Biology subjects

King, N.

Publications and source records attributed to King, N..

32 records · Page 2Linked to original sources

bioRxiv2020

Genome editing enables reverse genetics of multicellular development in the choanoflagellate Salpingoeca rosetta

In a previous study, we established a forward genetic screen to identify genes required for multicellular development in the choanoflagellate, Salpingoeca rosetta (Levin et al., 2014). Yet, the paucity of reverse genetic tools for choanoflagellates has hampered direct tests of gene function and impeded the establishment of choanoflagellates as a model for reconstructing the origin of their closest living relatives, the animals. Here we establish CRISPR/Cas9-mediated genome editing in S. rosetta by engineering a selectable marker to enrich for edited cells. We then use genome editing to disrupt the coding sequence of a S. rosetta C-type lectin gene, rosetteless, and thereby demonstrate its necessity for multicellular rosette development. This work advances S. rosetta as a model system in which to investigate how genes identified from genetic screens and genomic surveys function in choanoflagellates and evolved as critical regulators of animal biology.

evolutionary biology↗

bioRxiv2020

Structural and functional evaluation of de novo-designed, two-component nanoparticle carriers for HIV Env trimer immunogens

Two-component, self-assembling nanoparticles represent a versatile platform for multivalent presentation of viral antigens. Nanoparticles of different sizes and geometries can be designed and combined with appropriate antigens to fit the requirements of different immunization strategies. Here, we describe detailed antigenic, structural, and functional characterization of computationally designed tetrahedral, octahedral, and icosahedral nanoparticle immunogens displaying trimeric HIV envelope glycoprotein (Env) ectodomains. Env trimers, based on subtype A (BG505) or consensus group M (ConM) sequences and engineered with SOSIP stabilizing mutations, were fused to the underlying trimeric building block of each nanoparticle. Initial screening yielded one icosahedral and two tetrahedral nanoparticle candidates, capable of presenting twenty or four copies of the Env trimer. A number of analyses, including detailed structural characterization by cryo-EM, demonstrated that the nanoparticle immunogens possessed the intended structural and antigenic properties. Comparing the humoral responses elicited by ConM-SOSIP trimers presented on a two-component tetrahedral nanoparticle to the corresponding soluble protein revealed that multivalent presentation increased the proportion of the overall antibody response directed against autologous neutralizing Ab epitopes present on the ConM-SOSIP trimers. Author SummaryProtein constructs based on soluble ectodomains of HIV glycoprotein (Env) trimers are the basis of many current HIV vaccine platforms. Multivalent antigen display is one strategy applied to improve the immunogenicity of different subunit vaccine candidates. Here, we describe and comprehensively evaluate a library of de novo designed, protein nanoparticles of different geometries for their ability to present trimeric Env antigens. We found three nanoparticle candidates that can stably incorporate model Env trimer on their surface while maintaining its structure and antigenicity. Immunogenicity of the designed nanoparticles is assessed in vitro and in vivo. In addition to introducing a novel set of reagents for multivalent display of Env trimers, this work provides both guiding principles and a detailed experimental roadmap for the generation, characterization, and optimization of Env-presenting, self-assembling nanoparticle immunogens.

immunology↗

bioRxiv2020

Tailored Design of Protein Nanoparticle Scaffolds for Multivalent Presentation of Viral Glycoprotein Antigens

The adaptive immune system is highly sensitive to arrayed antigens, and multivalent display of viral glycoproteins on symmetric scaffolds has been found to substantially increase the elicitation of antigen-specific antibodies. Motivated by the considerable promise of this strategy for next-generation anti-viral vaccines, we set out to design new self-assembling protein nanoparticles with geometries specifically tailored to scaffold ectodomains of different viral glycoproteins. We first designed and characterized homo-trimers from designed repeat proteins with N-terminal helices positioned to match the C termini of several viral glycoprotein trimers. Oligomers found to experimentally adopt the designed configuration were then used to generate nanoparticles with tetrahedral, octahedral, or icosahedral symmetry. Examples of all three target symmetries were experimentally validated by cryo-electron microscopy and several were assessed for their ability to display viral glycoproteins via genetic fusion. Electron microscopy and antibody binding experiments demonstrated that the designed nanoparticles display conformationally intact native-like HIV-1 Env, influenza hemagglutinin, and prefusion RSV F trimers in the predicted geometries. This work demonstrates that novel nanoparticle immunogens can be designed from the bottom up with atomic-level accuracy and provides a general strategy for precisely controlling epitope presentation and accessibility.

