Biology subjects

King, N.

Publications and source records attributed to King, N..

At least 19 recordsLinked to original sources

bioRxiv2026

An adhesion GPCR regulates cell adhesion and mating in the closest living relatives of metazoans

The transition to metazoan multicellularity required the evolution of cell-surface receptors that coordinate adhesion and signaling under changing environmental conditions. We investigated potential regulators of cell interactions in the choanoflagellate Salpingoeca rosetta, one of the closest living relatives of metazoans. Here, we identify Cupidon, an adhesion G protein-coupled receptor that acts as a dual-function regulator of cell adhesion and mating. Under well-fed (i.e., nutrient-replete) conditions, Cupidon suppresses cell aggregation by inhibiting N-acetylglucosamine-dependent collar-mediated adhesion. Starvation of S. rosetta triggers gametogenesis, resulting in anisogametes: female gametes with an elongated collar and male gametes that form a basal protrusion, the "fertilopod." Cupidon undergoes concurrent changes in proteolytic processing and localization, ultimately concentrating at the gamete contact interface, where it promotes gamete fusion. Together, our findings reveal that aGPCR-mediated regulation of cell adhesion predates the origin of metazoans.

evolutionary biology↗

bioRxiv2026

Septins regulate cytokinesis and multicellular development in the closest living relatives of animals

Septins are cytoskeletal proteins that regulate cytokinesis in fungi and animals, yet their functions in choanoflagellates -- the closest living relatives of animals -- have remained unknown. Salpingoeca rosetta, a choanoflagellate that switches between unicellular and multicellular forms, encodes four septins closely related to animal and fungal septins. CRISPR/Cas9-mediated disruption of S. rosetta septins revealed that a subset regulate cell size, with two mutants exhibiting an elevated frequency of oversized cells and one exhibiting smaller cells. Three of the four septins were required for proper rosette colony development, while two also regulated rosette structural integrity. Characterization of Sros_septA, which showed the strongest phenotype, revealed a role in cytokinesis: mutant cells exhibited late-stage cytokinesis failure, resulting in enlarged, multinucleated cells. Cytokinesis failure rate increased in uninduced Sros_septA mutant cells and was further elevated upon rosette induction, suggesting that the multicellular context places heightened demands on the septin cytoskeleton. Endogenously tagged Sros_SeptA dynamically redistributed from the basal pole in interphase cells to the cleavage furrow and nascent intercellular bridge during cell division. These findings identify septins as regulators of cytokinesis and multicellular development in S. rosetta and offer a framework for exploring how cell division regulation contributed to the emergence of animal multicellularity. Significance StatementO_LISeptins are cytoskeletal proteins that regulate cell division in fungi and animals, but their functions in choanoflagellates - the closest living relatives of animals - were unknown. C_LIO_LIUsing CRISPR/Cas9 gene editing in Salpingoeca rosetta, we show that septins regulate both cell size and multicellular colony development. SeptA, whose gene disruption produced the strongest phenotype, localizes dynamically to the cleavage furrow and regulates cytokinesis, with cell size and division defects that are exacerbated during multicellular rosette development. C_LIO_LIThese findings raise the possibility that elaboration of the extracellular matrix during animal origins imposed new mechanical demands on dividing cells, linking the evolution of cell adhesion to the evolution of cytokinetic regulation. C_LI

evolutionary biology↗

bioRxiv2026

Herbarium genomics reveal signatures of colonization history, lineage turnover, and adaptation during invasion

O_LIA key challenge in invasion biology is dissecting the roles of colonization history, admixture, and selection on invasion success. However, it is often difficult to determine the sources and locations of introduction events as well as the timing and magnitude of gene flow and selection using genomics datasets from contemporary samples. C_LIO_LIWe disentangle the evolutionary processes acting during the North American invasion of Trifolium repens (white clover) using low-coverage whole genome sequencing from 513 herbarium specimens originally collected between 1838-2018. C_LIO_LIIntroduction history paralleled European colonization patterns. The earliest collected samples had predominantly Belgian and British ancestry, with increasing French and Spanish contributions over time in the Mid-Atlantic and South. Genomic diversity increased significantly through time likely driven by admixture between independently introduced lineages. In lowest latitude regions, there is a strong shift in dominant ancestry with limited admixture (i.e., lineage turnover). Putatively adaptive loci exhibited protracted shifts in allele frequency during the invasion, consistent with contemporary clines. C_LIO_LIOur study demonstrates the power of herbarium genomics to provide spatiotemporal insight into recent evolutionary history. We find a complex invasion history that includes multiple introductions, lineage turnover, and admixture that increased genetic diversity and likely facilitated adaptation in this now cosmopolitan plant. C_LI

plant biology↗

bioRxiv2025

G protein-coupled receptor diversity and evolution in the closest living relatives of Metazoa

