Biology subjects

Cook, S. J.

Publications and source records attributed to Cook, S. J..

At least 19 recordsLinked to original sources

bioRxiv2026

IKKα and IKKβ serve as kinase-independent scaffolds that prevent proteasomal degradation of NEMO

NF-{kappa}B essential modulator (NEMO) is the critical scaffold protein of the canonical I kappa B kinase (IKK) complex. Whilst studying the contribution of IKK and IKK{beta} to NF-{kappa}B signalling we noted that NEMO protein levels were reduced in IKK knockout (KO) HCT116 cells. Loss of NEMO was more pronounced in IKK KO cells than IKK{beta} KO, whilst IKK+IKK{beta} double knockout (DKO) cells exhibited a 90% reduction in NEMO abundance. In contrast, the abundance of IKKs was unaffected by NEMO KO, although this abolished canonical NF-{kappa}B activation. Loss of IKKs had no effect on NEMO mRNA levels but IKK DKO cells exhibited accelerated NEMO protein turnover that was reversed by the proteasome inhibitor, MG132, but not the NEDDylation inhibitor, MLN4924. TAK-243, a selective inhibitor of UBA1, the E1 ubiquitin-activating enzyme, increased basal NEMO levels in DKO cells but did not prevent NEMO turnover suggesting both ubiquitylation-dependent and independent pathways for degradation. Bioinformatic analysis identified substantial regions of intrinsic disorder within the N- and C-termini of NEMO. NEMO was rapidly degraded by isolated 20S proteasomes in vitro and this was inhibited by interaction with IKK or IKK{beta}. Finally, re-expression of IKK increased NEMO expression in IKK DKO cells and this required the C-terminal NEMO binding domain (NBD) but not a functional kinase domain. Thus the IKKs act as critical kinase-independent scaffolds that stabilise NEMO; loss of these interactions results in NEMO degradation via the proteasome. These results may be relevant to strategies for inhibiting NF-{kappa}B signalling by disrupting NEMO:IKK interactions.

biochemistry↗

bioRxiv2026

Terminal selector and subtype selector function across 200 million years of nematode evolution

The evolution of brains is subject to investigation in many different animal groups, each offering unique advantages to advance our understanding of the cellular, molecular and regulatory substrates of evolutionary change. Here, we use two nematode species, C. elegans and P. pacificus, separated by more than 200 million years of evolution to explore how neuronal cell types and the regulatory programs instructing the identity of these cell types have evolved over time. Using gene expression pattern analysis, we compare the differentiation programs of over half of all nematode neuron classes. To explore how the gene regulatory architecture of neuronal differentiation programs evolves, we apply our deep understanding of neuronal differentiation programs, controlled by terminal selectors and subtype selectors in C. elegans. Through mutant analysis of orthologous P. pacificus regulatory factors, we elucidate patterns of conservation and novelties over such substantial evolutionary distance. We discovered striking similarities in terminal selector expression and activities throughout the nervous system but also observed that terminal selectors can acquire novel sites of expression and distinct regulatory capabilities, manifested by changes in effector gene expression and, hence, neuronal phenotypes. Our mutant analysis argues for a buffering of terminal selector function and for an evolutionary lability of differences in closely related neuronal subtypes. Taken together, our analysis reveals molecular substrates of evolutionary change in nervous systems.

neuroscience↗

bioRxiv2026

T cell receptor-ligand affinity quantitatively tunes transcriptome remodelling in vivo, inversely regulating cell division and interferon response

