Biology subjects

Oliver, D.

Publications and source records attributed to Oliver, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Lipid versatility of the C2C domain of Extended Synaptotagmin 3 safeguards plasma membrane binding

Membrane contact sites between the endoplasmic reticulum (ER) and the plasma membrane (PM) are critical hubs for lipid homeostasis and signal transduction. Extended Synaptotagmins (E-Syts) tether the ER to the PM and mediate lipid transport from the PM to the ER following Ca2+ signaling. Although E-Syts are essential for maintaining ER-PM contact sites during PI(4,5)P2 resynthesis, the molecular mechanisms governing their membrane association remain poorly understood. In particular, it is unclear how these contact sites are maintained following PI(4,5)P2 hydrolysis, when their principal membrane-binding ligand is transiently depleted. Here, we characterize the membrane-binding mechanism of the C-terminal C2 domain of extended synaptotagmin 3 (E-Syt3) by combining coarse-grained molecular dynamics simulations with live-cell experiments. We identify distinct lipid binding sites with specific affinities for phosphoinositides as well as for phosphatidylinositol (PI) and phosphatidylserine (PS). While phosphoinositide interactions dominate membrane binding under resting conditions, PI and PS binding may provide an alternative membrane-binding mechanism that helps preserve ER-PM contacts during PI(4,5)P2 depletion. Our simulations further reveal that phosphoinositide binding is reinforced by the insertion of a tryptophan residue into the membrane hydrophobic core, providing a mechanistic basis for the high membrane affinity of the C2C domain. Together, our findings uncover the molecular determinants of E-Syt3 C2C-mediated membrane binding and provide mechanistic insight into the stability of E-Syt-mediated ER-PM contact sites during dynamic changes in membrane phosphoinositide composition.

molecular biology↗

bioRxiv2026

Longitudinal White Matter Microstructure and Transition to Psychosis in Individuals at Clinical High Risk

Altered white matter microstructure, commonly indexed by fractional anisotropy (FA), has been reported in individuals at clinical high-risk of psychosis (CHR-P) across multiple white matter tracts. However, the relationship between these alterations and later transition to psychosis remains unclear. To address this gap, we investigated whether baseline and longitudinally updated FA measurements were associated with risk of transition to psychosis in a large cohort of individuals at CHR-P. We analysed diffusion tensor imaging data from N = 205 participants at CHR-P, 23 of whom developed psychosis within 3 years of follow-up. Data were collected at baseline, 6 months, and 12 months. Associations between global and tract-specific FA and transition risk were examined using Cox proportional hazards models of baseline data and longitudinally updated data. Point estimates were directionally consistent, with hazard ratios <1 for FA across all white matter tracts in both baseline and longitudinal models. Associations reached nominal significance in the hippocampal cingulum, anterior thalamic radiation, and forceps minor, but none of these remained significant after correcting for multiple comparisons (pcorr > .05). The directionally consistent estimates are compatible with small, distributed effects across multiple white matter pathways being associated with transition to psychosis, although this requires replication. Longitudinally updated FA measurements did not yield stronger associations than were available from baseline imaging data alone. Studies at an even larger scale are needed to establish whether these associations are robust and clinically meaningful.

neuroscience↗

bioRxiv2026

Longitudinal White Matter Trajectories in Clinical High Risk and First-Episode Psychosis: Findings from the Multi-Centre PSYSCAN Study

Psychosis has been linked to changes in diffusion-derived fractional anisotropy (FA) across multiple white matter tracts, yet most evidence is cross-sectional. Longitudinal findings are inconsistent, and the clinical relevance of white matter changes over time remains unclear. To address this gap, we investigated longitudinal white matter changes and their clinical correlates in early psychosis in a large, multi-centre diffusion-tensor imaging study. Across 18 sites, 407 participants (healthy controls: n = 97, 59.8% men, mean age 23.9{+/-}4.6 years; clinical high risk of psychosis: n = 158, 53.5% men, mean age 23.0{+/-}4.9 years; first-episode psychosis: n = 152, 71.1% men, mean age 25.1{+/-}5.4 years) were scanned at up to three time points over 12 months. FA was assessed in the cingulum bundle, the superior longitudinal fasciculus, the inferior fronto-occipital fasciculus, and at the whole brain level. FA trajectories were analysed using linear mixed-effects models to test effects of group, social and occupational functioning, and (attenuated) psychotic symptoms, while controlling for demographic and socioeconomic covariates. No significant group differences in FA were observed, either globally or within tracts (p > .05). Longitudinal FA trajectories were not associated with changes in (attenuated) psychotic symptoms or functioning (p > .05). In contrast, higher baseline antipsychotic medication dose was significantly associated with lower FA (pcorr < .05). Overall, these findings indicate that white matter microstructure is relatively stable during the clinical-high-risk and first-episode phases of psychosis, and that it is not clearly associated with variation in symptom severity or functional outcomes over time.