G protein-coupled receptors (GPCRs) play a pivotal role in the perception of environmental cues across eukaryotic diversity. Although GPCRs have been relatively well characterized in metazoans, GPCR signaling is poorly understood in their sister group, the choanoflagellates, and in other close relatives of metazoans (CRMs). Here, we examine GPCR diversity and evolution in choanoflagellates by curating a catalog of 918 GPCRs, 141 G proteins, and 367 associated regulators from 23 choanoflagellate genomes and transcriptomes. We found that the repertoire of choanoflagellate GPCRs is larger and more diverse than previously anticipated, with 18 GPCR families found in choanoflagellates, of which 12 families are newly identified in these organisms. Comparative analyses revealed that most choanoflagellate GPCR families are conserved in metazoans and/or other eukaryotic lineages. Adhesion GPCRs and a class of GPCRs fused to kinases (the GPCR-TKL/Ks) are the most abundant GPCRs in choanoflagellates. The identification of GPCR repertoires in CRMs and other non-metazoans refines our understanding of metazoan GPCR evolution and reveals the existence of previously unreported GPCR families in metazoans and at the root of the eukaryotic tree.

evolutionary biology↗

bioRxiv2024

OrthoHMM: Improved Inference of Ortholog Groups using Hidden Markov Models

Accurate orthology inference is essential for comparative genomics and phylogenomics. However, orthology inference is challenged by sequence divergence, which is pronounced among anciently diverged organisms. We present OrthoHMM, an algorithm that infers orthologous gene groups using Hidden Markov Models parameterized from substitution matrices, which enables better detection of remote homologs. Benchmarking indicates OrthoHMM outperforms currently available methods; for example, using a curated set of Bilaterian orthogroups, OrthoHMM showed a 10.3 - 138.9% improvement in precision. Rank-based benchmarking using Bilaterian orthogroups and a novel dataset of orthogroups from organisms in three major eukaryotic kingdoms revealed OrthoHMM had the best overall performance (6.7 - 97.8% overall improvement). These findings suggest that Hidden Markov Models improve orthogroup inference.

bioinformatics↗

bioRxiv2024

Adaptation to climate in the native and introduced ranges of a cosmopolitan plant

Climate change and the global spread of non-native species are two of the most significant threats to biodiversity and ecosystem function. Both these phenomena subject populations to novel conditions, either in space (species introductions) or in time (climate change), yet the role of adaptation in how populations respond to these rapid environmental shifts is poorly understood. We conducted a large-scale trans-continental common garden experiment using white clover (Trifolium repens, Fabaceae) to test whether adaptive evolution to spatiotemporal variation in climate could contribute to the ecological success of one of the most widespread plant species in the world. Individuals from 96 populations of Trifolium repens (white clover) from both its native (Europe) and introduced (North America) ranges were planted into four experimental common gardens located in northern (Uppsala, Sweden) and southern (Montpellier, France) Europe, and northern (Mississauga, Canada) and southern (Lafayette, USA) North America. We recorded plant sexual and clonal fitness in each common garden and assessed whether the strength of local adaptation differed between the native and introduced ranges and whether populations are rapidly adapting to climate change. Results show that local adaptation was only evident when populations were transplanted into common gardens located in the same range (native or introduced) from which they originated and was driven by stronger selection (due to climatic factors rather than herbivory) at lower latitudes in both ranges. Our results indicate rapid local adaptation across a large latitudinal gradient in introduced T. repens populations, along with an associated adaptation cost when transplanted back into the native range. We also find evidence of an adaptation lag in the northern common garden in the introduced range, with plants from historically warmer climates exhibiting the greatest fitness. These findings support two major conclusions: 1) white clover can rapidly adapt to spatial variation in climate in its introduced range as well as the native range, and 2) despite rapid adaptation to novel environments, introduced white clover populations are not keeping pace with rapid climate change. Overall, our results provide insight into the role of adaptation in facilitating the ecological success of non-native species in a rapidly changing world. Open Research StatementData are provided for peer review. All data involved in this study is available on the GitHub page for LJA (https://github.com/ljalbano/transcontinental_common_garden).

