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The planulae larvae of cnidarians (jellyfish, hydras, anemones, corals) have attracted interest since Haeckels 150-year-old postulation of the gastrula developmental stage of sponges and corals as the terminal lifeform of primitive multicellular metazoans. Widely viewed as primarily particle feeders, the planulae larvae of the anemone Exaiptasia pallida (commonly Aiptasia) have not been reported to undergo settlement and metamorphosis to adult morphology, and the lack of a closed lifecycle has been a major obstacle in this increasingly popular model system for coral-dinoflagellate endosymbiosis. Here we studied Aiptasia larvae feeding behavior and show its indispensability to trigger the first reports of settlement and metamorphosis in the system, finally closing the Aiptasia lifecycle. Surprisingly, the young gastrula-like planulae at just two days post fertilization actively feed on living crustacean nauplii, preferentially to heat-killed ones. Predation is dependent on functional stinging cells (nematocytes), indicative of complex neuronal control. Larvae fed daily dramatically increase in size and at 14 days post fertilization begin to morphologically change prior to settlement at high efficiency. Strikingly, dinoflagellate endosymbionts are neither required for larval growth nor measurably affect settlement dynamics, but are essential for spawning of the mature adult polyps. Our data show for the first time that gastrula-like planulae were capable of prey capture, suggesting carnivory in addition to filter feeding as a common strategy of this terminal life form. These data are discussed in the context of recent revelations on the evolution of basal metazoans.

Animals with indeterminate growth can adapt their growth rate and body size to changing food availability throughout their lifetime. As the cellular basis underlying food-dependent growth plasticity is poorly understood, we quantified how the sea anemonesNematostella vectensisandExaiptasia diaphana(Aiptasia) respond to feeding and starvation on organismal and cellular levels. Using mathematical modelling to analyse growth phases, we found that growth and shrinkage rates inNematostellaare exponential, stereotypic and accompanied by high levels of cell gain or loss, respectively. During starvation and re-feeding, a considerable proportion of juvenile polyp cells (>7%) reversibly shift between S/G2/M and G1/G0cell cycle phases, suggesting a tight nutritional control of quiescence and cell cycle re-entry. In the facultative symbiotic sea anemone Aiptasia, we found that growth and cell proliferation rates are dependent on the symbiotic state and, in comparison toNematostella, respond less strongly to changes in food supply. Altogether, we provide a benchmark and resource to study the nutritional regulation of body plasticity on molecular, cellular and genomic levels using the rich functional toolkit available forNematostella.Summary statementFeeding and starvation in sea anemones induce growth and shrinkage, cell size changes and dynamic cell proliferation changes that support a nutritional control of quiescence and cell cycle re-entry.

Alveolata comprises diverse taxa of single-celled eukaryotes, many of which are renowned for their ability to live inside animal cells. Notable examples are apicomplexan parasites and dinoflagellate symbionts, the latter of which power coral reef ecosystems. Although functionally distinct, they evolved from a common, free-living ancestor and must evade their host's immune response for persistence. Both the initial cellular events that gave rise to this intracellular lifestyle and the role of host immune modulation in coral-dinoflagellate endosymbiosis are poorly understood. Here, we use a comparative approach in the cnidarian endosymbiosis model Aiptasia, which re-establishes endosymbiosis with free-living dinoflagellates every generation. We find that uptake of microalgae is largely indiscriminate, but non-symbiotic microalgae are expelled by vomocytosis, while symbionts induce host cell innate immune suppression and form a lysosomal-associated membrane protein 1-positive niche. We demonstrate that exogenous immune stimulation results in symbiont expulsion and, conversely, inhibition of canonical Toll-like receptor signalling enhances infection of host animals. Our findings indicate that symbiosis establishment is dictated by local innate immune suppression, to circumvent expulsion and promote niche formation. This work provides insight into the evolution of the cellular immune response and key steps involved in mediating endosymbiotic interactions.