neuroscience↗

bioRxiv2026

Convergent Multimodal Evidence of Cortical Excitation-Inhibition Imbalance in Psychosis

Psychosis is increasingly understood as a disorder of disrupted cortical excitation-inhibition balance, yet robust non-invasive translational biomarkers remain lacking. The resting-state fMRI Hurst exponent (HE) and EEG aperiodic spectral exponent are promising complementary biomarkers, with lower values in each proposed to reflect a shift towards cortical hyperexcitability, but they have not been jointly examined in psychosis, and the spatial and molecular architecture of HE alterations remains poorly defined. We therefore tested for convergent systems-level signatures across independent cohorts and modalities, using resting-state fMRI (107 patients, 53 controls) and EEG (547 patients, 363 controls). Whole-brain and regional HE were estimated using wavelet methods, and EEG aperiodic exponents were quantified using spectral parameterisation. Compared with healthy controls, individuals with psychosis showed reduced whole-brain HE and widespread regional reductions. Regional HE case-control differences were associated with cortical gene-expression patterns, with enrichment for potassium channel and GABA receptor pathways, and correlated with noradrenergic, muscarinic, serotonergic, glutamatergic and dopaminergic receptor density maps, but not with cortical thickness or symptom or cognitive measures. In the independent EEG cohort, psychosis was similarly associated with a reduced aperiodic spectral exponent. Together, these findings provide cross-modal evidence for altered cortical resting-state dynamics in psychosis, consistent with a shift towards cortical hyperexcitability. Integration with receptor-density and transcriptomic maps implicates biologically plausible molecular pathways and supports HE and EEG aperiodic activity as scalable translational biomarkers in psychosis.

neuroscience↗

bioRxiv2026

TACE reprograms RANKL-mediated differentiation of macrophages by activating the non-canonical pathway of IRF3

Inflammation is associated with an influx of inflammatory macrophages and increased activation and differentiation of osteoclasts. Receptor activator of NF-kB ligand (RANKL) is a key driver for osteoclast differentiation. However, the pathogenic mechanisms augmenting RANKL-induced osteoclast differentiation in inflammatory conditions are not fully elucidated. Here, we show that TNF- converting enzyme (TACE) plays a critical role in pathological bone erosion and enhances osteoclast differentiation in inflammatory conditions. Myeloid cell-specific TACE deletion in a murine arthritis model attenuates joint inflammation and bone destruction. TACE deficiency suppresses distal RANKL signaling in macrophages by increasing IRF3 activation, while enhancing proximal RANKL signaling, leading to the suppression of osteoclast differentiation. Mechanistically, IRF3 activation limits macrophage reprogramming by suppressing NFATc1 and HB-EGF in response to RANKL through a non-canonical pathway. HB-EGF, a TACE substrate, activates EGFR signaling and promotes osteoclastogenesis by inhibiting IRF3 activation. TACE regulates the reciprocal inhibition of the IRF3-HBEGF axis. Our study highlights a role for TACE as a rheostat, balancing both pro- and anti-osteoclastogenic signals.

cell biology↗

bioRxiv2025

Somatic function of the Argonaute protein Aubergine is essential for neuromuscular development and function in Drosophila

