Biology subjects

Wagner, E.

Publications and source records attributed to Wagner, E..

At least 19 recordsLinked to original sources

bioRxiv2026

Patient-Derived Melanoma Organoids Preserve Tumor-Immune Heterogeneity and Reveal Context-Dependent Responses to Immune Checkpoint Blockade

Abstract Background: Experimental models that retain endogenous tumor immune complexity are needed to investigate heterogeneous responses to immune checkpoint inhibitors (ICIs) in melanoma. We established patient-derived melanoma organoids (PDMOs) to examine retention of parental tumor cellular components and investigate patient-specific and context-dependent responses to checkpoint blockade. Methods: Fresh melanoma specimens (n=50), including primary tumors, neoadjuvant-treated tumors and tumor-infiltrating lymphocyte (TIL) associated samples were processed for Matrigel-embedded culture of patient-derived melanoma organoids (PDMOs). Characterization and functional studies were performed in subsets of established cultures. Immunofluorescence assessed tumor, stromal, immune, and checkpoint-marker expression in PDMOs and matched parental tissues. NGFR expression was evaluated in relation to organoid establishment and growth. Responses to anti PD1 alone or combined with anti-LAG3 or anti-CTLA4 therapies were evaluated using viability and morphological analyses, with selected models undergoing immune phenotyping and multiplex cytokine profiling. Available clinical outcomes were used for exploratory comparison. PDMOs and matched two-dimensional cultures were evaluated under 21% and 5% oxygen conditions. Results: PDMOs were established from 30 of 50 specimens (60%) and retained melanoma, stromal, lymphoid, and myeloid components identified in matched parental tissues during early culture, together with immune checkpoint expression. Higher NGFR (CD271) expression was associated with greater organoid-forming capacity in an exploratory subset. Responses to checkpoint blockade varied across patient-derived models and regimens, with combination blockade not uniformly producing lower viability than PD1 monotherapy. Ex vivo responses showed parallels with available clinical outcomes, with discordant cases also observed. In three selected PDMOs, divergent PD-1 responses were accompanied by differences in T-cell and myeloid representation, MHCII and AXL expression, and inflammatory versus immunoregulatory cytokine profiles. Under normoxic conditions, PDMOs and matched two-dimensional cultures exhibited divergent treatment sensitivities, including clinically responding cases in which sensitivity was retained in PDMOs but attenuated in monolayers. Changing oxygen tension further altered checkpoint sensitivity within individual PDMOs, with the direction and magnitude of the observed shifts varying by model and regimen. Conclusions: Early passage PDMOs retain key endogenous tumor, immune, and stromal components and support functional investigation of patient-derived melanoma biology. Their heterogenous responses across checkpoint regimens and oxygen conditions provide a platform for investigating how tumor-immune composition and environmental context influence treatment sensitivity.

immunology↗

bioRxiv2026

Environmental factors and microbe-microbe interactions drive the structure of the core microbiota of terrestrial microalgae

Plants and other photosynthetic organisms interact with their environment and surrounding microbiota through specialized associations. A global core microbiota has been proposed at high taxonomic levels, such as the order level. However, it remains unclear which environmental factors and how microbe-microbe interactions drive variation of this core microbiota at lower taxonomic resolution. Here, we leveraged the environmental diversity of 141 sites across the southwest of France to characterize algal populations, and their associated bacterial and fungal microbiota. We then performed a meta-analysis, combining these data with published datasets to formally identify the global core microbiota of terrestrial photosynthetic organisms, which comprises seven bacterial and five fungal orders. We next investigated diversity within this core microbiota and the environmental drivers shaping site-specific community composition. While environmental factors have a low impact on the total relative abundance of core orders, the core microbiota at the ASV-level is impacted by climatic factors, edaphic factors, and plant community descriptors. Using interaction network analysis, we finally explored how microbe-microbe interactions contribute to the assembly of stable core communities. Our results show that core ASVs occupy central positions in algal-associated microbial networks and that distinct core orders drive site-specific variation in core microbiota structure. Together, these findings highlight the importance of both environmental context and microbial interactions in shaping the composition and stability of the core microbiota associated with photosynthetic organisms.