Emergence of the symbiotic lifestyle fostered the immense diversity of all ecosystems on Earth, but symbiosis plays a particularly remarkable role in marine ecosystems. Photosynthetic dinoflagellate endosymbionts power reef ecosystems by transferring vital nutrients to their coral hosts. The mechanisms driving this symbiosis, specifically those which allow hosts to discriminate between beneficial symbionts and pathogens, are not well understood. Here, we uncover that host immune suppression is key for dinoflagellate endosymbionts to avoid elimination by the host using a comparative, model systems approach. Unexpectedly, we find that the clearance of non-symbiotic microalgae occurs by non-lytic expulsion (vomocytosis) and not intracellular digestion, the canonical mechanism used by professional immune cells to destroy foreign invaders. We provide evidence that suppression of TLR signalling by targeting the conserved MyD88 adapter protein has been co-opted for this endosymbiotic lifestyle, suggesting that this is an evolutionarily ancient mechanism exploited to facilitate symbiotic associations ranging from coral endosymbiosis to the microbiome of vertebrate guts.

Cnidarians such as reef-building corals depend upon nutrient transfer from intracellular symbionts, but the mechanisms and evolution of this process remain unknown. Homologues of the conserved cholesterol binder Niemann-Pick Type C2 (NPC2) in cnidarians are implicated in the transfer of sterol from symbionts. Here, we show that symbionts transfer bulk sterols to the host, host sterol utilization is plastic, and pharmacological inhibition of sterol trafficking disrupts symbiosis. Having undergone an anthozoan-specific expansion, “non-canonical” NPC2s respond to symbiosis and accumulate over time at the lysosomal-like organelle in which the symbiont resides (“symbiosome”). We demonstrate that both a non- and canonicalAiptasiaNPC2 bind symbiont-produced sterols, yet only the non-canonical homologue exhibits increased stability at low pH. We propose that symbiotic cnidarians adapted pre-existing cholesterol-trafficking machinery to function in the highly acidic symbiosome environment, allowing corals to dominate nutrient-poor shallow tropical seas worldwide.

Reef-building corals depend on an intracellular symbiosis with photosynthetic dinoflagellates for their survival in nutrient-poor oceans. Symbionts are phagocytosed by coral larvae from the environment and transfer essential nutrients to their hosts. Aiptasia, a small tropical marine sea anemone, is emerging as a tractable model system for coral symbiosis; however, to date functional tools and genetic transformation are lacking. Here we have established an efficient workflow to collect Aiptasia eggs for in vitro fertilization and microinjection as the basis for experimental manipulations in the developing embryo and larvae. We demonstrate that protein, mRNA, and DNA can successfully be injected into live Aiptasia zygotes to label actin with recombinant Lifeact-eGFP protein; to label nuclei and cell membranes with NLS-eGFP and farnesylated mCherry translated from injected mRNA; and to transiently drive transgene expression from an Aiptasia-specific promoter, respectively, in embryos and larvae. These proof-of-concept approaches pave the way for future functional studies of development and symbiosis establishment in Aiptasia, a powerful model to unravel the molecular mechanisms underlying intracellular coral-algal symbiosis.Summary StatementToolkit extension: development of microinjection for cellular labelling, expression of exogenous genes and live imaging in Aiptasia, an emerging model for intracellular coral-algal symbiosis.

Symbiosis, defined as the persistent association between two distinct species, is an evolutionary and ecologically critical phenomenon facilitating survival of both partners in diverse habitats. The biodiversity of coral reef ecosystems depends on a functional symbiosis with photosynthetic dinoflagellates of the highly diverse genus Symbiodinium, which reside in coral host cells and continuously support their nutrition. The mechanisms underlying symbiont selection to establish a stable endosymbiosis in non-symbiotic juvenile corals are unclear. Here we show for the first time that symbiont selection patterns for larvae of two Acropora coral species and the model anemone Aiptasia are similar under controlled conditions. We find that Aiptasia larvae distinguish between compatible and incompatible symbionts during uptake into the gastric cavity and phagocytosis. Using RNA-Seq, we identify a set of candidate genes potentially involved in symbiosis establishment. Together, our data complement existing molecular resources to mechanistically dissect symbiont phagocytosis in cnidarians under controlled conditions, thereby strengthening the role of Aiptasia larvae as a powerful model for cnidarian endosymbiosis establishment. published