The strength of T cell receptor (TCR) engagement by antigenic ligands governs naive T cell activation, expansion and differentiation. However, the molecular changes underpinning this process in vivo remain incompletely understood. To address this, we coupled an influenza infection model of varied TCR-ligand affinity with high-dimensional protein and RNA measurements. As previously reported, high affinity stimulation drove greater expansion, with disproportionately abundant effector subsets. Experiments using a KLRG1-fate reporter showed that differentiation state biases affected cells from both direct and indirect memory differentiation trajectories. Early post-infection, these biases manifested in differential metabolic and proliferative activities. Examination of T cells showing signs of initial priming in vivo revealed that TCR-ligand affinity primarily changed the magnitude of TCR-induced transcriptome remodelling, not the genes involved. This quantitative tuning drove affinity-dependent amplification of ribosome biogenesis and suppression of interferon response genes before mitogenic diversion, and was associated with elevated TCR-induced transcription factor activity. Together, these data demonstrate the in vivo underpinnings of affinity-dependent responses and reveal how accumulation of TCR-induced signalling outputs translates binding properties into appropriate differentiation outcomes.

immunology↗

bioRxiv2026

Phosphoproteomics identifies the DYRK1B protein kinase as a regulator of processing bodies

Dual-specificity tyrosine-phosphorylation-regulated kinase 1B (DYRK1B) modulates the cell cycle, cell fate during development, and is deregulated in cancer and metabolic syndrome. However, only a few DYRK1B substrates have been defined, so we undertook a phosphoproteomics screen in cells that exhibit inducible DYRK1B expression. Motif analysis revealed enrichment for proline-directed serine or threonine phosphorylation sites (pSer/pThr-Pro), consistent with the consensus motif of class I DYRKs. Gene ontology analysis revealed enrichment of proteins involved in mRNA binding, mRNA processing and ribonucleoprotein complexes. Several processing body (PB) components, including DCP1A, PATL1(PAT1B), EDC3 and 4E-T, were identified as DYRK1B-inducible phosphoproteins. DYRK1B also co-immunoprecipitated with DCP1A, PAT1B, EDC3, EDC4, DDX6 and XRN1. Super-resolution microscopy demonstrated that DYRK1B co-localised with DCP1A, DCP1B and DDX6 in PBs. Activation of DYRK1B increased PB abundance, whereas inhibition, depletion or knockout of DYRK1B reduced phosphorylation of DCP1A and 4E-T and decreased PB number. Re-expression of wild type, but not kinase-dead, DYRK1B restored PB numbers in knockout cells. These findings reveal novel DYRK1B targets and establish DYRK1B as a regulator of processing body abundance. HighlightsO_LIDYRK1B induces phosphorylation of a cluster of RNA binding and processing body associated proteins. C_LIO_LIDYRK1B localises to PBs and associates with multiple PB components. C_LIO_LIDYRK1B controls P-body abundance in a kinase-dependent manner. C_LI

cell biology↗

bioRxiv2026

Heterogeneity of white matter structure in the human brain

White-matter, which contains the long-range axons connecting different brain regions, makes up nearly half the human brain, yet the three-dimensional organization of individual axons has not been characterized. While advances in diffusion MRI have enabled macroscopic mapping of major WM pathways1, these methods are unable to resolve individual axons: their trajectories, density, and relative orientations2. Here, we present a histological and imaging pipeline optimized for post-mortem human white-matter that shows the 3D trajectories of densely stained large (diameter greater than ~1 m) projection axons. Applying our approach to multiple centimeter-scale WM regions in an adult human brain, we observed striking regional diversity in axonal organization. Specifically, we identified distinct architectural motifs ranging from loosely packed, multi-orientation meshworks (in most superficial white matter), to laminar lattice-like structures (near the basal ganglia), to tightly packed bundles of fibers (e.g. in the corpus callosum). We speculate that these patterns reflect local adaptations to spatial constraints, axonal density, and the diversity of axonal sources and targets, offering region-specific solutions to anatomical optimization problems. These findings offer new insights into the principles shaping brain connectivity and underscore the need for regionally detailed atlases of human white matter..

neuroscience↗

bioRxiv2025

Identity and functions of monoaminergic neurons in the predatory nematode Pristionchus pacificus reveal nervous system conservation and divergence

