Biology subjects

Gilmore, T. D.

Publications and source records attributed to Gilmore, T. D..

14 recordsLinked to original sources

bioRxiv2025

Algae-specific Immune Modulation Influences Responses to Heat and Pathogen Challenge in a Symbiotic Coral

The role of symbiotic algae in coral life history and host health is well documented, but the immune and physiological trade-offs of hosting these symbionts remain less explored. While association with the algal symbionts of the genus Durusdinium is known to confer thermotolerance, it has also been linked to coral tissue loss under stress. We investigated whether algal type influences host immunity and stress responses in the tropical coral Pocillopora acuta. Durusdinium-hosting (D-hosting) P. acuta have distinct transcriptomic profiles, higher immune-related gene expression, and elevated baseline levels of immunity transcription factor NF-{kappa}B as compared to corals hosting Cladocopium (C-hosting). Under heat challenge, D-hosting P. acuta exhibited tissue loss, oxidative stress, immune and microbial dysregulation, whereas C-hosting P. acuta were more susceptible to bleaching, metabolic dysregulation, and decline in nitrogen-fixing and antioxidant-producing bacteria. Finally, infection with the the bacterium Vibrio coralliilyticus caused high tissue loss in D-hosting corals, but not in C-hosting corals. Our results suggest a mechanism for how Durusdinium association enhances thermotolerance yet predisposes corals to tissue damage under stress, suggesting immune trade-offs that can compromise host survival under multiple stressors.

genomics↗

bioRxiv2025

Symbiotic State Affects Microbiome Recovery in a Facultatively Symbiotic Cnidarian

The cnidarian holobiont consists of host cells, algal symbionts, and a complex microbiome that resides in and on the host tissue and the algal symbionts. To investigate the interactions between a host cnidarian, its algal symbionts, and its bacterial microbiome, we used antibiotics to deplete the microbiome of the facultatively symbiotic sea anemone Exaiptasia pallida (Aiptasia) in both symbiotic and aposymbiotic states. To understand the dynamics of microbiome reestablishment following disruption, we used 16S gene sequencing of the bacterial microbiome to characterize communities during recovery following antibiotic treatment. We assessed the host molecular response to microbiome depletion and recovery using RNA-seq and Western blotting of the immune transcription factor NF-{kappa}B. 16S results demonstrate that, following depletion, symbiotic Aiptasia can reestablish bacterial communities (based on diversity metrics and community composition) alike to those of controls before microbiome depletion. However, the microbiomes of aposymbiotic Aiptasia fail to reestablish to control conditions even after seven days of recovery. Bacteria in the Family Endozoicomonadaceae reestablish to control levels in symbiotic, but not aposymbiotic, Aiptasia, suggesting an association between Endozoicomonadaceae and algal symbionts. Our molecular analyses show that host immune system gene expression is downregulated during recovery from antibiotic treatment, but that NF-{kappa}B protein levels increase during recovery, suggesting mechanisms for microbiome reestablishment following disruption. This study shows the dynamics of microbiome recovery following depletion and the influence of microbiome community members on host gene expression in a cnidarian model, providing a foundation for future research involving pairwise interactions between microorganisms and cnidarian hosts.