plant biology↗

bioRxiv2026

Preserving Native Cellulose-Xylan Architecture Enables Structure-Property Control in Holocellulose Nanofibrils and High-Performance Sustainable Materials

The hierarchical organization of cellulose microfibrils and their intimate interactions with hemicelluloses such as xylan underpin the exceptional mechanical performance of plant cell walls. However, translating these biological design principles into sustainable nanocellulosic materials remains limited by conventional cellulose nanofibril production routes, which rely on harsh chemical treatments that disrupt the native cellulose-hemicellulose architecture. Here, we present an optimized isolation strategy for holocellulose nanofibrils (hCNFs) that preserves native cellulose structure, xylan substitution and conformation, and cellulose-xylan interactions. Using wild-type Arabidopsis thaliana, a xylan glucuronidation-deficient gux1/2 mutant, and Brassica napus straw as model systems, we systematically elucidate how xylan content and substitution pattern govern nanofibril isolation, interfacial interactions, and macroscopic properties. Two-dimensional 13C magic-angle spinning NMR demonstrates retention of native cellulose glucosyl environments, the presence of two-fold and three-fold helical xylan conformations, and cellulose-associated two-fold helical xylan. Cryogenic transmission electron microscopy reveals fibril widths of [~]3 nm, consistent with elementary cellulose I{beta} microfibrils. We show that xylan glucuronidation regulates colloidal stability, hydration behavior, and interfibrillar cohesion, whereas xylan content controls nanofibrillation efficiency. These multiscale structural features translate directly into moisture sorption, thermal behavior, and mechanical performance. Notably, Brassica napus hCNF films exhibit exceptional strength and extensibility, surpassing many chemically modified CNF systems. This work demonstrates that preserving the native cellulose-hemicellulose architecture enables high-performance, sustainable nanocellulosic materials without chemical reconstruction.

biochemistry↗

bioRxiv2026

Integrated Spatial Multi-omic Profiling Identifies HSV-associated Inflammatory Macrophage Niches Linked to Oncolytic Virotherapy Response in Melanoma

BackgroundPrimary and secondary resistance to immune checkpoint blockade (ICB) remains a critical challenge in advanced melanoma. Oncolytic Viruses (OV) selectively lyse tumor cells while generating systemic anti-tumor immune responses with minimal side effects. Yet their clinical use is limited to refractory melanoma patients and are only given in combination with second-line ICB regimens. ICB can both help and hinder OV efficacy depending on the source of checkpoint interactions across the tumor-immune microenvironment (TiME). However, functional checkpoint interactions are typically inferred from gene or protein expression and rarely contextualized within myeloid- and antigen presenting cell-associated immune niches during OV therapy, despite these populations dominating melanoma TiMEs and serving as key regulators of anti-viral immunity. MethodsAn integrated multi-omics framework combining Nanostring GeoMx digital spatial profiling (DSP), COMET sequential immunofluorescence (seqIF) and functional oncology mapping (FuncOmap) was applied to melanoma patient tissues collected pre- and post-neoadjuvant Talimogene Laherparepvec (T-VEC) to characterize immune remodeling and directly quantify checkpoint interaction dynamics associated with pathologic responses to OV therapy. ResultsT-VEC induced broad lymphocyte- and myeloid-associated immune transcriptional activation across melanoma TiMEs; however, pathologic responses could not be defined by bulk transcriptomics or cellular deconvolution alone. COMET seqIF analysis identified that HSV-associated M1/APC-like tumor-associated macrophages (TAMs) were enriched in complete pathologic response (CR) tissues and were a major source of PD-1/PD-L1 interaction niches. While partial (PR) and non-pathologic response (NR) tissues retained melanoma-centered PD-1/PD-L1 interaction niches and were enriched for B cell and M2-like TAM populations. FuncOmap analysis indicated that post-T-VEC PD-1/PD-L1 interaction states were consistently elevated in tumor bed, but not in lymph node tissues, across all pathologic response groups. Suggesting that immune checkpoint interactions may benefit T-VEC therapeutic responses depending on their spatial and immune context relative to OV infection. ConclusionsThese findings highlight the importance of integrated transcriptomic and functional proteomic analyses for resolving the spatial distribution and functional status of immune niches during OV therapy. Resolving PD-1/PD-L1 interaction states to specific M1/APC-like TAM and B cell niches may define mechanisms of responses and resistance to OV therapy.