evolutionary biology↗

bioRxiv2024

Structural variants underlie parallel adaptation following global invasion

Rapid adaptation during invasion has historically been considered limited and unpredictable. We leverage whole-genome sequencing of >2600 plants across six continents to investigate the relative roles of colonization history and adaptation during the worldwide invasion of Trifolium repens. Introduced populations contain high levels of genetic variation with independent colonization histories evident on different continents. Five large structural variants on three chromosomes exist as standing genetic variation within the native range, and exhibit strong signatures of parallel climate-associated adaptation across continents. Common gardens in the native and introduced ranges demonstrate that three structural variants exhibit patterns of selection consistent with local adaptation across each range. Our results provide strong evidence that rapid and parallel adaptation during invasion is caused by large-effect structural variants introduced throughout the world. Significance StatementBiological invasions occur over short timescales and introductions are often hypothesized to include limited genetic diversity, making the role of adaptation in invasion success controversial. We demonstrate that the invasion of a human-commensal species, Trifolium repens, likely stems from multiple, diverse introductions with significant evidence of climate-associated adaptation following introduction. The genetic basis of adaptation is most strongly linked to five chromosomal rearrangements that each span hundreds of genes - matching theoretical predictions that large-effect variants are key to the initial stages of adaptation to novel environments. Chromosomal rearrangements have remarkably parallel signatures of adaptation across different introductions despite initial colonization from different areas of Europe. Our study highlights the impact of globalization and rapid adaptation for the invasion success of human commensal species.

evolutionary biology↗

bioRxiv2024

Mixed clonal-aggregative multicellularity entrained by extreme salinity fluctuations in a close relative of animals

Multicellularity evolved multiple times independently during eukaryotic diversification1-4. Two distinct mechanisms underpin multicellularity5: clonality (serial cell division without sister-cell separation) and aggregation (whereby independent cells assemble into a multicellular entity). Clonal and aggregative multicellularity are traditionally considered mutually exclusive1,6-9, with rare exceptions10, and evolutionary hypotheses have addressed why multicellularity might diverge toward one or the other extreme3,4. Both animals and their sister group, the choanoflagellates, are currently only known to acquire multicellularity clonally4,11-13. Here, we show that the choanoflagellate Choanoeca flexa14 forms motile and contractile cell monolayers (or "sheets") through multiple mechanisms: C. flexa sheets can form purely clonally, purely aggregatively, or by a combination of both processes. We characterise the life history of C. flexa in its natural environment - ephemeral splash pools on the island of Curacao - and show that C. flexa undergoes reversible transitions between unicellularity and multicellularity during cycles of evaporation and refilling. Different splash pools house genetically distinct strains of C. flexa, between which aggregation is constrained by kin recognition15-18. We show that clonal-aggregative multicellularity serves as a versatile strategy for the robust re-establishment of multicellularity in this variable and fast-fluctuating environment. Our findings challenge former generalisations about choanoflagellates and expand the option space of choanozoan multicellularity.

evolutionary biology↗

bioRxiv2023

Hierarchical design of pseudosymmetric protein nanoparticles

Discrete protein assemblies ranging from hundreds of kilodaltons to hundreds of megadaltons in size are a ubiquitous feature of biological systems and perform highly specialized functions 1-3. Despite remarkable recent progress in accurately designing new self-assembling proteins, the size and complexity of these assemblies has been limited by a reliance on strict symmetry 4,5. Inspired by the pseudosymmetry observed in bacterial microcompartments and viral capsids, we developed a hierarchical computational method for designing large pseudosymmetric self-assembling protein nanomaterials. We computationally designed pseudosymmetric heterooligomeric components and used them to create discrete, cage-like protein assemblies with icosahedral symmetry containing 240, 540, and 960 subunits. At 49, 71, and 96 nm diameter, these nanoparticles are the largest bounded computationally designed protein assemblies generated to date. More broadly, by moving beyond strict symmetry, our work represents an important step towards the accurate design of arbitrary self-assembling nanoscale protein objects.