microbiology↗

bioRxiv2025

Comparison of Activities of Transcription Factor NF-κB from Two Jellyfish Models

Herein, transcription factor Nuclear Factor-kappaB (NF-{kappa}B) is characterized as the downstream effector of putative innate immune signaling pathways from two model jellyfish, Aurelia aurita (Aa) and Clytia hemisphaerica (Ch). Both jellyfish NF-{kappa}B proteins consist primarily of the N-terminal DNA-binding/dimerization domain, and they lack the C-terminal ankyrin repeat inhibitory domain found in vertebrate NF-{kappa}B proteins. Both jellyfish NF-{kappa}B proteins bind to a consensus mammalian NF-{kappa}B binding site, and their AlphaFold3-predicted structures on DNA are similar to that of mouse NF-{kappa}B p50. Neither Aa- nor Ch-NF-{kappa}B activated transcription of an NF-{kappa}B-site reporter gene in human cells; however, Aa-NF-{kappa}B did activate transcription from a GAL4-site reporter gene in yeast, whereas Ch-NF-{kappa}B did not. Aa- and Ch-NF-{kappa}B were both constitutively located in the nucleus when expressed in vertebrate cells. Homologs of I{kappa}B-like proteins from both species interacted with their corresponding NF-{kappa}B proteins in co-immunoprecipitation assays in HEK 293T cells. These I{kappa}B-like proteins influenced the subcellular localization of their cognate NF-{kappa}B proteins in vertebrate cells, and their ankyrin repeat domains were predicted to interact with Ch-NF-{kappa}B in a manner similar to mammalian I{kappa}B and NF-{kappa}B. RNA-sequencing data from Ch animals indicate that Ch-NF-{kappa}B is expressed at high levels in early developmental stages, when Ch-I{kappa}B expression is low, suggesting that active Ch-NF-{kappa}B controls an early developmental process. In contrast, in adult animals the expression of Ch-I{kappa}B is high, suggesting that Ch-NF-{kappa}B requires a signal in order to become active. Overall, these results provide comparative information on the structure, activity, and mRNA expression of jellyfish NF-{kappa}B pathway proteins among jellyfish, and they suggest roles for NF-{kappa}B in developing and adult jellyfish.

molecular biology↗

bioRxiv2024

Antibiotic treatment and microbiome depletion slow cnidarian regeneration

Cnidarian microbiomes play an essential role in physiology and development, but how these communities influence tissue regeneration is poorly understood. Here, we examined the effects of antibiotic exposure on regeneration and microbial communities in two cnidarian models, the sea anemones Nematostella vectensis (non-symbiotic, hereafter, Nematostella) and Exaiptasia pallida (symbiotic, hereafter, Aiptasia). Bisected animals were incubated in either sterile or antibiotic-containing artificial seawater for seven days and regeneration was monitored daily. After seven days, tentacle number and length were measured, and microbial communities were profiled using metabarcoding of the V4 region of the 16S rRNA. Microbiome disruption was observed under antibiotic treatment in both species, resulting in decreased microbial load and shifts in relative abundances of some microbial taxa. However, Nematostella exhibited a greater reduction in microbial diversity and community shifts under antibiotic exposure, whereas Aiptasia showed only moderate changes in diversity. In both species, microbiome disruption was associated with slower regeneration rates and reduced tentacle number and length, suggesting a functional role for the microbiome in cnidarian regeneration. Our findings suggest that host-microbiome interactions in both symbiotic and aposymbiotic anemones are important for the maintenance of regenerative processes. These findings provide insight into how cnidarians and their microbiomes respond to environmental stressors, with implications for predicting cnidarian resilience in the context of emerging threats to the marine environment.

developmental biology↗

bioRxiv2024

AutoMorFi: Automated Whole-image Morphometry in Fiji/ImageJ for Diverse Image Analysis Needs

Running on the highly popular and accessible ImageJ/Fiji platform for biological image analysis, we have established AutoMorFi as a streamlined interface for automated whole-image morphometric analysis that generates at least 47 measurements per cell or object in under 1 minute. We performed multiple validated cluster and principal component analyses on nonredundant morphometric parameters derived from AutoMorFi for various cell types, objects, and organisms. We used images of rodent macrophages, human postmortem brain tissues from multiple sclerosis (MS) and Alzheimers disease (AD) patients, iPSC/animal models for Downs syndrome and autism spectrum disorder (ASD), and organisms such as sea anemone and corals. AutoMorFis adaptability extends across diverse imaging modalities including brightfield, confocal, or widefield fluorescence microscopy as well as underwater photography. Due to its unlimited and unbiased sampling across any image and high potential for modification and customization, using AutoMorFi has led to the discovery of new distinguishing features in previously studied cell types and organisms as well as the development of rapid diagnostic approaches. AutoMorFi represents a transformative tool that will accelerate morphometric analysis and offer broad relevance in biological studies.