This protocol describes a cell-free system for studying vertebrate centromere and kinetochore formation. We reconstitute tandem arrays of centromere protein A (CENP-A) nucleosomes as a substrate for centromere and kinetochore assembly. These chromatin substrates are immobilized on magnetic beads and then incubated in Xenopus egg extracts that provide a source for centromere and kinetochore proteins and that can be cycled between mitotic and interphase cell cycle states. This cell-free system lends itself to use in protein immunodepletion, complementation and drug inhibition as a tool to perturb centromere and kinetochore assembly, cytoskeletal dynamics, DNA modification and protein post-translational modification. This system provides a distinct advantage over cell-based investigations in which perturbing centromere and kinetochore function often results in lethality. After incubation in egg extract, reconstituted CENP-A chromatin specifically assembles centromere and kinetochore proteins, which locally stabilize microtubules and, on microtubule depolymerization with nocodazole, activate the mitotic checkpoint. A typical experiment takes 3 d.

During cell division, chromosomes are segregated to nascent daughter cells by attaching to the microtubules of the mitotic spindle through the kinetochore. Kinetochores are assembled on a specialized chromatin domain, called the centromere that is characterized by the replacement of nucleosomal histone H3 with the histone H3 variant centromere protein A (CENP-A). CENP-A is essential for centromere and kinetochore formation in all eukaryotes but it is unknown how CENP-A chromatin directs centromere and kinetochore assembly 1. Here we generate synthetic CENP-A chromatin that recapitulates essential steps of centromere and kinetochore assembly in vitro. We show that reconstituted CENP-A chromatin when added to cell free extracts is sufficient for the assembly of centromere and kinetochore proteins, microtubule binding and stabilization, and mitotic checkpoint function. Using chromatin assembled from histone H3/CENP-A chimeras, we demonstrate that the conserved C-terminus of CENP-A is necessary and sufficient for centromere and kinetochore protein recruitment and function but that the CENP-A targeting domain (CATD), required for new CENP-A histone assembly 2, is not. These data show that two of the primary requirements for accurate chromosome segregation, the assembly of the kinetochore and the propagation of CENP-A chromatin are specified by different elements in the CENP-A histone. Our unique cell-free system enables complete control and manipulation of the chromatin substrate and thus presents a powerful tool to study centromere and kinetochore assembly in higher eukaryotes.

Endosymbiosis is widespread among cnidarians and is of high ecological relevance. The tropical sea anemone Aiptasia sp. is a laboratory model system for endosymbiosis between reef-building corals and photosynthetic dinoflagellate algae of the genus Symbiodinium. Here we identify the key environmental cues to induce reproducible spawning in Aiptasia under controlled laboratory conditions. We find that simulating a lunar cycle with blue-wavelength light is necessary to promote abundant gamete production and synchronous release in well-fed animals. Sexual reproduction rates are genetically determined and differ among clonal lines under similar conditions. We also find the inverse difference in rates of asexual reproduction. This study provides the requisite basis for further development of the Aiptasia model system, allowing analysis of basic cellular and molecular mechanisms in the laboratory as well as investigations of broad questions of ecological and evolutionary relevance.

The most diverse marine ecosystems, coral reefs, depend upon a functional symbiosis between a cnidarian animal host (the coral) and intracellular photosynthetic dinoflagellate algae. The molecular and cellular mechanisms underlying this endosymbiosis are not well understood, in part because of the difficulties of experimental work with corals. The small sea anemone Aiptasia provides a tractable laboratory model for investigating these mechanisms. Here we report on the assembly and analysis of the Aiptasia genome, which will provide a foundation for future studies and has revealed several features that may be key to understanding the evolution and function of the endosymbiosis. These features include genomic rearrangements and taxonomically restricted genes that may be functionally related to the symbiosis, aspects of host dependence on alga-derived nutrients, a novel and expanded cnidarian-specific family of putative pattern-recognition receptors that might be involved in the animal-algal interactions, and extensive lineage-specific horizontal gene transfer. Extensive integration of genes of prokaryotic origin, including genes for antimicrobial peptides, presumably reflects an intimate association of the animal-algal pair also with its prokaryotic microbiome. published