BackgroundThe PIWI-interacting RNA (piRNA) pathway is the primary defense against the deleterious activity of transposable elements (TEs), a role classically assigned to the germline. We recently discovered that the retrotransposon Copia is a negative regulator of synaptogenesis at the Drosophila larval neuromuscular junction (LNMJ) [1]. Here, we investigated whether the piRNA pathway regulates Copia in this somatic context. MethodsAnalysis of existing sequencing data revealed the expression of piRNA pathway components in somatic tissues [2]. We focused on Aubergine (aub), a core PIWI-clade Argonaute. We utilized CRISPR generated aub reporter lines and confocal microscopy to confirm the enrichment of AUB at the LNMJ and next generation sequencing coupled with digital PCR to validate the upregulation of TEs in aub knockdown larvae and adult tissues. ResultsData from genetic reporters and antibody staining show that AUB is expressed and localized to the LNMJ. Tissue-specific knockdown of aub at the LNMJ resulted in increased TE expression, including Copia. In contrast to the synaptic overgrowth seen with Copia depletion [1], aub reduction caused a decrease in synapse number and impaired motor function and lifespan. These phenotypes are consistent with the upregulation of Copia, a negative regulator of synapse growth. ConclusionsOur findings demonstrate that AUB functions somatically at the LNMJ to repress TEs, thereby ensuring proper neuromuscular development and function. This work establishes a physiological role for the piRNA pathway in a somatic tissue, linking TE repression to neuromuscular development.

neuroscience↗

bioRxiv2025

Beyond Antagonism: IL-4 Exploits TNF signaling to Shape Its Gene Expression Signature in Monocytes and Macrophages.

Investigation of crosstalk between antagonistic pro- and anti-inflammatory cytokines has focused on mechanisms and functional consequences of cross-inhibition. We investigated cross-regulation between proinflammatory TNF and anti-inflammatory IL-4 in primary human monocytes and in a skin wound-healing model. Surprisingly, TNF functioned mainly as a costimulator of IL-4-induced gene expression, whereas IL-4 selectively inhibited the TNF-induced IFN response, leaving inflammatory gene expression mostly intact. TNF and IL-4 synergistically induced gene sets important for regulating inflammation and tissue repair, which were highly induced during the phase of wound healing when these cytokines are co-expressed. Crosstalk between TNF and IL-4 was mediated by epigenetic chromatin-mediated mechanisms associated with cooperation between NF-{kappa}B and STAT6 transcription factors, erasure of negative histone mark H3K27me3, and selective inhibition of IRF1. These results identify a long-sought mechanism for expansion of the IL-4 response, and highlight the complexity of crosstalk between antagonistic cytokines that includes cooperation for select gene responses important in immune response and tissue repair.

immunology↗

bioRxiv2025

Paralemmin-3 sustains the integrity of the lateral plasma membrane and subsurface cisternae of auditory hair cells

In the mammalian inner ear, cochlear inner hair cells (IHCs) enable accurate and faithful synaptic sound encoding, while outer hair cells (OHCs) perform frequency-specific sound amplification and fine-tuning through their intrinsic voltage-dependent somatic electromotility. This latter process is facilitated by the unique trilaminate structure of the OHC lateral wall, which consists of the plasma membrane that is densely occupied by the transmembrane motor protein Prestin, the submembrane actin- and spectrin-based cytoskeleton, and the endomembranous subsurface cisternae. This complex system provides mechanical resilience while allowing for cell expansion and contraction during electromotility. Whereas the ultrastructure of the lateral wall is well described, its molecular architecture remains largely elusive. Here, we identified Paralemmin-3 (Palm3) as a novel protein specifically localized to the lateral walls of auditory HCs to play a crucial role in connecting the plasma membrane to the underlying cytoskeleton and subsurface cisternae. Palm3-KO mice display early-onset and progressive hearing impairment that results from diminished cochlear amplification. Subsequent multiscale morphological analyses revealed structural collapse of OHCs that led to progressive and extensive OHC loss along the tonotopic axis. Palm3-KO OHCs exhibited disrupted expression and distribution of several membrane-associated proteins - including spectrin isoforms and Prestin - suggesting an essential role of Palm3 in plasma membrane scaffolding. Electron tomography of OHC lateral walls revealed significantly fewer and structurally perturbed subsurface cisternae. Finally, adeno-associated virus (AAV)-mediated rescue of Palm3 during early postnatal development partly restored hearing function, enhanced OHC survival, and restored OHC cell shape as well as membrane protein expression levels. In summary, Palm3 is a key component of the submembrane cytoskeleton in cochlear hair cells, playing a fundamental role in hair cell biology and hearing, and emerges as an attractive candidate for the long-elusive "pillar" component of the hair cell lateral wall ultrastructure.