cell biology↗

bioRxiv2024

Single-cell RNA-sequencing reveals immune system compartmentalization under symbiosis in the stony coral Oculina arbuscula

Many cnidarians host single-celled algae within gastrodermal cells, yielding a mutually beneficial exchange of nutrients between host and symbiont, and dysbiosis can lead to host mortality. Previous research has uncovered symbiosis tradeoffs, including suppression of immune pathways in cnidarians hosting intracellular algae and correlations between symbiotic state and pathogen susceptibility. Here, we used a multiomic approach to characterize symbiotic states of the facultatively symbiotic coral Oculina arbuscula by generating genotype-controlled fragments of symbiotic and aposymbiotic tissue. 16S metabarcoding showed no difference in bacterial communities between symbiotic states. Whole-organism proteomics revealed differential abundance of proteins related to immunity, confirming immune suppression during symbiosis. Finally, single-cell RNAseq identified diverse cell clusters within seven cell types across symbiotic states. Specifically, the gastrodermal cell clusters containing algal-hosting cells from symbiotic tissue had higher expression of nitrogen cycling and sugar transport genes than aposymbiotic gastrodermal cells. Furthermore, differential enrichment of immune system gene pathways and lower expression of genes involved in immune regulation were observed in these gastrodermal cells from symbiotic tissue. However, no differences in immune gene expression in the immune cell cluster were observed between symbiotic states. This work reveals a compartmentalization of immune system regulation in specific gastrodermal cells in symbiosis, which may limit symbiosis tradeoffs by simultaneously dampening immunity in algal-hosting cells while maintaining general organismal immunity.

genomics↗

bioRxiv2024

Rapid profiling of transcription factor-cofactor interaction networks reveals principles of epigenetic regulation

Transcription factor (TF)-cofactor (COF) interactions define dynamic, cell-specific networks that govern gene expression; however, these networks are understudied due to a lack of methods for high-throughput profiling of DNA-bound TF-COF complexes. Here we describe the Cofactor Recruitment (CoRec) method for rapid profiling of cell-specific TF-COF complexes. We define a lysine acetyltransferase (KAT)-TF network in resting and stimulated T cells. We find promiscuous recruitment of KATs for many TFs and that 35% of KAT-TF interactions are condition specific. KAT-TF interactions identify NF-{kappa}B as a primary regulator of acutely induced H3K27ac. Finally, we find that heterotypic clustering of CBP/P300-recruiting TFs is a strong predictor of total promoter H3K27ac. Our data supports clustering of TF sites that broadly recruit KATs as a mechanism for widespread co-occurring histone acetylation marks. CoRec can be readily applied to different cell systems and provides a powerful approach to define TF-COF networks impacting chromatin state and gene regulation.

molecular biology↗

bioRxiv2023

Nutrient deprivation differentially affects gene expression, immunity, and pathogen susceptibility across symbiotic states in a model cnidarian

