phosphatase inhibitor Calyculin A (CalyA). The effects of this drug were studied using digitally-enhanced video microscopy of living cells combined with immunofluorescent localization and scanning electron microscopy. Under the influence of CalyA, the coelomocyte actin cytoskeleton undergoes a radical reorganization from a dense network to one displaying an array of tangential arcs and radial rivulets in which actin and the Arp2/3 complex concentrate. In addition, the structure and dynamics of the cell center are transformed due to the accumulation of actin and membrane in this region and the constriction of the central actomyosin ring. Physiological evidence of an increase in actomyosin-based contractility following CalyA treatment was demonstrated in experiments in which cells generated tears in their cell centers in response to the drug. Western blotting and immunofluorescent localization with antibodies against the phosphorylated form of the myosin regulatory light chain (MRLC) suggested that the demonstrated constriction of actomyosin distribution was the result of CalyA-induced phosphorylation of MRLC. Overall, the results suggest that there is significant cross talk between the two underlying mechanisms of actin polymerization and actomyosin contraction, and indicate that changes in actomyosin tension may be translated into alterations in the structural organization of the actin cytoskeleton.