Changes in neurotransmitter usage in homologous neurons may drive evolutionary adaptations in neural circuits across animal phylogeny. The predatory nematode Pristionchus pacificus can be used as a model system to examine nervous system evolution by comparing neurotransmitter expression with that of C. elegans and other nematodes. Here we characterize P. pacificus neurotransmitter expression and function in specific neurons, focusing on its complete set of monoaminergic neurons. We discover patterns of conservation as well as novelties. We examine the roles of monoamines in specific behaviors using neurotransmitter synthesis and vesicular transporter mutants, finding possible differences in the control of host-finding and dispersal behavior.

neuroscience↗

bioRxiv2025

Whole-brain chemosensory responses of both C. elegans sexes

Sexually-dimorphic neural circuits play a critical role in shaping sex-specific animal behaviors. Maps of the structural dimorphisms in these circuits have been explored by analyzing "synaptic connectomes", electron micrograph reconstructions of synaptic connectivity. Nevertheless, recent studies in the model organism C. elegans have shown little to no correlation between the synaptic connectome and dynamic neural activity. Therefore, the extent of sexual dimorphism in functional neural activity remains unknown. To determine the extent of functional sexual-dimorphisms in C. elegans we compared activity, neuron-by-neuron, across all neurons in the heads of both sexes. To sample a broad view of responses to different sensory modalities, we tested a diverse panel of ethologically-relevant olfactory, gustatory, and chemical stimuli, representing both attractive and aversive cues. We found that nearly every sensory neuron responded dimorphically to at least one cue and monomorphically to other cues, indicating that sexually-dimorphic circuits are pervasive and stimulus dependent. This dimorphic and monomorphic activity was present to a lesser extent in downstream interneurons and even less so in motoneurons, implicating sensory neurons as the primary source and location of sexually-dimorphic activity. Comparing the functional activity we measured to the published synaptic connectomes of both sexes revealed that sexual dimorphism in functional connectivity was distinct from and complementary to sexual dimorphism in synaptic connectivity. Our results provide a first-of-its-kind comparison of whole-brain dynamics between sexes at the level of single neurons, serving as an extensive resource for further investigations of functional sex differences.

neuroscience↗

bioRxiv2024

KRAS G12 mutant alleles differentially control glutamine metabolism via FOXO1

Mutations in KRAS, particularly at codon 12, are frequent in adenocarcinomas of the colon, lungs and pancreas, driving carcinogenesis by altering cell signalling and reprogramming metabolism. However, the specific mechanisms by which different KRAS G12 alleles initiate distinctive patterns of metabolic reprogramming is unclear. Using isogenic panels of colorectal cell lines harbouring the G12A, G12C, G12D and G12V heterozygous mutations and employing transcriptomics, metabolomics, and extensive biochemical validation, we demonstrate distinctive features of each allele. We also demonstrate that cells harbouring the common G12D and G12V oncogenic mutations significantly alter glutamine metabolism and nitrogen recycling through FOXO1-mediated regulation compared to parental lines. Moreover, with a combination of small molecule inhibitors targeting glutamine and glutamate metabolism, we also identify a common vulnerability that eliminates mutant cells selectively. These results highlight a previously unreported mutant-specific effect of KRAS alleles on metabolism and signalling that could be potentially harnessed for cancer therapy. In briefBer et al. reveal common and distinct features of oncogenic KRAS mutant isogenic cell lines. Mutant lines upregulate different nitrogen-recycling metabolic pathways and show sensitivity to combinatorial drugging of glutamine metabolism. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/618135v1_ufig1.gif" ALT="Figure 1"> View larger version (101K): org.highwire.dtl.DTLVardef@1459558org.highwire.dtl.DTLVardef@14fcd43org.highwire.dtl.DTLVardef@2d22ccorg.highwire.dtl.DTLVardef@147b068_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDifferent KRAS mutations trigger distinct metabolic phenotypes in colorectal cell lines C_LIO_LIGlutamine metabolism and nitrogen recycling are upregulated in KRAS mutant cells C_LIO_LIFOXO1 is a key regulator of KRAS-induced rewiring of glutamine and nitrogen metabolism C_LIO_LIInhibition of glutamine synthetase and glutaminase selectively kills mutant KRAS lines C_LI