Mutualistic symbioses between cnidarians and photosynthetic algae are modulated by complex interactions between host immunity and environmental conditions. Here, we investigate how symbiosis interacts with nutrient limitation to influence gene expression and stress response programming in the sea anemone Exaiptasia pallida (Aiptasia). Transcriptomic responses to starvation were similar between symbiotic and aposymbiotic Aiptasia; however, aposymbiotic anemone responses were stronger. Starved Aiptasia of both symbiotic states exhibited increased protein levels of immune-related transcription factor NF-{kappa}B, its associated gene pathways, and putative target genes. However, this starvation-induced increase in NF-{kappa}B only correlated with increased immunity in symbiotic anemones. Furthermore, starvation had opposite effects on Aiptasia susceptibility to pathogen and oxidative stress challenges, suggesting distinct energetic priorities under nutrient scarce conditions. Finally, when we compared starvation responses in Aiptasia to those of a facultative coral and nonsymbiotic anemone, "defense" responses were similarly regulated in Aiptasia and the facultative coral, but not in the nonsymbiotic anemone. This pattern suggests that capacity for symbiosis influences immune responses in cnidarians. In summary, expression of certain immune pathways - including NF-{kappa}B - does not necessarily predict susceptibility to pathogens, highlighting the complexities of cnidarian immunity and the influence of symbiosis under varying energetic demands.

genomics↗

bioRxiv2023

A Conserved Core Region of the Scaffold NEMO is Essential for Signal-induced Conformational Change and Liquid-liquid Phase Separation

Scaffold proteins help mediate interactions between protein partners, often to optimize intracellular signaling. Herein, we use comparative, biochemical, biophysical, molecular, and cellular approaches to investigate how the scaffold protein NEMO contributes to signaling in the NF-{kappa}B pathway. Comparison of NEMO and the related protein optineurin from a variety of evolutionarily distant organisms revealed that a central region of NEMO, called the Intervening Domain (IVD), is conserved between NEMO and optineurin. Previous studies have shown that this central core region of the IVD is required for cytokine-induced activation of I{kappa}B kinase (IKK). We show that the analogous region of optineurin can functionally replace the core region of the NEMO IVD. We also show that an intact IVD is required for the formation of disulfide-bonded dimers of NEMO. Moreover, inactivating mutations in this core region abrogate the ability of NEMO to form ubiquitin-induced liquid-liquid phase separation droplets in vitro and signal-induced puncta in vivo. Thermal and chemical denaturation studies of truncated NEMO variants indicate that the IVD, while not intrinsically destabilizing, can reduce the stability of surrounding regions of NEMO, due to the conflicting structural demands imparted on this region by flanking upstream and downstream domains. This conformational strain in the IVD mediates allosteric communication between N- and C-terminal regions of NEMO. Overall, these results support a model in which the IVD of NEMO participates in signal-induced activation of the IKK/NF-{kappa}B pathway by acting as a mediator of conformational changes in NEMO.

molecular biology↗

bioRxiv2022

Starvation Decreases Immunity and Immune Regulatory Factor NF-κB in the Starlet Sea Anemone Nematostella vectensis

Lack of proper nutrition (malnutrition) or the complete absence of all food (starvation) have important consequences on the physiology of all organisms. In many cases, nutritional status affects immunity, but, for the most part, the relationship between nutrition and immunity has been limited to studies in vertebrates and terrestrial invertebrates. Herein, we describe a positive correlation between nutrition and immunity in the sea anemone Nematostella vectensis. Gene expression profiling of adult fed and starved anemones showed downregulation of many genes involved in nutrient metabolism and cellular respiration, as well as immune-related genes, in starved animals. Starved adult anemones also had reduced protein levels and DNA-binding activity of immunity-related transcription factor NF-{kappa}B. Starved juvenile anemones had increased sensitivity to bacterial infection and also had lower NF-{kappa}B protein levels, as compared to fed controls. Weighted Gene Correlation Network Analysis (WGCNA) revealed significantly correlated gene networks that were inversely associated with starvation. Based on the WGCNA and a reporter gene assay, we identified TRAF3 as a likely NF-{kappa}B target gene in N. vectensis. Overall, these experiments demonstrate a correlation between nutrition and immunity in a basal marine metazoan, and the results have implications for the survival of marine organisms as they encounter changing environments. Significance StatementAdequate nutrition is required to sustain proper biological function. One factor threatening many marine organisms, as a result of modern day anthropogenic environmental changes, is nutrient availability. Here, we characterize transcriptional changes following food deprivation in the cnidarian model sea anemone Nematostella vectensis. We show that starvation is correlated with decreased expression of genes associated with nutrient metabolism and immunity, among others. Moreover, starvation reduces the level of expression and activity of immune regulatory transcription factor NF-{kappa}B and causes anemones to have increased susceptibility to bacterial infection. These results demonstrate that this basal organism responds at the transcriptional level to the absence of food, and that, in addition to changes in metabolic factors, starvation leads to a reduction in immunity.