cancer biology↗

bioRxiv2026

KIF18A Maintains Kinetochore-Microtubule Attachments in CIN Cells by Limiting Microtubule Polymerization

Chromosomal instability (CIN) generates vulnerabilities that can be therapeutically exploited, including sensitivity to inhibition of the kinesin motor KIF18A. However, the mechanistic basis for why a subset of CIN tumor cells depend on KIF18A remains unclear. Here, we compare mitotic phenotypes across KIF18A-sensitive and -insensitive cell models. In sensitive CIN cells, KIF18A inhibition leads to formation of polar chromosomes with unattached kinetochores, recruitment of spindle assembly checkpoint proteins, and prolonged mitotic arrest. Although KIF18A loss reduces kinetochore-microtubule stability in all cell lines, sensitive cells exhibit lower baseline attachment stability and heightened microtubule polymerization rates, predisposing them to attachment failure. Acute KIF18A inhibition disrupts maintenance of attachments after metaphase alignment, while reducing microtubule polymerization suppresses mitotic defects. These findings support a model in which CIN tumor cells rely on KIF18A to restrain excessive microtubule dynamics and maintain attachment.

cancer biology↗

bioRxiv2026

Calcium interaction with Nav1.5 via FGF12A and CaM binding

Voltage-gated Na+ (Nav) channels, including Nav1.5, are responsible for the initiation of cardiac and neuronal action potentials. Regulation of Nav1.5 inactivation is linked to multiple accessory proteins that bind its C-terminal domain (CTD) including calmodulin (CaM) and intracellular fibroblast growth factors (iFGF). Previous results demonstrate that Ca2+-bound CaM preferentially binds to iFGF12A. The role of intracellular Ca2+ ([Ca2+]i) in regulating Nav1.5 gating, either directly or via auxiliary proteins like CaM, is controversial. We hypothesize that CaM binding to the Nav1.5 CTD and iFGF12A synergistically alters channel inactivation in a previously unobserved calcium-dependent manner. We performed Fluorescence Resonance Energy Transfer (FRET) imaging in live cells to observe the interaction between the Nav1.5 alpha subunit, CaM and iFGF12A. At resting [Ca2+]i, a 2-fold difference between acceptor and donor FRET efficiency was observed, implying that a single CaM acceptor is present on the Nav1.5 CTD even in the presence of FGF12A. After increasing [Ca2+]i, the donor and acceptor FRET efficiencies equalize, suggesting a 2:1:1 ratio between CaM, FGF12A, and the Nav1.5 CTD. We then compared the voltage-dependent gating kinetics of Nav1.5 with FGF12A in the presence/absence of calcium. With low [Ca2+]i, the steady-state inactivation of Nav1.5 with FGF12A was significantly shifted toward hyperpolarized potential compared to resting [Ca2+]i. Thus, the FGF12A:CaM complex confers a Ca2+-dependent mechanism enabling FGF12A modulates the Nav1.5 steady-state inactivation. Additionally, the ability of multiple subunits to bring CaM to the Nav1.5 CTD implies biological redundancy to prevent major alteration to Nav1.5 inactivation in the absence of CaM.