cell biology↗

bioRxiv2024

Versatile Functional Interaction between Electrically Silent KV Subunits and KV7 Potassium Channels

Voltage-gated K+ (KV) channels govern K+-ion flux across cell membranes in response to changes in membrane potential. They are formed by the assembly of four subunits, typically from the same family. Electrically silent KV channels (KVS), however, are unable to conduct currents on their own. It has been assumed that these KVS must obligatorily assemble with subunits from the KV2 family into heterotetrameric channels, thereby giving raise to currents distinct from those of homomeric KV2 channels. Herein, we show that KVS subunits indeed also modulate the activity, biophysical properties and surface expression of recombinant KV7 isoforms in a subunit-specific manner. Employing co-immunoprecipitation, and proximity labelling, we unveil the spatial coexistence of KVS and KV7 within a single protein complex. Electrophysiological experiments further indicate functional interaction and probably heterotetramer formation. Finally, single-cell transcriptomic analyses identify native cell types in which this KVS and KV7 interaction may occur. Our finding demonstrate that KV cross-family interaction is much more versatile than previously thought - possibly serving nature to shape potassium conductance to the needs of individual cell types.

neuroscience↗

bioRxiv2024

Mechanisms of Epigenomic and Functional Convergence Between Glucocorticoid and IL4-Driven Macrophage Programming

Macrophages adopt distinct phenotypes in response to environmental cues, with type-2 cytokine interleukin-4 promoting a tissue-repair homeostatic state (M2IL4). Glucocorticoids, widely used anti-inflammatory therapeutics, reportedly impart a similar phenotype (M2GC), but how such disparate pathways may functionally converge is unknown. We show using integrative functional genomics that M2IL4 and M2GC transcriptomes share a striking overlap mirrored by a shift in chromatin landscape in both common and signal-specific gene subsets. This core homeostatic program is enacted by transcriptional effectors KLF4 and the GC receptor, whose genome-wide occupancy and actions are integrated in a stimulus-specific manner by the nuclear receptor cofactor GRIP1. Indeed, many of the M2IL4:M2GC-shared transcriptomic changes were GRIP1-dependent. Consistently, GRIP1 loss attenuated phagocytic activity of both populations in vitro and macrophage tissue-repair properties in the murine colitis model in vivo. These findings provide a mechanistic framework for homeostatic macrophage programming by distinct signals, to better inform anti-inflammatory drug design.

immunology↗

bioRxiv2024

Chloride binding and cholesterol effects on high frequency complex nonlinear capacitance (cNLC) in the mouse outer hair cell: experiment and molecular dynamics

Prestin (SLC26a5) function evolved to enhance auditory sensitivity and frequency selectivity by providing mechanical feedback via outer hair cells (OHC) into the organ of Corti. Its effectiveness is governed by the voltage-dependent kinetics of the proteins charge movements, namely, nonlinear capacitance (NLC). We study the frequency response of NLC in the mouse OHC, a species with ultrasonic hearing. We find that the characteristic frequency cut-off (Fis) for the mouse in near 27 kHz. Single point mutations within the chloride binding pocket of prestin (e.g., S396E, S398E) lack the proteins usual anion susceptibility. In agreement, we now show absence of anion binding in these mutants through molecular dynamics (MD) simulations. NLC Fis in the S396E knock-in mouse is unaltered, indicating that high frequency activity is not governed by chloride, but more likely by viscoelastic loads within the membrane. We also show that the allosteric action of chloride does not underlie piezoelectric-like behavior in prestin, since tension sensitivity of S396E NLC is comparable to that of WT. Because prestin structures of all species studied to-date are essentially indistinguishable, with analogous chloride binding pockets, auditory requirements of individual species for cochlear amplification likely evolved to enhance prestin performance by modifying, not its protein-anion interaction, but instead external mechanical loads on the protein. SignificancePrestin is believed to provide cochlear amplification in mammals that possess a wide range of frequency sensitivities. Previously, chloride anions have been shown to control prestin kinetics at frequencies below 10 kHz. However, now we find that chloride binding is not influential for prestin kinetics in the very high range of the mouse. We suggest that such high frequency prestin performance is governed by impinging mechanical loads within the membrane, and not interactions with anions.