immunology↗

bioRxiv2021

Diversification of Transcription Factor NF-κB in Protists

In this report, we investigate the evolution of transcription factor NF-{kappa}B by examining its structure, activity, and regulation in two protists using phylogenetic, cellular, and biochemical techniques. In Capsaspora owczarzaki (Co), we find that full-length NF-{kappa}B has an N-terminal DNA-binding domain and a C-terminal Ankyrin (ANK) repeat inhibitory domain, and its DNA-binding activity is more similar to metazoan NF-{kappa}B rather than Rel proteins. As with mammalian NF-{kappa}B proteins, removal of the ANK repeats is required for Co-NF-{kappa}B to enter the nucleus, bind DNA, and activate transcription. However, C-terminal processing of Co-NF-{kappa}B is not induced by co-expression of IKK in human cells. Exogenously expressed Co-NF-{kappa}B localizes to the nucleus in Co cells. NF-{kappa}B mRNA and DNA-binding levels differ across three life stages of Capsaspora, suggesting distinct roles for NF-{kappa}B in these life stages. RNA-seq and GO analyses identify possible gene targets and biological functions of Co-NF-{kappa}B. We also show that three NF-{kappa}B-like proteins from the choanoflagellate Acanthoeca spectabilis (As) all consist of primarily the N-terminal conserved Rel Homology domain sequences of NF-{kappa}B, and lack C-terminal ANK repeats. All three As-NF-{kappa}B proteins constitutively enter the nucleus of human and Co cells, but differ in their DNA-binding and transcriptional activation activities. Furthermore, all three As-NF-{kappa}B proteins can form heterodimers, indicating that NF-{kappa}B diversified into multi-subunit families at least two times during evolution. Overall, these results present the first functional characterization of NF-{kappa}B in a taxonomic kingdom other than Animalia and provide information about the evolution and diversification of this biologically important transcription factor. SignificanceThese results represent the first functional characterization of the biologically important transcription factor NF-{kappa}B in a taxonomic kingdom other than Animalia. As such, they provide information on the evolutionary origins and basal diversification of NF-{kappa}B outside of metazoans. These results suggest that NF-{kappa}B plays life stage-specific roles in Capsaspora, the closest unicellular ancestor to all metazoans. Finally, the analysis of three NF-{kappa}B proteins in a single choanoflagellate indicates that choanoflagellates have subclasses of NF-{kappa}Bs, which can form heterodimers, suggesting that NF-{kappa}B subunit expansion and diversification has occurred at least twice in evolution.

cell biology↗

bioRxiv2019

Transcription factor NF-κB in a basal metazoan, the sponge, has conserved and unique sequences, activities, and regulation