biophysics↗

bioRxiv2026

Common Genetic Variants of The Cardiac Sodium Channel Alter Patient Response to Class 1b Antiarrhythmics

Several common variants have been identified in SCN5A, which encodes the cardiac sodium channel -subunit Nav1.5 and is targeted by class I antiarrhythmics. Lidocaine and its analog mexiletine both have a primary amine that blocks Na+ current. While lidocaine is highly effective in terminating ventricular tachycardia after acute myocardial infarction, mexiletine has been shown to prevent arrhythmia induction in only [~]20% of patients. The factors underlying this inconsistent drug response are unclear. Here, we use cardiomyocytes that are derived from induced pluripotent stem cells to observe that a common polymorphism in the SCN5A gene, S1103Y, exhibits an altered pharmacological response to mexiletine, with enhanced use-dependent and tonic block of peak sodium current. In addition, an unexpected increase in late sodium current causes action potential prolongation. This paradoxical proarrhythmic phenotype shifts the paradigm of conventional antiarrhythmic therapy with mexiletine, suggesting that background variants may alter pharmacological responses leading to unanticipated consequences. Our results suggest that the unique genetic background of patients should inform therapeutic approaches to treat and prevent arrhythmias associated with common cardiac pathologies.

physiology↗

bioRxiv2025

AXL tyrosine kinase inhibition rescues immune checkpoint blockade-resistant melanoma in a tumor microenvironment-dependent fashion

BackgroundImmune checkpoint blockade (ICB) achieves durable responses in approximately half of patients with advanced melanoma, but the mechanistic basis for resistance in the remaining patients remains incompletely defined. AXL tyrosine kinase has emerged as a candidate resistance mediator, yet clinical development of AXL inhibitors has yielded heterogeneous results in unselected patient populations, suggesting that cell-type and tumor microenvironment context may critically govern therapeutic response. MethodsWe characterized AXL expression across cell types in ICB-resistant melanoma using publicly available single-cell RNA sequencing datasets and The Cancer Genome Atlas (TCGA). In vivo efficacy was assessed in the YUMM1.7 PD-1-resistant syngeneic melanoma model using three pharmacologically distinct AXL inhibitors (warfarin, bemcentinib, and cabozantinib) as monotherapy and in combination with anti-PD-1 therapy. Tumor-associated macrophage (TAM) context-dependency was established using anti-CSF1R and anti-F4/80 depletion strategies. Functional PD-L1:PD-1 checkpoint interactions were quantified in tumor sections by immune Forster Resonance Energy Transfer (iFRET). TAM secretome reprogramming was characterized by 40-plex Luminex immunoassay in polarized RAW264.7 macrophages. ResultsAXL expression in ICB-resistant melanoma was predominantly localized to TAMs rather than tumor cells by single-cell analysis, with AXL+ TAMs distributed across both M1-like and M2-like phenotypic compartments. AXL inhibition significantly reduced tumor burden and synergized with anti-PD-1 therapy in vivo; however, efficacy was abolished by depletion of the monocyte-derived myeloid compartment (anti-CSF1R) and enhanced by depletion of tissue-resident TAMs (anti-F4/80), establishing TAM-context dependency. iFRET revealed a paradoxical gain of PD-L1:PD-1 interaction efficiency in anti-PD-1-treated resistant tumors, a functional resistance signature detectable by iFRET but not by PD-L1 expression that was reversed by AXL combination therapy. In vitro secretome profiling demonstrated that combination therapy reprograms macrophage secretome in a polarization-context-dependent manner, amplifying pro-inflammatory cytokines including IL-6 and GM-CSF in M2-like macrophages while selectively dampening T cell chemokine production. ConclusionsThese findings establish AXL as a TAM-resident immune target in ICB-resistant melanoma whose therapeutic relevance is governed by tumor-immune micronenvironment (TiME) macrophage composition rather than tumor cell AXL expression. TAM polarization contexture represents a candidate stratification axis for AXL inhibitor-based combination strategies, and iFRET-measured checkpoint interaction offers a functional complement to PD-L1 expression for monitoring resistance and response. Key MessagesO_ST_ABSWhat is already known on this topicC_ST_ABSO_LIAXL tyrosine kinase has been studied predominantly as a tumor-intrinsic mesenchymal marker and therapeutic target in melanoma, with prior clinical development of AXL inhibitors predicated on tumor cell AXL expression as the primary biomarker; the contribution of TAM-expressed AXL to ICB resistance and the dependence of therapeutic response on TiME composition have not been defined. C_LI What this study addsO_LIAXL expression in ICB-resistant melanoma is predominantly localized to tumor-associated macrophages distributed across both M1-like and M2-like phenotypic compartments, reframing AXL as a TAM immune target whose therapeutic relevance is governed by TiME macrophage composition rather than tumor cell AXL expression. C_LIO_LICombination AXL inhibition and anti-PD-1 reprograms the macrophage secretome in a polarization-context-dependent manner and reverses a paradoxical gain of functional PD-L1:PD-1 checkpoint interaction in resistant tumors -- a resistance signature detectable by spatial iFRET but not by PD-L1 expression. C_LI How this study might affect research, practice or policyO_LIThe context-dependent effects of AXL inhibition (immunostimulatory in M2-heavy TiMEs, potentially counterproductive in M1-heavy TiMEs) indicate that TAM polarization profiling should be incorporated into clinical trial design for AXL inhibitor-based combinations, and that unselected enrollment may obscure meaningful efficacy signals in the subset most likely to benefit. C_LIO_LIiFRET-based quantification of functional checkpoint interaction represents a candidate dynamic biomarker for ICB resistance monitoring that is orthogonal to PD-L1 immunohistochemistry and may identify resistant patients who retain immunosuppressive checkpoint interactions that can be disrupted by AXL targeting. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=170 HEIGHT=200 SRC="FIGDIR/small/667666v2_ufig1.gif" ALT="Figure 1"> View larger version (90K): org.highwire.dtl.DTLVardef@521de1org.highwire.dtl.DTLVardef@127a933org.highwire.dtl.DTLVardef@d56132org.highwire.dtl.DTLVardef@e03ef9_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Functional Spatial Mapping of the Tumour Immune Microenvironment In Advanced Melanoma Patients