biophysics↗

bioRxiv2023

Modulation of cannabinoid receptor 2 alters neuroinflammation and reduces formation of alpha-synuclein aggregates in a rat model of nigral synucleinopathy

Research into the disequilibrium of microglial phenotypes has become an area of intense focus in neurodegenerative disease as a potential mechanism that contributes to chronic neuroinflammation and neuronal loss in Parkinsons disease (PD). There is growing evidence that neuroinflammation accompanies and may promote progression of alpha-synuclein (Asyn)-induced nigral dopaminergic (DA) degeneration. From a therapeutic perspective, development of immunomodulatory strategies that dampen overproduction of pro-inflammatory cytokines from chronically activated immune cells and induce a pro-phagocytic phenotype is expected to promote Asyn removal and protect vulnerable neurons. Cannabinoid receptor-2 (CB2) is highly expressed on activated microglia and peripheral immune cells, is upregulated in the substantia nigra of individuals with PD and in mouse models of nigral degeneration. Furthermore, modulation of CB2 protects against rotenone-induced nigral degeneration; however, CB2 has not been pharmacologically and selectively targeted in an Asyn model of PD. Here, we report that 7 weeks of peripheral administration of CB2 inverse agonist SMM-189 reduced phosphorylated (pSer129) alpha-synuclein in the substantia nigra compared to vehicle treatment. Additionally, SMM-189 delayed Asyn-induced immune cell infiltration into the brain as determined by flow cytometry, increased CD68 protein expression, and elevated wound-healing-immune-mediator gene expression. Additionally, peripheral immune cells increased wound-healing non-classical monocytes and decreased pro-inflammatory classical monocytes. In vitro analysis of RAW264.7 macrophages treated with lipopolysaccharide (LPS) and SMM-189 revealed increased phagocytosis as measured by the uptake of fluorescence of pHrodo E. coli bioparticles. Together, results suggest that targeting CB2 with SMM-189 skews immune cell function toward a phagocytic phenotype and reduces toxic aggregated species of Asyn. Our novel findings demonstrate that CB2 may be a target to modulate inflammatory and immune responses in proteinopathies.

immunology↗

bioRxiv2023

The Rapidly Evolving X-linked miR-506 Family Finetunes Spermatogenesis to Enhance Sperm Competition

Despite rapid evolution across eutherian mammals, the X-linked miR-506 family miRNAs are located in a region flanked by two highly conserved protein-coding genes (Slitrk2 and Fmr1) on the X chromosome. Intriguingly, these miRNAs are predominantly expressed in the testis, suggesting a potential role in spermatogenesis and male fertility. Here, we report that the X-linked miR-506 family miRNAs were derived from the MER91C DNA transposons. Selective inactivation of individual miRNAs or clusters caused no discernable defects, but simultaneous ablation of five clusters containing nineteen members of the miR-506 family led to reduced male fertility in mice. Despite normal sperm counts, motility and morphology, the KO sperm were less competitive than wild-type sperm when subjected to a polyandrous mating scheme. Transcriptomic and bioinformatic analyses revealed that these X-linked miR-506 family miRNAs, in addition to targeting a set of conserved genes, have more targets that are critical for spermatogenesis and embryonic development during evolution. Our data suggest that the miR-506 family miRNAs function to enhance sperm competitiveness and reproductive fitness of the male by finetuning gene expression during spermatogenesis. Significance StatementThe X-linked miR-506 family has rapidly evolved in mammals, but their physiological significance remains elusive. Given their abundant and preferential expression in the testis and sperm, these X-linked miRNAs likely play a functional role in spermatogenesis and/or early embryonic development. However, the deletion of either individual miRNA genes or all of the five miRNA clusters encoding 38 mature miRNAs did not cause major fertility defects in mice. When these mutant males were subjected to conditions resembling polyandrous mating, the mutant sperm were much less competitive than the wild-type sperm, rendering the mutant males "functionally sub-fertile". Our data suggest that the miR-506 family of miRNAs regulates sperm competition and the reproductive fitness of the male.

developmental biology↗

bioRxiv2022

Converging pathways direct synapse elimination through the homeodomain transcriptional regulator DVE-1