synthetic biology↗

bioRxiv2019

Synergistic cues from diverse bacteria enhance multicellular development in a choanoflagellate

Bacteria regulate the life histories of diverse eukaryotes, but relatively little is known about how eukaryotes interpret and respond to multiple bacterial cues encountered simultaneously. To explore how a eukaryote might respond to a combination of bioactive molecules from multiple bacteria, we treated the choanoflagellate Salpingoeca rosetta with two sets of bacterial cues, one that induces mating and the other that induces multicellular development. We found that simultaneous exposure to both sets of cues enhanced multicellular development in S. rosetta, eliciting both larger multicellular colonies and an increase in the number of colonies. Thus, rather than conveying conflicting sets of information, these distinct bacterial cues synergize to augment multicellular development. This study demonstrates how a eukaryote can integrate and modulate its response to cues from diverse bacteria, underscoring the potential impact of complex microbial communities on eukaryotic life histories. ImportanceEukaryotic biology is profoundly influenced by interactions with diverse environmental and host-associated bacteria. However, it is not well understood how eukaryotes interpret multiple bacterial cues encountered simultaneously. This question has been challenging to address because of the complexity of many eukaryotic model systems and their associated bacterial communities. Here, we studied a close relative of animals, the choanoflagellate Salpingoeca rosetta, to explore how eukaryotes respond to diverse bacterial cues. We found that a bacterial chondroitinase that induces mating on its own can also synergize with bacterial lipids that induce multicellular "rosette" development. When encountered together, these cues enhance rosette development, resulting in the formation of more rosettes, each containing more cells than rosettes that form in the absence of the chondroitinase. These findings highlight how synergistic interactions among bacterial cues can influence the biology of eukaryotes.

microbiology↗

bioRxiv2019

Potential of genomic selection for improvement of resistance to Ostreid Herpes virus in Pacific oyster (Crassostrea gigas)

In genomic selection (GS), genome-wide SNP markers are used to generate genomic estimated breeding values (gEBVs) for selection candidates. The application of GS in shellfish looks promising and has the potential to help in dealing with one of the main issues currently affecting Pacific oyster production worldwide, which is the \"summer mortality syndrome\". This causes periodic mass mortality in farms worldwide and has mainly been attributed to a specific variant of the Ostreid herpesvirus (OsHV-1-var). In the current study, we evaluated the potential of genomic selection for host resistance OsHV in Pacific oysters, and compared it to pedigree-based approaches. An OsHV-1 disease challenge was performed using an immersion-based virus exposure treatment for oysters for seven days. 768 samples were genotyped using the medium density SNP array for oysters. GWAS was performed for the survival trait using a GBLUP approach in BLUPF90 software. Heritability ranged from 0.25{+/-}0.05 to 0.37{+/-}0.05 (mean{+/-}s.e) based on pedigree and genomic information, respectively. Genomic prediction was more accurate than pedigree prediction, and SNP density reduction had little impact on prediction accuracy until marker densities dropped below [~]500 SNPs. This demonstrates the potential for GS in Pacific oyster breeding programs and importantly, demonstrates that a low number of SNPs might suffice to obtain accurate gEBVs, thus potentially making the implementation of GS more cost effective.