bioengineering↗

bioRxiv2022

An RFX transcription factor regulated ciliogenesis in the progenitors of choanoflagellates and animals

Little is known about the origins of the transcriptional modules that coordinate cell-type specific functions in animals. The controlled expression of one cellular feature - the cilium - was likely critical during early animal evolution. Two key transcription factors, RFX and FoxJ1, coordinate ciliogenesis in animals but are absent from the genomes of most other ciliated eukaryotes, raising the question of how the transcriptional regulation of ciliogenesis has evolved. To reconstruct the evolution of the RFX/FoxJ1 transcriptional module and its role in the regulation of ciliogenesis, we investigated RFX and FoxJ1 function in one of the closest living relatives of animals, the choanoflagellate Salpingoeca rosetta. Targeted disruption of the S. rosetta RFX homolog cRFXa resulted in delayed cell proliferation and aberrant ciliogenesis, marked by the collapse and resorption of nascent cilia. Ciliogenesis genes and foxJ1 were significantly down-regulated in cRFXa mutants, consistent with a pre-animal ancestry for this transcriptional module. We also found that cRFXa protein preferentially binds to a sequence motif that is enriched in the promoters of S. rosetta ciliary genes and matches the sequence motif bound by animal RFX proteins. These findings suggest that RFX coordinated ciliogenesis before the divergence of animals and choanoflagellates, and that the deployment of this module may have provided a mechanism to differentiate ciliated and non-ciliated cell types in early animal evolution.

evolutionary biology↗

bioRxiv2022

A small molecule screen reveals the essentiality of p38 kinase for choanoflagellate cell proliferation

Animal kinases regulate cellular responses to environmental stimuli, including cell differentiation, migration, survival, and response to stress, but the ancestry of these functions is poorly understood. Choanoflagellates, the closest living relatives of animals, encode homologs of diverse animal kinases and have emerged as model organisms for reconstructing animal origins. However, efforts to identify key kinase regulators in choanoflagellates have been constrained by the limitations of currently available genetic tools. Here, we report on a framework that combines small molecule-driven kinase discovery with targeted genetics to reveal kinase function in choanoflagellates. To study the physiological roles of choanoflagellate kinases, we established two high-throughput platforms to screen the model choanoflagellate Salpingoeca rosetta with a curated library of human kinase inhibitors. We identified 95 diverse kinase inhibitors that disrupt S. rosetta cell proliferation. By focusing on one inhibitor, sorafenib, we identified a p38 kinase as a regulator of the heat shock response in S. rosetta. This finding reveals a conserved p38 function between choanoflagellates, animals, and fungi. Moreover, this study demonstrates that existing kinase inhibitors can serve as powerful tools to examine the ancestral roles of kinases that regulate modern animal development.

biochemistry↗

bioRxiv2022

Nitric oxide signaling controls collective contractions in a colonial choanoflagellate

Although signaling by the gaseous molecule nitric oxide (NO) regulates key physiological processes in animals, including contractility1-3, immunity4,5, development6-9 and locomotion10,11, the early evolution of animal NO signaling remains unclear. To reconstruct the role of NO in the animal stem lineage, we set out to study NO signaling in choanoflagellates, the closest living relatives of animals12. In animals, NO produced by the nitric oxide synthase (NOS) canonically signals through cGMP by activating soluble guanylate cyclases (sGCs)13,14. We surveyed the distribution of the NO signaling pathway components across the diversity of choanoflagellates and found three species that express NOS, sGCs, and downstream genes previously shown to be involved in the NO/cGMP pathway. One of these, Choanoeca flexa, forms multicellular sheets that undergo collective contractions controlled by cGMP15. We found that treatment with NO induces sustained contractions in C. flexa by signaling through an sGC/cGMP pathway. Biochemical assays show that NO directly binds C. flexa sGC1 and stimulates its cyclase activity. The NO/cGMP pathway acts independently from other inducers of C. flexa contraction, including mechanical stimuli and heat, but sGC activity is required for contractions induced by light-to-dark transitions. The output of NO signaling in C. flexa - contractions resulting in a switch from feeding to swimming - resembles the effect of NO in sponges1-3 and cnidarians11,16,17, where it interrupts feeding and activates contractility. These data provide insights into the biology of the first animals and the evolution of NO signaling.