An important step in brain development is the remodeling of juvenile neural circuits to establish mature connectivity. The elimination of juvenile synapses is a critical step in this process; however, the molecular mechanisms directing synapse elimination activities and their timing are not fully understood. We identify here a conserved transcriptional regulator, DVE-1, that shares homology with mammalian special AT-rich sequence-binding (SATB) family members and directs the elimination of juvenile synaptic inputs onto remodeling C. elegans GABAergic neurons. Dorsally localized juvenile acetylcholine receptor clusters and apposing presynaptic sites are eliminated during maturation of wild type GABAergic neurons but persist into adulthood in dve-1 mutants. The persistence of juvenile synapses in dve-1 mutants does not impede synaptic growth during GABAergic remodeling and therefore produces heightened motor connectivity and a turning bias during movement. DVE-1 is localized to GABAergic nuclei prior to and during remodeling and DVE-1 nuclear localization is required for synapse elimination to proceed, consistent with DVE-1s function as a transcriptional regulator. Pathway analysis of DVE-1 targets and proteasome inhibitor experiments implicate transcriptional control of the ubiquitin-proteasome system in synapse elimination. Together, our findings demonstrate a new role for a SATB family member in the control of synapse elimination during circuit remodeling through transcriptional regulation of ubiquitin-proteasome signaling. Contributions SummaryKDA generated strains, transgenic lines, molecular constructs, confocal microscopy images and analysis, performed optogenetic behavioral experiments, photoconversion experiments, modencode ChIP-seq analysis and pathway analysis. SR performed all calcium imaging experiments/analysis and conducted single worm tracking. KB performed all Bortezomib inhibitor experiments and analysis. CL generated most vectors and constructs. JR assisted with generation of CRISPR/Cas9 generated strains. WA and MR assisted with aldicarb behavioral assay. DO assisted with EMS screen and isolation of dve-1 mutant. CB and MD aided in CloudMap bioinformatic analysis of the uf171 mutant. MMF and KDA designed and interpreted results of all experiments and wrote the manuscript.

neuroscience↗

bioRxiv2022

Chemically modified dsRNA induces RNAi effects in insects in vitro and in vivo: A potential new tool for improving RNA-based plant protection

Global agriculture loses over $100 billion of produce annually to crop pests such as insects. Many of these crop pests either have no current means of control or have developed resistance against chemical pesticides. Long dsRNAs are capable of inducing RNA interference (RNAi) in insects and are emerging as novel highly selective alternatives for sustainable insect management strategies. However, there are significant challenges associated with RNAi efficacy in insects. In this study, we synthesised a range of chemically modified long dsRNA in an approach to improve nuclease resistance and RNAi efficacy in insects. The results showed that dsRNA containing phosphorothioate modifications demonstrated increased resistance to southern green stink bug saliva nucleases. Phosphorothioate and 2-fluoro modified dsRNA also demonstrated increased resistance to degradation by soil nucleases and increased RNAi efficacy in Drosophila melanogaster cell cultures. In live insects, chemically modified long dsRNA successfully resulted in mortality in both stink bug and corn rootworm. The results provide further mechanistic insight of RNAi efficacy dependence on modifications in the sense or antisense strand of the dsRNA in insects and demonstrate for the first time that RNAi can successfully be triggered by chemically modified long dsRNA in insect cells or live insects.

bioengineering↗

bioRxiv2021

Kinesin-3 mediated delivery of presynaptic neurexin stabilizes growing dendritic spines and postsynaptic components in vivo

The functional properties of neural circuits are defined by the patterns of synaptic connections between their partnering neurons, but the mechanisms that stabilize circuit connectivity are poorly understood. We systemically examined this question at synapses onto newly characterized dendritic spines of C. elegans GABAergic motor neurons. We show that the presynaptic adhesion protein, neurexin/NRX-1, is required for stabilization of postsynaptic structure. We find that early postsynaptic developmental events proceed without a strict requirement for synaptic activity and are not disrupted by deletion of neurexin/nrx-1. However, in the absence of presynaptic NRX-1, dendritic spines and receptor clusters become destabilized and collapse prior to adulthood. We demonstrate that kinesin-3/UNC-104 delivers NRX-1 to presynaptic terminals and show that ongoing delivery is required for postsynaptic maintenance in mature animals. By defining the dynamics and temporal order of synapse formation events in vivo, we describe a mechanism for stabilizing mature circuit connectivity through neurexin-based adhesion.