Biological and biochemical functions of immunity transcription factor NF-{kappa}B in basal metazoans are largely unknown. Herein, we characterize transcription factor NF-{kappa}B from the demosponge Amphimedon queenslandica (Aq), in the phylum Porifera. Structurally and phylogenetically, the Aq-NF-{kappa}B protein is most similar to NF-{kappa}B p100 and p105 among vertebrate proteins, with an N-terminal DNA-binding/dimerization domain, a C-terminal Ankyrin (ANK) repeat domain, and a DNA binding-site profile more similar to human NF-{kappa}B proteins than Rel proteins. Aq-NF-{kappa}B also resembles the mammalian NF-{kappa}B protein p100 in that C-terminal truncation results in translocation of Aq-NF-{kappa}B to the nucleus and increases its transcriptional activation activity. Overexpression of a human or sea anemone I{kappa}B kinase (IKK) can induce C-terminal processing of Aq-NF-{kappa}B in vivo, and this processing requires C-terminal serine residues in Aq-NF-{kappa}B. Unlike human NF-{kappa}B p100, however, the C-terminal sequences of Aq-NF-{kappa}B do not effectively inhibit its DNA-binding activity when expressed in human cells. Tissue of another demosponge, a black encrusting sponge, contains NF-{kappa}B site DNA-binding activity and an NF-{kappa}B protein that appears mostly processed and in the nucleus of cells. NF-{kappa}B DNA-binding activity and processing is increased by treatment of sponge tissue with LPS. By transcriptomic analysis of A. queenslandica we identified likely homologs to many upstream NF-{kappa}B pathway components. These results present a functional characterization of the most ancient metazoan NF-{kappa}B protein to date, and show that many characteristics of mammalian NF-{kappa}B are conserved in sponge NF-{kappa}B, but the mechanism by which NF-{kappa}B functions and is regulated in the sponge may be somewhat different.

cell biology↗

bioRxiv2019

Varied effects of algal symbionts on transcription factor NF-κB in a sea anemone and a coral: possible roles in symbiosis and thermotolerance

Many cnidarians, including the reef-building corals, undergo symbiotic mutualisms with photosynthetic dinoflagellate algae of the family Symbiodiniaceae. These partnerships are sensitive to temperature extremes, which cause symbiont loss and increased coral mortality. Previous studies have implicated host immunity and specifically immunity transcription factor NF-{kappa}B as having a role in the maintenance of the cnidarian-algal symbiosis. Here we have further investigated a possible role for NF-{kappa}B in establishment and loss of symbiosis in various strains of the anemone Exaiptasia (Aiptasia) and in the coral Pocillopora damicornis. Our results show that NF-{kappa}B expression is reduced in Aiptasia larvae and adults that host certain algae strains. Treatment of Aiptasia larvae with a known symbiosis-promoting cytokine, transforming growth factor {beta}, also led to decreased NF-{kappa}B expression. We also show that aposymbiotic Aiptasia (with high NF-{kappa}B expression) have increased survival following infection with the pathogenic bacterium Serratia marcescens as compared to symbiotic Aiptasia (low NF-{kappa}B expression). Furthermore, a P. damicornis coral colony hosting Durusdinium spp. (formerly clade D) symbionts had higher basal NF-{kappa}B expression and decreased heat-induced bleaching as compared to two individuals hosting Cladocopium spp. (formerly clade C) symbionts. Lastly, genome-wide gene expression profiling and genomic promoter analysis identified putative NF-{kappa}B target genes that may be involved in thermal bleaching, symbiont maintenance, and/or immune protection in P. damicornis. Our results provide further support for the hypothesis that modulation of NF-{kappa}B and immunity plays a role in some, but perhaps not all, cnidarian-Symbiodiniaceae partnerships as well as in resistance to pathogens and bleaching.

molecular biology↗

bioRxiv2018

CRISPR/Cas9-based Editing of a Sensitive Transcriptional Regulatory Element to Achieve Cell Type-Specific Knockdown of the NEMO Scaffold Protein

The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-{kappa}B signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter A) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support canonical NF-{kappa}B signaling in response to treatment with tumor necrosis factor (TNF). Nevertheless, non-canonical NF-{kappa}B signaling is intact in these NEMO-deficient cells. Expression of ectopic NEMO in the edited cells restores downstream NF-{kappa}B signaling in response to TNF. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter A) in the human SNU-423 liver cancer cell line. We have also used homology directed repair (HDR) to fix the promoter B element in a 293T cell clone. Overall, we have created a strategy for selectively eliminating cell type-specific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-{kappa}B signaling are discussed.

molecular biology↗