evolutionary biology↗

bioRxiv2021

Widespread distribution of collagens and collagen-associated domains in eukaryotes

The origin of collagen, the dominant structural component of metazoan extracellular matrix, has long been cited as a critical step in the evolution of metazoan multicellularity. While collagens were once thought to be found only in metazoans, scattered reports of collagen domains in Fungi, and more recently in close relatives of metazoans, have called into question whether collagens are truly unique to metazoans. Here, we take advantage of recently sequenced genomes and transcriptomes of diverse holozoans (the clade encompassing metazoans and their close relatives), as well as publicly available proteomes from diverse non-holozoan eukaryotes, to conduct a systematic search for collagen domains across eukaryotic diversity. We find that collagen domains are ubiquitous in choanoflagellates, the sister group of metazoans, and widespread across many other major eukaryotic taxa. Many predicted collagens in non-metazoans are comparable to metazoan collagens in length and proline content. Moreover, most are present in species that also encode putative prolyl 4-hydroxylase domains, suggesting that, like metazoan collagens, they may be stabilized through the hydroxylation of prolines. Fibrillar collagen and collagen IV appear to be unique to metazoans, and we posit that their ability to assemble into superstructures may have contributed to the origin of metazoan multicellularity.

evolutionary biology↗

bioRxiv2021

STING mediates immune responses in a unicellular choanoflagellate

Animals have evolved unique repertoires of innate immune genes and pathways that provide their first line of defense against pathogens. To reconstruct the ancestry of animal innate immunity, we have developed the choanoflagellate Monosiga brevicollis, one of the closest living relatives of animals, as a model for studying mechanisms underlying pathogen recognition and immune response. We found that M. brevicollis is killed by exposure to Pseudomonas aeruginosa bacteria. Moreover, M. brevicollis expresses STING, which, in animals, activates innate immune pathways in response to cyclic dinucleotides during pathogen sensing. M. brevicollis STING increases the susceptibility of M. brevicollis to P. aeruginosa-induced cell death and is required for responding to the cyclic dinucleotide 23 cGAMP. Furthermore, similar to animals, autophagic signaling in M. brevicollis is induced by 23 cGAMP in a STING-dependent manner. This study provides evidence for a pre-animal role for STING in antibacterial immunity and establishes M. brevicollis as a model system for the study of immune responses.

microbiology↗

bioRxiv2021

Colonial choanoflagellate isolated from Mono Lake harbors a microbiome

Choanoflagellates offer key insights into bacterial influences on the origin and early evolution of animals. Here we report the isolation and characterization of a new colonial choanoflagellate species, Barroeca monosierra, that, unlike previously characterized species, harbors a microbiome. B. monosierra was isolated from Mono Lake, California and forms large spherical colonies that are more than an order of magnitude larger than those formed by the closely related Salpingoeca rosetta. By designing fluorescence in situ hybridization probes from metagenomic sequences, we found that B. monosierra colonies are colonized by members of the halotolerant and closely related Saccharospirillaceae and Oceanospirillaceae, as well as purple sulfur bacteria (Ectothiorhodospiraceae) and non-sulfur Rhodobacteraceae. This relatively simple microbiome in a close relative of animals presents a new experimental model for investigating the evolution of stable interactions among eukaryotes and bacteria. IMPORTANCEThe animals and bacteria of Mono Lake (California) have evolved diverse strategies for surviving the hypersaline, alkaline, arsenic-rich environment. We sought to investigate whether the closest living relatives of animals, the choanoflagellates, exist among the relatively limited diversity of organisms in Mono Lake. We repeatedly isolated members of a single species of choanoflagellate, which we have named Barroeca monosierra, suggesting that it is a stable and abundant part of the ecosystem. Characterization of B. monosierra revealed that it forms large spherical colonies that each contain a microbiome, providing an opportunity to investigate the evolution of stable physical associations between eukaryotes and bacteria.