IntroductionCurrent spatial proteomic approaches quantify immune checkpoint expression but do not directly measure functional receptor/ligand (PD-1/PD-L1) interactions within the tumor immune microenvironment (TiME). Therapeutic antibodies disrupt receptor-ligand interactions and do not target protein abundance. Methods that resolve functional checkpoint interactions provide biologically distinct insight beyond expression-based assays MethodsWe combined computation and quantitative spatial imaging, FuncO:TiME, [Functional Oncology Mapping (FuncOmap)], to map PD-1/PD-L1 interaction states to spatially defined regions of the TiME, in clinically annotated melanoma specimens, collected before and after neoadjuvant immune checkpoint blockade (ICB), ResultsFuncOmap spatially quantified millions of per-pixel PD-1/PD-L1 interactions demonstrated spatial heterogeneity in checkpoint interaction, not reflected by PD-1 expression levels alone. Post-treatment tissues exhibited increased PD-1/PD-L1 interaction states despite no corresponding increase in expression, indicating persistent or augmented functional checkpoint interaction despite therapy. Integration with spatial immune profiling further demonstrated that checkpoint interaction intensity can be contextualized within distinct immune cell populations. ConclusionWe have established the feasibility of spatially resolved functional checkpoint mapping in human melanoma tissues. We demonstrate that receptor-ligand interactions diverge from protein expression patterns. By enabling direct interrogation of functional checkpoint interaction dynamics within intact tissue architecture, FuncO:TiME advances a functional paradigm for studying immune regulation in cancer.

