Annika Guse

Phosphorylation of ZEN-4/MKLP1 by Aurora B Regulates Completion of Cytokinesis

The 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.

In vitro centromere and kinetochore assembly on defined chromatin templates

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, which 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. 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 chimaeras, we demonstrate that the conserved carboxy terminus of CENP-A is necessary and sufficient for centromere and kinetochore protein recruitment and function but that the CENP-A targeting domain—required for new CENP-A histone assembly—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.

The genome of Aiptasia, a sea anemone model for coral symbiosis

Significance Coral reefs form marine-biodiversity hotspots of enormous ecological, economic, and aesthetic importance that rely energetically on a functional symbiosis between the coral animal and a photosynthetic alga. The ongoing decline of corals worldwide due to anthropogenic influences, including global warming, ocean acidification, and pollution, heightens the need for an experimentally tractable model system to elucidate the molecular and cellular biology underlying the symbiosis and its susceptibility or resilience to stress. The small sea anemone Aiptasia is such a system, and our analysis of its genome provides a foundation for research in this area and has revealed numerous features of interest in relation to the evolution and function of the symbiotic relationship. 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.

CSC-1: a subunit of the aurora b kinase complex that binds to the survivin-like protein BIR-1 and the incenp-like protein ICP-1

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.

CENP-A confers a reduction in height on octameric nucleosomes

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

A cell-free system for functional centromere and kinetochore assembly

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.

Functional knockdown of VCAM-1 at the posttranslational level with ER retained antibodies

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.

Similar specificities of symbiont uptake by adults and larvae in an anemone model system for coral biology.

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 used 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.

Aiptasia sp. larvae as a model to reveal mechanisms of symbiont selection in cnidarians.

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.

Induction of Gametogenesis in the Cnidarian Endosymbiosis Model Aiptasia sp.

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.