genomics↗

bioRxiv2019

Light-regulated collective contractility in a multicellular choanoflagellate

Collective cell contractions that generate global tissue deformations are a signature feature of animal movement and morphogenesis. Nonetheless, the ancestry of collective contractility in animals remains mysterious. While surveying the Caribbean island of Curacao for choanoflagellates, the closest living relatives of animals, we isolated a previously undescribed species (here named Choanoeca flexa sp. nov.), that forms multicellular cup-shaped colonies. The colonies rapidly invert their curvature in response to changing light levels, which they detect through a rhodopsin-cGMP pathway. Inversion requires actomyosin-mediated apical contractility and allows alternation between feeding and swimming behavior. C. flexa thus rapidly converts sensory inputs directly into multicellular contractions. In this respect, it may inform reconstructions of hypothesized animal ancestors that existed before the evolution of specialized sensory and contractile cells.\n\nOne Sentence SummaryA newly described choanoflagellate species forms cup-shaped colonies that reversibly invert their curvature in response to light.

cell biology↗

bioRxiv2019

Biophysical principles of choanoflagellate self-organization

Inspired by the patterns of multicellularity in choanoflagellates, the closest living relatives of animals, we quantify the biophysical processes underlying the morphogenesis of rosette colonies in the choanoflagellate Salpingoeca rosetta. We find that rosettes reproducibly transition from an early stage of 2D growth to a later stage of 3D growth, despite the underlying stochasticity of the cell lineages. We postulate that the extracellular matrix (ECM) exerts a physical constraint on the packing of proliferating cells, thereby sculpting rosette morphogenesis. Our perturbative experiments coupled with biophysical simulations demonstrates the fundamental importance of a basally-secreted ECM for rosette morphogenesis. In addition, this yields a morphospace for the shapes of these multicellular colonies, consistent with observations of a range of choanoflagellates. Overall, our biophysical perspective on rosette development complements previous genetic perspectives and thus helps illuminate the interplay between cell biology and physics in regulating morphogenesis.\n\nSignificance statementComparisons among animals and their closest living relatives, the choanoflagellates, have begun to shed light on the origin of animal multicellularity and development. Here we complement previous genetic perspectives on this process by focusing on the biophysical principles underlying colony morphology and morphogenesis. Our study reveals the crucial role of the extracellular matrix in shaping the colonies and leads to a phase diagram that delineates the range of morphologies as a function of the biophysical mechanisms at play.

biophysics↗

bioRxiv2018

The architecture of cell differentiation in choanoflagellates and sponge choanocytes

Collar cells are ancient animal cell types which are conserved across the animal kingdom [1] and their closest relatives, the choanoflagellates [2]. However, little is known about their ancestry, their subcellular architecture, or how they differentiate. The choanoflagellate Salpingoeca rosetta [3] expresses genes necessary for animal multicellularity and development [4] and can alternate between unicellular and multicellular states [3,5], making it a powerful model to investigate the origin of animal multicellularity and mechanisms underlying cell differentiation [6,7]. To compare the subcellular architecture of solitary collar cells in S. rosetta with that of multicellular \"rosettes\" and collar cells in sponges, we reconstructed entire cells in 3D through transmission electron microscopy on serial ultrathin sections. Structural analysis of our 3D reconstructions revealed important differences between single and colonial choanoflagellate cells, with colonial cells exhibiting a more amoeboid morphology consistent with relatively high levels of macropinocytotic activity. Comparison of multiple reconstructed rosette colonies highlighted the variable nature of cell sizes, cell-cell contact networks and colony arrangement. Importantly, we uncovered the presence of elongated cells in some rosette colonies that likely represent a distinct and differentiated cell type. Intercellular bridges within choanoflagellate colonies displayed a variety of morphologies and connected some, but not all, neighbouring cells. Reconstruction of sponge choanocytes revealed both ultrastructural commonalities and differences in comparison to choanoflagellates. Choanocytes and colonial choanoflagellates are typified by high amoeboid cell activity. In both, the number of microvilli and volumetric proportion of the Golgi apparatus are comparable, whereas choanocytes devote less of their cell volume to the nucleus and mitochondria than choanoflagellates and more of their volume to food vacuoles. Together, our comparative reconstructions uncover the architecture of cell differentiation in choanoflagellates and sponge choanocytes and constitute an important step in reconstructing the cell biology of the last common ancestor of the animal kingdom.