cancer biology↗

bioRxiv2025

Amino acids activate parallel chemosensory pathways in Drosophila

Amino acids (AAs) are essential dietary macronutrients that impact an organisms fitness in a concentration-dependent manner, but the mechanisms mediating AA detection to drive consumption are less clear. In Drosophila, we identified the full repertoire of taste cells and receptors involved in feeding initiation towards a glutamate-rich AA mixture, tryptone, using in vivo calcium imaging and the proboscis extension response (PER). We found that AA attraction occurs through sweet cells, whereas feeding aversion is mediated through Ionotropic Receptor 94e (IR94e) cells and bitter cells, dependent on concentration. Further, our results corroborate previous findings that ionotropic receptors IR76b, IR51b, and IR94e detect AAs in their respective cell types. Additionally, we describe a new role for the appetitive IR56d receptor and bitter gustatory receptors in detecting AAs. This work establishes a cellular and molecular framework of AA feeding initiation and highlights redundancy in aversive pathways that regulate AA feeding.

neuroscience↗

bioRxiv2025

Olfactory receptor 2 drives abdominal aortic aneurysm by promoting CX3CR1-mediated monocyte recruitment

BackgroundAbdominal aortic aneurysms (AAA) are characterized by an intricate interplay of extracellular matrix degradation and inflammation. Macrophages are centrally involved in these processes. The mechanisms underlying macrophage activation in AAA remain incompletely understood. Vascular macrophages have been shown to express olfactory receptor 2 (Olfr2), a G-protein coupled receptor involved in mediating the sense of smelling and regulating inflammatory activity in macrophages. Whether Olfr2 plays a role in modulating macrophage responses in AAA formation remains unknown. Methods & ResultsIn silico micro-array analysis showed increased expression of the human Olfr2 orthologue OR6A2 in AAA tissue compared to healthy aorta. Flow cytometric analysis revealed increased expression of OR6A2 on classical, non-classical, and intermediate monocytes of patients with a large AAA (> 5cm) in comparison to patients with smaller AAA (< 5cm). Up to 30% of vascular macrophages expressed OR6A2 in human AAA and Olfr2 in mouse AAA tissue. Olfr2 expression peaked on major histocompatibility complex II-high (MHCIIhigh) and C-C chemokine receptor type 2-low (CCR2low) aortic monocytes and macrophages on day 7 following experimental AAA initiation and decreased to baseline expression on day 28. Olfr2 gene (Olfr2-/-) deficiency protected mice from AAA formation, which was accompanied by lowered ECM degradation, reduced macrophage infiltration and increased smooth muscle cell content. Conversely, treatment with the Olfr2 agonist octanal exacerbated AAA formation and inflammation, while the antagonist citral reduced AAA formation in comparison to vehicle treated mice. Bulk transcriptome analysis of aortic tissue revealed reduced inflammatory gene expression in Olfr2-/- mice at day 7 following AAA initiation. Spectral flow cytometry resolved 20 aortic immune cell populations, which were largely reduced in quantity by Olfr2-deficiency at day 7 and day 28 post experimental AAA formation, while circulating leukocyte counts and monocyte subset distribution were not altered between Olfr2+/+ and Olfr2-/- mice. Circulating Ly6Chigh-monocytes exhibited reduced expression of the CX3C motif chemokine receptor 1 (CX3CR1) and CCR2 during AAA formation. Transcriptional analysis of monocytes confirmed downregulation of pathways associated with cell adhesion, motility and migration. In vitro, Olfr2-/- monocytes showed impaired migration towards the CX3CR1 ligand CX3CL1. Competitive transfer of Olfr2+/+ and Olfr2-/- monocytes confirmed reduced migratory capacity of Olfr2-/- monocytes into the developing AAA. ConclusionWe demonstrate a critical relevance for Olfr2 in the modulation of the inflammatory response underlying AAA, which is mediated by enhanced monocyte recruitment.