neuroscience↗

bioRxiv2021

GEM-DeCan: Improving tumor immune microenvironment profiling by the integration of novel gene expression and DNA methylation deconvolution signatures

Quantifying the proportion of the different cell types present in tumor biopsies remains a priority in cancer research. So far, a number of deconvolution methods have emerged for estimating cell composition using reference signatures, either based on gene expression or on DNA methylation from purified cells. These two deconvolution approaches could be complementary to each other, leading to even more performant signatures, in cases where both data types are available. However, the potential relationship between signatures based on gene expression and those based on DNA methylation remains underexplored. Here we present five new deconvolution signature matrices, based RNAseq data or on DNA methylation, which can estimate the proportion of immune cells and cancer cells in a tumour sample. We test these signature matrices on available datasets for in-silico and in-vitro mixtures, peripheral blood, cancer samples from TCGA, and a single-cell melanoma dataset. Cell proportions estimates based on deconvolution performed using our signature matrices, implemented within the EpiDISH framework, show comparable or better correlation with FACS measurements of immune cell-type abundance and with various estimates of cancer sample purity and composition than existing methods. Using publicly available data of 3D chromatin structure in haematopoietic cells, we expanded the list of genes to be included in the RNAseq signature matrices by considering the presence of methylated CpGs in gene promoters or in genomic regions which are in 3D contact with these promoters. Our expanded signature matrices have improved performance compared to our initial RNAseq signature matrix. Finally, we show the value of our signatures in predicting patient response to immune checkpoint inhibitors in three melanoma cancer cohorts, based on bulk tumour sample gene expression. We also provide GEM-DeCan: a snakemake pipeline, able to run an analysis from raw sequencing data to deconvolution based on various gene expression signature matrices, both for bulk RNASeq and DNA methylation data.

bioinformatics↗

bioRxiv2020

Local PI(4,5)P2 pool dynamics detected by the coincidence biosensor tubbyCT maintain the integrity of ER-PM junctions during PLC signaling

Phosphoinositides (PIs) are important signaling molecules and determinants of membrane identity in the eukaryotic plasma membrane, where they multi-task in divergent signaling pathways. Signaling pleiotropy likely depends on distinct PI pools in the same membrane, although the physical definition of such pools has remained ambiguous. PI(4,5)P2, specifically, is also the precursor for the second messengers in the Gq/PLC pathway, IP3 and DAG, and is broken down by PLC{beta} during signaling. Endoplasmic reticulum-plasma membrane contact sites (ER-PM junctions) have emerged as central hubs for lipid transport between both membranes, and specifically for PI homeostasis by supplying the PM with phosphatidylinositol. Here we show that the tubby protein, by virtue of its C-terminal tubby-domain, preferentially localizes to ER-PM junctions by binding to both PI(4,5)P2 and the ER-PM tether E-Syt3. Under conditions of vigorous PI(4,5)P2 consumption by PLC{beta}, additional recruitment of tubby revealed an increase of a local PI(4,5)P2 pool fed by local synthesis through PI kinases. Inhibition of this pool-filling process led to the release of the ER-PM tethers, E-Syts, from the membrane and hence to loss of integrity of the ER-PM contact sites. We conclude that spatiotemporal metabolic channeling of PI synthesis initiated by non-vesicular transport in the ER-PM junctions specifies a local pool of PI(4,5)P2 that is pivotal for the maintenance of homeostatic functions during global depletion of PI(4,5)P2. The findings further suggest that the tubby-like proteins (TULPs), so far known to impact on energy homeostasis and obesity through primary cilia signaling, have an additional function at ER-PM junctions. HIGHLIGHTSO_LIThe tubby domain preferentially assembles into ER-PM junctions due to coincidence detection of PI(4,5)P2 and E-Syt3 C_LIO_LITubby recruitment reveals an increase of a local pool of PI(4,5)P2 in ER-PM junctions during PLC{beta} signaling C_LIO_LIJunctional PI(4,5)P2 dynamics require local synthesis of PI(4,5)P2 C_LIO_LILocal PI(4,5)P2 supply is required for integrity of ER-PM junctions during PLC{beta} activity. C_LI

cell biology↗