microbiology↗

bioRxiv2020

Dietary protein increases T cell independent sIgA production through changes in gut microbiota-derived extracellular vesicles

Secretory IgA (sIgA) is a key mucosal component ensuring host-microbiota mutualism. Using nutritional geometry modelling in mice fed 10 different macronutrient-defined, isocaloric diets, we identified dietary protein as the major driver of sIgA production. Protein-driven sIgA induction was not mediated by T cell-dependent pathways or changes in gut microbiota composition. Instead, the microbiota of high-protein fed mice produced significantly higher quantities of extracellular vesicles (EV), compared to those of mice fed high-carbohydrate or high-fat diets. These EV activated TLR4 to increase the epithelial expression of IgA-inducing cytokine, APRIL, B cell chemokine, CCL28, and the IgA transporter, PIGR. We showed that succinate, produced in high concentrations by microbiota of high-protein fed animals, increased the generation of reactive oxygen species by bacteria, which in turn promoted EV production. This is the first report establishing a causal link between dietary macronutrient composition, gut microbial EV release and host sIgA response.

immunology↗

bioRxiv2020

A flagellate-to-amoeboid switch in the closest living relatives of animals

The evolution of different cell types was a key process of early animal evolution1-3. Two fundamental cell types, epithelial cells and amoeboid cells, are broadly distributed across the animal tree of life4,5 but their origin and early evolution are unclear. Epithelial cells are polarized, have a fixed shape and often bear an apical cilium and microvilli. These features are shared with choanoflagellates - the closest living relatives of animals - and are thought to have been inherited from their last common ancestor with animals1,6,7. The deformable amoeboid cells of animals, on the other hand, seem strikingly different from choanoflagellates and instead evoke more distantly related eukaryotes, such as diverse amoebae - but it has been unclear whether that similarity reflects common ancestry or convergence8. Here, we show that choanoflagellates subjected to spatial confinement differentiate into an amoeboid phenotype by retracting their flagella and microvilli, generating blebs, and activating myosin-based motility. Choanoflagellate cell crawling is polarized by geometrical features of the substrate and allows escape from confined microenvironments. The confinement-induced amoeboid switch is conserved across diverse choanoflagellate species and greatly expands the known phenotypic repertoire of choanoflagellates. The broad phylogenetic distribution of the amoeboid cell phenotype across animals9-14 and choanoflagellates, as well as the conserved role of myosin, suggests that myosin-mediated amoeboid motility was present in the life history of their last common ancestor. Thus, the duality between animal epithelial and crawling cells might have evolved from a temporal phenotypic switch between flagellate and amoeboid forms in their single-celled ancestors3,15,16.

evolutionary biology↗

bioRxiv2020

In silico detection of SARS-CoV-2 specific B-cell epitopes and validation in ELISA for serological diagnosis of COVID-19

Rapid generation of diagnostics is paramount to understand epidemiology and to control the spread of emerging infectious diseases such as COVID-19. Computational methods to predict serodiagnostic epitopes that are specific for the pathogen could help accelerate the development of new diagnostics. A systematic survey of 27 SARS-CoV-2 proteins was conducted to assess whether existing B-cell epitope prediction methods, combined with comprehensive mining of sequence databases and structural data, could predict whether a particular protein would be suitable for serodiagnosis. Nine of the predictions were validated with recombinant SARS-CoV-2 proteins in the ELISA format using plasma and sera from patients with SARS-CoV-2 infection, and a further 11 predictions were compared to the recent literature. Results appeared to be in agreement with 12 of the predictions, in disagreement with 3, while a further 5 were deemed inconclusive. We showed that two of our top five candidates, the N-terminal fragment of the nucleoprotein and the receptor-binding domain of the spike protein, have the highest sensitivity and specificity and signal-to-noise ratio for detecting COVID-19 sera/plasma by ELISA. Mixing the two antigens together for coating ELISA plates led to a sensitivity of 94% (N=80 samples from persons with RT-PCR confirmed SARS-CoV2 infection), and a specificity of 97.2% (N=106 control samples).

bioinformatics↗