immunology↗

bioRxiv2024

Investigating the Functional Role of the DI-DII Linker in Nav1.5 Channel Function

The cardiac voltage-gated sodium channel, Nav1.5 initiates the cardiac action potential. Its dysfunction can lead to dangerous arrhythmias, sudden cardiac arrest, and death. The functional Nav1.5 core consists of four homologous repeats (I, II, III, and IV), each formed from a voltage sensing and a pore domain. The channel also contains three cytoplasmic linkers (I-II, II-III, and III-IV). While Nav1.5 structures have been published, the I-II and II-III linkers have remained absent, are predicted to be disordered, and their functional role is not well understood. We divided the I-II linker into eight regions ranging in size from 32 to 52 residues, chosen based on their distinct properties. Since these regions had unique sequence properties, we hypothesized that they may have distinct effects on channel function. We tested this hypothesis with experiments with individual Nav1.5 constructs with each region deleted. These deletions had small effects on channel gating, though two (430 - 457del and 556 - 607del) reduced peak current. Phylogenetic analysis of the I-II linker revealed five prolines (P627, P628, P637, P640, P648) that were conserved in mammals but absent from the Xenopus sequence. We created mutant channels, where these were replaced with their Xenopus counterparts. The only mutation that had a significant effect on channel gating was P627S, which depolarized channel activation (10.13 +/- 2.28 mV). Neither a phosphosilent (P627A) nor a phosphomimetic (P627E) mutation had a significant effect, suggesting that either phosphorylation or another specific serine property is required. Since deletion of large regions had little effect on channel gating while a point mutation had a conspicuous impact, the I-II linker role may be to facilitate interactions with other proteins. Variants may have a larger impact if they create or disrupt these interactions, which may be key in evaluating pathogenicity of variants.

physiology↗

bioRxiv2024

Targeting DNA-PK is a highly conserved poxvirus innate immune evasion mechanism

The sensing of viral nucleic acid by pattern recognition receptors (PRRs) is essential for initiation of a type-I interferon response against infection. Intracellular DNA sensing PRRs are responsible for initiating innate immune responses to poxviruses and other double-stranded DNA viruses. Poxviruses, in turn, encode an armoury of immunomodulators that inhibit this host defence mechanism. DNA-dependent protein kinase (DNA-PK) is an essential component of the cGAS/STING-dependent viral DNA sensing machinery that leads to the initiation of a type-I interferon response during poxvirus infection. Poxviruses counter this host sensing mechanism using the C4/C10 family of proteins that target DNA-PK, interfering with its ability to bind viral DNA. Although the DNA-PK complex, known also for its role in double strand break repair, is conserved across multiple taxa, its function in innate immunity outside mammals is unexplored. Here we analysed the contribution of DNA-PK to poxvirus DNA sensing in chickens, a species that is evolutionarily distant from mammals, but that is also infected by poxviruses. We found that DNA-PK functions as a DNA sensor in chickens, and this process is countered by C4/C10 family members found in fowlpox virus. This host/pathogen interaction is conserved across a broader range of species than other mechanisms of poxvirus antagonism of innate immune sensing, which may reflect the difficulty of the host in evolving escape mechanisms that interfere with a protein that is essential for maintenance of genomic stability.

immunology↗

bioRxiv2024

Movement of the endoplasmic reticulum is driven by multiple classes of vesicles marked by Rab-GTPases