cancer biology↗

bioRxiv2024

Evolution of lateralized gustation in nematodes

Animals with small nervous systems have a limited number of sensory neurons that must encode information from a changing environment. This problem is particularly exacerbated in nematodes that populate a wide variety of distinct ecological niches but only have a few sensory neurons available to encode multiple modalities. How does sensory diversity prevail within this constraint in neuron number? To identify the genetic basis for patterning different nervous systems, we demonstrate that sensory neurons in Pristionchus pacificus respond to various salt sensory cues in a manner that is partially distinct from that of the distantly related nematode Caenorhabditis elegans. By visualizing neuronal activity patterns, we show that contrary to previous expectations based on its genome sequence, the salt responses of P. pacificus are encoded in a left/right asymmetric manner in the bilateral ASE neuron pair. Our study illustrates patterns of evolutionary stability and change in the gustatory system of nematodes. Animals with small nervous systems have evolved left and right asymmetry in their neurons to process various salts at different concentrations.

neuroscience↗

bioRxiv2024

RAF inhibitors activate the integrated stress response by direct activation of GCN2

Paradoxical RAF activation by chemical RAF inhibitors (RAFi) is a well-understood on-target biological and clinical response. In this study, we show that a range of RAFi drive ERK1/2-independent activation of the Unfolded Protein Response (UPR), including expression of ATF4 and CHOP, that required the translation initiation factor eIF2. RAFi-induced ATF4 and CHOP expression was not reversed by inhibition of PERK, a known upstream activator of the eIF2-dependent Integrated Stress Response (ISR). Rather, we found that RAFi exposure activated GCN2, an alternate eIF2 kinase, leading to eIF2-dependent (and ERK1/2-independent) ATF4 and CHOP expression. The GCN2 kinase inhibitor A-92, GCN2 RNAi, GCN2 knock-out or ISRIB (an eIF2 antagonist) all reversed RAFi-induced expression of ATF4 and CHOP indicating that RAFi require GCN2 to activate the ISR. RAFi also activated full-length recombinant GCN2 in vitro and in cells, generating a characteristic bell-shaped concentration-response curve, reminiscent of RAFi-driven paradoxical activation of WT RAF dimers. Activation of the ISR by RAFi was abolished by GCN2 kinase dead mutations and M802A or M802G gatekeeper mutations, suggesting that RAFi bind directly to the GCN2 kinase domain; this was supported by mechanistic structural models of RAFi interaction with GCN2. Since the ISR is a critical pathway for determining cell survival or death, our observations may be relevant to the clinical use of RAFi, where paradoxical GCN2 activation may be a previously unappreciated off-target effect that may modulate tumour cell responses.

biochemistry↗

bioRxiv2024

Paradoxical Activation of GCN2 by ATP-competitive inhibitors via allosteric activation and autophosphorylation

Recently it has been found that General Control Non-derepressible 2 (GCN2) can be activated by an array of small molecule ATP-competitive inhibitors, including clinically relevant compounds such as Ponatinib, and compounds specifically designed to be GCN2 inhibitors, such as GCN2iB. Furthermore, we recently showed that GCN2 can be activated in cells by clinically approved small molecule RAF inhibitors. GCN2 is a drug target, specifically in cancers such as mesothelioma, and a better understanding of this paradoxical activation is required to develop drugs which truly inhibit the enzyme. Using biochemical assays and structural mass spectrometry, we present a model for how GCN2 is activated by these compounds by promoting an active conformation in the HisRS domain while competitively inhibiting the kinase domain. This conformation promotes activating phosphorylation of GCN2, potentially through phosphorylation of other activated GCN2 molecules which are not bound to compound. Together this model suggests that efforts to inhibit GCN2 would benefit from exploring allosteric routes rather than targeting the ATP-binding pocket of the kinase domain.

biochemistry↗

bioRxiv2024

Comparative connectomics of two distantly related nematode species reveals patterns of nervous system evolution