SummaryThe central spindle regulates the formation and positioning of the contractile ring and is essential for completion of cytokinesis [1]. Central spindle assembly begins in early anaphase with the bundling of overlapping, antiparallel, nonkinetochore microtubules [2, 3], and these bundles become compacted and mature into the midbody. Prominent components of the central spindle include aurora B kinase and centralspindlin, a complex containing a Kinesin-6 protein (ZEN-4/MKLP1) and a Rho family GAP (CYK-4/MgcRacGAP) that is essential for central spindle assembly [4]. Centralspindlin localization depends on aurora B kinase [5]. Aurora B concentrates in the midbody and persists between daughter cells. Here, we show that in C. elegans embryos and in cultured human cells, respectively, ZEN-4 and MKLP1 are phosphorylated by aurora B in vitro and in vivo on conserved C-terminal serine residues. In C. elegans embryos, a nonphosphorylatable mutant of ZEN-4 localizes properly but does not efficiently support completion of cytokinesis. In mammalian cells, an inhibitor of aurora kinase acutely attenuates phosphorylation of MKLP1. Inhibition of aurora B in late anaphase causes cytokinesis defects without disrupting the central spindle. These data indicate a conserved role for aurora-B-mediated phosphorylation of ZEN-4/MKLP1 in the completion of cytokinesis.

Nucleosomes in which histone H3 is replaced by CENP-A direct kinetochore assembly. CENP-A nucleosomes extracted from human and Drosophila cells have been reported to have reduced heights relative to canonical octameric H3 nucleosomes, suggesting a unique tetrameric, hemisomal composition. We demonstrate that even octameric CENP-A nucleosomes assembled in vitro exhibit a reduced height, indicating that they are physically distinct from H3 nucleosomes, and negating the need to invoke the presence of hemisomes.

The Aurora B kinase complex is a critical regulator of chromosome segregation and cytokinesis. In Caenorhabditis elegans, AIR-2 (Aurora B) function requires ICP-1 (Incenp) and BIR-1 (Survivin). In various systems, Aurora B binds to orthologues of these proteins. Through genetic analysis, we have identified a new subunit of the Aurora B kinase complex, CSC-1. C. elegans embryos depleted of CSC-1, AIR-2, ICP-1, or BIR-1 have identical phenotypes. CSC-1, BIR-1, and ICP-1 are interdependent for their localization, and all are required for AIR-2 localization. In vitro, CSC-1 binds directly to BIR-1. The CSC-1/BIR-1 complex, but not the individual subunits, associates with ICP-1. CSC-1 associates with ICP-1, BIR-1, and AIR-2 in vivo. ICP-1 dramatically stimulates AIR-2 kinase activity. This activity is not stimulated by CSC-1/BIR-1, suggesting that these two subunits function as targeting subunits for AIR-2 kinase.

Reef-building corals depend for much of their energy on photosynthesis by symbiotic dinoflagellate algae (genus Symbiodinium) that live within their gastrodermal cells. However, the cellular mechanisms underpinning this ecologically critical symbiosis, including those governing the specificity of symbiont uptake by the host, remain poorly understood, in part because of the difficulties of working with corals in the laboratory. Here we use the small symbiotic sea anemone Aiptasia as an experimentally tractable model system to analyze the specificity and timing of symbiosis onset in larval and adult animals under controlled laboratory conditions. Using four clonal, axenic Symbiodinium strains, we found no difference in uptake specificity between larvae (even when very young) and adults. Although both compatible and incompatible algal strains were found within the larval guts, only the former appeared to be internalized by gastrodermal cells, and they (but not incompatible algae) proliferated rapidly within the larvae in the absence of detectable exchange with other larvae. Older larvae showed reduced ingestion of both compatible and incompatible algae, and the addition of food failed to promote the uptake of an incompatible algal strain. Thus, Aiptasia adults and larvae appear to have similar mechanisms for discriminating between compatible and incompatible dinoflagellate types prior to phagocytosis by host gastrodermal cells. Whether a particular algal strain is compatible or incompatible appears to be stable during years of axenic culture in the absence of a host. These studies provide a foundation for future analyses of the mechanisms of symbiont-uptake specificity in this emerging model system.