evolutionary biology↗

bioRxiv2018

Glycosyltransferase homologs prevent promiscuous cell aggregation and promote multicellular development in the choanoflagellate S. rosetta

The mechanisms underlying multicellular development in the animal stem lineage may be reconstructed through the study of choanoflagellates, the closest living relatives of animals. To determine the genetic underpinnings of multicellularity in the emerging model choanoflagellate S. rosetta, we performed a screen for mutants with defects in multicellular rosette development. In two of the mutants, Jumble and Couscous, single cells failed to develop into orderly rosettes but instead aggregated promiscuously into amorphous clumps of cells. Both mutants mapped to lesions in genes encoding glycosyltransferases and the mutations perturbed glycosylation patterns in the extracellular matrix (ECM). In animals, glycosyltransferases transfer activated sugars to donor molecules and thereby sculpt the polysaccharide-rich ECM, regulate integrin and cadherin activity, and, when disrupted, contribute to tumorigenesis. The finding that glycosyltransferases promote proper rosette development and prevent cell aggregation in S. rosetta suggests a pre-metazoan role for glycosyltransferases in regulating development and preventing abnormal tumor-like multicellularity.\n\nIMPACT STATEMENTA genetic screen reveals that glycosyltransferases are required for proper rosette development and the prevention of cell clumping in one of the closest living relatives of animals, the choanoflagellate S. rosetta.

genetics↗

bioRxiv2018

A robust method for transfection in choanoflagellates illuminates their cell biology and the ancestry of animal septins

As the closest living relatives of animals, choanoflagellates offer unique insights into animal origins and core mechanisms underlying animal cell biology. However, unlike traditional model organisms, such as yeast, flies and worms, choanoflagellates have been refractory to DNA delivery methods for expressing foreign genes. Here we report the establishment of a robust method for expressing transgenes in the choanoflagellate Salpingoeca rosetta, overcoming barriers that have previously hampered DNA delivery and expression. To demonstrate how this method accelerates the study of S. rosetta cell biology, we engineered a panel of fluorescent protein markers that illuminate key features of choanoflagellate cells. We then investigated the localization of choanoflagellate septins, a family of GTP-binding cytoskeletal proteins that are hypothesized to regulate the multicellular rosette development in S. rosetta. Fluorescently tagged septins localized to the basal pole of S. rosetta single cells and rosettes in a pattern resembling septin localization in animal epithelia. The establishment of transfection in S. rosetta and its application to the study of septins represent critical advances in the growth of S. rosetta as an experimental model for investigating choanoflagellate cell biology, core mechanisms underlying animal cell biology, and the origin of animals.

cell biology↗

bioRxiv2017

The ancestral animal genetic toolkit revealed by diverse choanoflagellate transcriptomes

The changes in gene content that preceded the origin of animals can be reconstructed by comparison with their sister group, the choanoflagellates. However, only two choanoflagellate genomes are currently available, providing poor coverage of their diversity. We sequenced transcriptomes of 19 additional choanoflagellate species to produce a comprehensive reconstruction of the gains and losses that shaped the ancestral animal gene repertoire. We find roughly 1,700 gene families with origins on the animal stem lineage, of which only a core set of 36 are conserved across animals. We find more than 350 gene families that were previously thought to be animal-specific actually evolved before the animal-choanoflagellate divergence, including Notch and Delta, Toll-like receptors, and glycosaminoglycan hydrolases that regulate animal extracellular matrix (ECM). In the choanoflagellate Salpingoeca helianthica, we show that a glycosaminoglycan hydrolase modulates rosette colony size, suggesting a link between ECM regulation and morphogenesis in choanoflagellates and animals.\n\nData AvailabilityRaw sequencing reads: NCBI BioProject PRJNA419411 (19 choanoflagellate transcriptomes), PRJNA420352 (S. rosetta polyA selection test)\n\nTranscriptome assemblies, annotations, and gene families: https://dx.doi.org/10.6084/m9.figshare.5686984\n\nProtocols: https://dx.doi.org/10.17504/protocols.io.kwscxee

evolutionary biology↗

bioRxiv2017

Lessons from simple marine models on the bacterial regulation of eukaryotic development