Understanding the evolution of the bilaterian brain requires a detailed exploration of the precise nature of cellular and subcellular differences between related brains. To define the anatomical substrates of evolutionary change in the nervous system, we undertook an electron micrographic reconstruction of the brain of the predatory nematode Pristionchus pacificus. A comparison with the brain of Caenorhabditis elegans, which diverged at least 100 million years ago, reveals a conserved nematode core connectome and a wide range of specific substrates of evolutionary change. These changes include differences in neuronal cell death, neuronal cell position, axo-dendritic projection patterns and many changes in synaptic connectivity of homologous neurons that display no obvious changes in overall neurite morphology and projection patterns. Arguing against specific hot spots of evolutionary change, connectivity differences are distributed throughout the nervous system and extend to glia as well. We observed examples of apparent circuit drift, where changes in morphology and connectivity of a neuron do not appear to alter its behavioral output. In conclusion, our comprehensive comparison of distantly related nematode species provides novel vistas on patterns of conservation as well as the substrates of evolutionary change in the brain that span multiple organizational levels.

neuroscience↗

bioRxiv2023

Restructuring of an asymmetric neural circuit during associative learning

Asymmetric brain function is common across the animal kingdom and involved in language processing, and likely in learning and memory. What regulates asymmetric brain function remains elusive. Here, we show that the nematode Caenorhabditis elegans restructures an asymmetric salt sensing neural circuit during associative learning. Worms memorize and prefer the salt concentration at which they were raised in the presence of food through a left-biased network architecture. When conditioned at elevated salt concentrations, animals change the left-biased to a right-biased network, which explains the changed salt-seeking behavior. The changes in circuit architecture require new synapse formation induced through asymmetric, paracrine insulin-signaling. Therefore, experience-dependent changes in asymmetric network architecture rely on paracrine insulin signaling and are fundamental to learning and behavior.

neuroscience↗

bioRxiv2022

Neuronal contact predicts connectivity in the C. elegans brain

Axons must project to particular brain regions, contact adjacent neurons, and choose appropriate synaptic targets to form a nervous system. Multiple mechanisms have been proposed to explain synaptic partnership choice. In a lock-and-key mechanism, first proposed by Sperrys chemoaffinity model1, a neuron selectively chooses a synaptic partner among several different, adjacent target cells, based on a specific molecular recognition code2. Alternatively, Peters rule posits that neurons indiscriminately form connections with other neuron types in their proximity; hence, neighborhood choice, dictated by initial neuronal process outgrowth and position, is the sole predictor of connectivity3,4. However, whether Peters rule plays an important role in synaptic wiring remains unresolved5. To assess the nanoscale relationship between neuronal adjacency and connectivity, we evaluate the expansive set of C. elegans connectomes. We find that synaptic connectivity can be accurately modeled as a path-length-dependent process of neuronal adjacency and brain strata, offering strong support for Peters rule as an organizational principle of C. elegans brain wiring.

neuroscience↗

bioRxiv2021

DNA replication during acute MEK inhibition drives acquisition of resistance through amplification of the BRAF oncogene

Mutations and gene amplifications that confer drug resistance emerge frequently during chemotherapy, but their mechanism and timing is poorly understood. Here, we investigate BRAFV600E amplification events that underlie resistance to the MEK inhibitor selumetinib (AZD6244/ARRY-142886) in COLO205 cells, a well-characterised model for reproducible emergence of drug resistance, and show that de novo amplification of BRAF is the primary path to resistance irrespective of pre-existing amplifications. Selumetinib causes long-term G1 arrest accompanied by reduced expression of DNA replication and repair genes, but cells stochastically re-enter the cell cycle during treatment despite continued repression of pERK1/2. Most DNA replication and repair genes are re-expressed as cells enter S and G2, however, mRNAs encoding a subset of factors important for error-free replication and chromosome segregation including TIPIN, PLK2 and PLK3 remain at low abundance. This suggests that DNA replication in drug is more error prone and provides an explanation for the DNA damage observed under long-term RAF-MEK-ERK1/2 pathway inhibition. To test the hypothesis that DNA replication in drug promotes de novo BRAF amplification, we exploited the combination of palbociclib and selumetinib. Combined treatment with selumetinib and a dose of palbociclib sufficient to reinforce G1 arrest in selumetinib-sensitive cells, but not to impair proliferation of resistant cells, delays the emergence of resistant colonies, meaning that escape from G1 arrest is critical in the formation of resistant clones. Our findings demonstrate that acquisition of MEK inhibitor resistance often occurs through de novo gene amplification and can be suppressed by impeding cell cycle entry in drug.