Vascular cell adhesion molecule 1 (VCAM-1) is involved in the recruitment of leukocytes to inflammatory sites. In this study we present the first functional knockdown of VCAM-1 using an ER retained antibody construct. We generated a knockdown construct encoding the VCAM-1 specific single chain variable fragment scFv6C7.1 fused to the C-terminal ER retention sequence KDEL. HEK-293:VCAM-YFP cells stably expressing a VCAM-YFP fusion protein were transiently transfected with the knockdown construct and showed down-regulation of surface VCAM-1. Knockdown efficiency of the system is time-dependent due to used transient transfection of the intrabody construct. Furthermore, intrabody mediated knockdown of HEK-293:VCAM-YFP cells also impaired cell–cell interaction with Jurkat cells that are endogenously expressing VLA-4, the physiological partner of VCAM-1. Posttranslational knockdown with ER retained antibodies seems to be a promising technique, as shown here for VCAM-1.

Molybdopterin guanine dinucleotide (MGD) is the form of the molybdenum cofactor that is required for the activity of most bacterial molybdoenzymes. MGD is synthesized from molybdopterin (MPT) and GTP in a reaction catalyzed by the MobA protein. Here we report that wild type MobA can be copurified along with bound MPT and MGD, demonstrating a tight binding of both its substrate and product. To study structure-function relationships, we have constructed a number of site-specific mutations of the most highly conserved amino acid residues of the MobA protein family. Variant MobA proteins were characterized for their ability to support the synthesis of active molybdenum enzymes, to bind MPT and MGD, to interact with the molybdenum cofactor biosynthesis proteins MobB and MoeA. They were also characterized by x-ray structural analysis. Our results suggest an essential role for glycine 15 of MobA, either for GTP binding and/or catalysis, and an involvement of glycine 82 in the stabilization of the product-bound form of the enzyme. Surprisingly, the individual and double substitution of asparagines 180 and 182 to aspartate did not affect MPT binding, catalysis, and product stabilization.

The Aurora B kinase complex is a critical regulator of chromosome segregation and cytokinesis. In Caenorhabditis elegans, AIR-2 (Aurora B) function requires ICP-1 (Incenp) and BIR-1 (Survivin). In various systems, Aurora B binds to orthologues of these proteins. Through genetic analysis, we have identified a new subunit of the Aurora B kinase complex, CSC-1. C. elegans embryos depleted of CSC-1, AIR-2, ICP-1, or BIR-1 have identical phenotypes. CSC-1, BIR-1, and ICP-1 are interdependent for their localization, and all are required for AIR-2 localization. In vitro, CSC-1 binds directly to BIR-1. The CSC-1/BIR-1 complex, but not the individual subunits, associates with ICP-1. CSC-1 associates with ICP-1, BIR-1, and AIR-2 in vivo. ICP-1 dramatically stimulates AIR-2 kinase activity. This activity is not stimulated by CSC-1/BIR-1, suggesting that these two subunits function as targeting subunits for AIR-2 kinase.

Light represents a key environmental factor, which shapes the physiology and evolution of most organisms. Notable illustrations of this are reef-building corals (Anthozoa), which adapted to shallow, oligotrophic, tropical oceans by exploiting light from the sun and the moon to regulate various aspects of physiology including sexual reproduction, phototaxis and photosymbiosis. Together with the Medusozoa, (including jellyfish), the Anthozoa constitute the ancestral metazoan phylum cnidaria. While light perception in Medusozoa has received attention, the mechanisms of light sensing in Anthozoa remain largely unknown. Cnidaria express two principle groups of light-sensing proteins: opsins and photolyases/cryptochromes. By inspecting the genomic loci encoding these photoreceptors in over 35 cnidarian species, we reveal that Anthozoa have substantially expanded and diversified their photoreceptor repertoire. We confirm that, in contrast to Medusozoa, which retained one opsin class, anthozoans possess all three urmetazoan opsin classes. We show that anthozoans also evolved an extra sub-group (actinarian ASO-IIs). Strikingly, we reveal that cryptochromes including CRY-IIs are absent in Medusozoa, while the Anthozoa retained these and evolved an additional, novel cryptochrome class (AnthoCRYs), which contain unique tandem duplications of up to 6 copies of the PHR region. We explored the functionality of these photoreceptor groups by structure-function and gene expression analysis in the anthozoan model species Exaiptasia pallida (Aiptasia), which recapitulates key photo-behaviors of corals. We identified an array of features that we speculate reflect adaptations to shallow aquatic environments, moonlight-induced spawning synchronization and photosymbiosis. We further propose that photoreceptor complexity and diversity in Anthozoa reflects adaptation to challenging habitats.