Molecular cues from environmental bacteria influence important developmental decisions in diverse marine eukaryotes. Yet, relatively little is understood about the mechanisms underlying these interactions, in part because marine ecosystems are dynamic and complex. With the help of simple model systems, including the choanoflagellate Salpingoeca rosetta, we have begun to uncover the bacterial cues that shape eukaryotic development in the ocean. Here, we review how diverse bacterial cues - from lipids to macromolecules - regulate development in marine eukaryotes. It is becoming clear that there are networks of chemical information circulating in the ocean, with both eukaryotes and bacteria acting as nodes; one eukaryote can precisely respond to cues from several diverse environmental bacteria, and a single environmental bacterium can regulate the development of different eukaryotes.\n\nHighlightsO_LICues from environmental bacteria influence the development of many marine eukaryotes\nC_LIO_LIThe molecular cues produced by environmental bacteria are structurally diverse\nC_LIO_LIEukaryotes can respond to many different environmental bacteria\nC_LIO_LISome environmental bacteria act as \"information hubs\" for diverse eukaryotes\nC_LIO_LIExperimentally tractable systems, like the choanoflagellate S. rosetta, promise to reveal molecular mechanisms underlying these interactions\nC_LI

microbiology↗

bioRxiv2017

The origin of animal multicellularity and cell differentiation

How animals evolved from their single-celled ancestors over 600 million years ago is poorly understood. Comparisons of genomes from animals and their closest relatives - choanoflagellates, filastereans and ichthyosporeans - have recently revealed the genomic landscape of animal origins. However, the cell and developmental biology of the first animals have been less well examined. Using principles from evolutionary cell biology, we reason that the last common ancestor of animals and choanoflagellates (the Urchoanozoan) used a collar complex - a flagellum surrounded by a microvillar collar - to capture bacterial prey. The origin of animal multicellularity likely occurred through the modification of pre-existing mechanisms for extracellular matrix synthesis and regulation of cytokinesis. The progenitors of animals likely developed clonally through serial division of flagellated cells, giving rise to sheets of cells that folded into spheres by a morphogenetic process comparable to that seen in modern choanoflagellate rosettes and calcareous sponge embryos. Finally, we infer that cell differentiation evolved in the animal stem-lineage by a combination of three mechanisms: division of labor from ancient plurifunctional cell types, conversion of temporally segregated phenotypes into spatially segregated cell types, and functional innovation.

evolutionary biology↗

bioRxiv2017

An Aphrodisiac Produced By Vibrio fischeri Stimulates Mating In The Closest Living Relatives Of Animals

We serendipitously discovered that the marine bacterium Vibrio fischeri induces sexual reproduction in one of the closest living relatives of animals, the choanoflagellate Salpingoeca rosetta. Although bacteria influence everything from nutrition and metabolism to cell biology and development in eukaryotes, bacterial regulation of eukaryotic mating was unexpected. Here we show that a single V. fischeri protein, the previously uncharacterized EroS, fully recapitulates the aphrodisiac activity of live V. fischeri. EroS is a chondroitin lyase; although its substrate, chondroitin sulfate, was previously thought to be an animal synapomorphy, we demonstrate that S. rosetta produces chondroitin sulfate and thus extend the ancestry of this important glycosaminoglycan to the premetazoan era. Finally, we show that V. fischeri, purified EroS, and other bacterial chondroitin lyases induce S. rosetta mating at environmentally-relevant concentrations suggesting that bacterially-produced aphrodisiacs likely regulate choanoflagellate mating in nature.

microbiology↗