molecular biology↗

bioRxiv2020

Molecular topography of an entire nervous system

Nervous systems are constructed from a deep repertoire of neuron types but the underlying gene expression programs that specify individual neuron identities are poorly understood. To address this deficit, we have produced an expression profile of all 302 neurons of the C. elegans nervous system that matches the single cell resolution of its anatomy and wiring diagram. Our results suggest that individual neuron classes can be solely identified by combinatorial expression of specific gene families. For example, each neuron class expresses unique codes of [~]23 neuropeptide-encoding genes and [~]36 neuropeptide receptors thus pointing to an expansive "wireless" signaling network. To demonstrate the utility of this uniquely comprehensive gene expression catalog, we used computational approaches to (1) identify cis-regulatory elements for neuron-specific gene expression across the nervous system and (2) reveal adhesion proteins with potential roles in synaptic specificity and process placement. These data are available at cengen.org and can be interrogated at the web application CengenApp. We expect that this neuron-specific directory of gene expression will spur investigations of underlying mechanisms that define anatomy, connectivity and function throughout the C. elegans nervous system.

neuroscience↗

bioRxiv2020

TCR affinity controls the dynamics but not the functional specification of the Th1 response to mycobacteria

The quality of T cell responses depends on the lymphocytes ability to undergo clonal expansion, acquire effector functions and traffic to the site of infection. Although TCR signal strength is thought to dominantly shape the T cell response, by using TCR transgenic CD4+ T cells with different pMHC binding affinity, we reveal that TCR affinity does not control Th1 effector function acquisition nor the functional output of individual effectors following mycobacterial infection. Rather, TCR affinity calibrates the rate of cell division to synchronize the distinct processes of T cell proliferation, differentiation and trafficking. By timing cell division-dependent IL-12R expression, TCR affinity controls when T cells become receptive to Th1-imprinting IL-12 signals, determining the emergence and magnitude of the Th1 effector pool. These findings reveal a distinct yet cooperative role for IL-12 and TCR signalling in Th1 differentiation and suggests that the temporal activation of clones with different TCR affinity is a major strategy to coordinate immune surveillance against persistent pathogens.

immunology↗

bioRxiv2020

Beyond the connectome: A map of a brain architecture derived from whole-brain volumetric reconstructions

Animal nervous system organization is crucial for all body functions and its disruption can manifest in severe cognitive and behavioral impairment. This organization relies on features across scales, from nano-level localization of synapses, through multiplicities of neuronal morphologies and their contribution to circuit organization, to the high level stereotyped connections between different regions of the brain. The sheer complexity of this organ means that to date, we have yet to reconstruct and model the structure of a complete nervous system that is integrated across all these scales. Here, we present a complete structure-function model of the nematode C. elegans main neuropil, the nerve ring, which we derive by integrating the volumetric reconstruction from two animals with corresponding synaptic and gap junctional connectomes. Whereas previously the nerve ring was considered a densely packed tract of axons, we uncover internal organization into 5 functional bundles and show how they spatially constrain and support the synaptic connectome. We find that the C. elegans connectome is not invariant, but that a precisely wired core circuit is embedded in a background of variable connectivity, and propose a corresponding reference connectome for the core circuit. Using this reference, we show that the architecture of the C. elegans brain can be viewed as a modular Residual Network that supports sensory computation and integration, sensory-motor convergence, and brain-wide coordination. These findings point to scalable and robust features of brain organization that are likely universal across phyla.

neuroscience↗