Peripheral endoplasmic reticulum (ER) tubules move along microtubules to interact with various organelles through membrane contact sites (MCS). Traditionally, ER moves by either sliding along stable microtubules via molecular motors or attaching to the plus ends of dynamic microtubules through tip attachment complexes (TAC). A recently discovered third process, hitchhiking, involves motile vesicles pulling ER tubules along microtubules. Previous research showed that ER hitchhikes on Rab5- and Rab7-marked endosomes, but it is uncertain if other Rab-vesicles can do the same. In U2OS cells, we screened Rabs for their ability to cotransport with ER tubules and found that ER hitchhikes on post-Golgi vesicles marked by Rab6 (isoforms a and b). Rab6-ER hitchhiking occurs independently of ER-endolysosome contacts and TAC-mediated ER movement. Disrupting either Rab6 or the motility of Rab6-vesicles reduces overall ER movement. Conversely, relocating these vesicles to the cell periphery causes peripheral ER accumulation, indicating that Rab6-vesicle motility is crucial for a subset of ER movements. Proximal post-Golgi vesicles marked by TGN46 are involved in Rab6-ER hitchhiking, while other post-Golgi vesicles (Rabs 8/10/11/13/14) are not essential for ER movement. Our further analysis finds that ER to Golgi vesicles marked by Rab1 are also capable of driving a subset of ER movements. Taken together, our findings suggest that ER hitchhiking on Rab-vesicles is a significant mode of ER movement. SIGNIFICANCE STATEMENTO_LIPeripheral endoplasmic reticulum tubules move on microtubules by either attaching to motors (cargo adaptor-mediated), dynamic microtubule-plus ends (tip attachment complexes) or motile vesicles (hitchhiking) but the prevalence of each mode is not clear C_LIO_LIPost-Golgi vesicles marked by Rab6/TGN46 and ER to Golgi vesicles marked by Rab1 drive ER movements C_LIO_LIER hitchhiking on multiple classes of vesicles (endolysosomal, post-Golgi and ER to Golgi) marked by Rabs plays a prominent role in ER movement C_LI

cell biology↗

bioRxiv2024

How to measure functional connectivity using resting-state fMRI? A comprehensive empirical exploration of different connectivity metrics

BackgroundFunctional connectivity in the context of functional magnetic resonance imaging is typically quantified by Pearso[n]s or partial correlation between regional time series of the blood oxygenation level dependent signal. However, a recent interdisciplinary methodological work proposes more than 230 different metrics to measure similarity between different types of time series. ObjectiveHence, we systematically evaluated how the results of typical research approaches in functional neuroimaging vary depending on the functional connectivity metric of choice. We further explored which metrics most accurately detect neural decline induced by age and malignant brain tumors, aiming to initiate a debate on how best assessing brain connectivity in functional neuroimaging research. MethodsWe addressed both research questions using four independent neuroimaging datasets, comprising multimodal data from a total of 1187 individuals. We analyzed resting-state functional sequences to calculate functional connectivity using 20 representative metrics from four distinct mathematical domains. We further used T1- and T2-weighted images to compute regional brain volumes, diffusion-weighted imaging data to build structural connectomes, and pseudo-continuous arterial spin labeling to measure regional brain perfusion. ResultsFirst, our results demonstrate that the results of typical functional neuroimaging approaches differ fundamentally depending on the functional connectivity metric of choice. Second, we show that correlational and distance metrics are most appropriate to cover neural decline induced by age. In this context, partial correlation performs worse than other correlational metrics. Third, our findings suggest that the FC metric of choice depends on the utilized scanning parameters, the regions of interest, and the individual investigated. Lastly, beyond the major objective of this study, we provide evidence in favor of brain perfusion measured via pseudo-continuous arterial spin labeling as a robust neural entity mirroring age-related neural and cognitive decline. ConclusionOur empirical evaluation supports a recent theoretical functional connectivity framework. Future functional imaging studies need to comprehensively define the study-specific theoretical property of interest, the methodological property to assess the theoretical property, and the confounding property that may bias the conclusions.

neuroscience↗

bioRxiv2024

Visualizing sarcomere and cellular dynamics in skeletal muscle to improve cell therapies

The giant striated muscle protein titin integrates into the developing sarcomere to form a stable myofilament system that is extended as myocytes fuse. The logistics underlying myofilament assembly and disassembly have started to emerge with the possibility to follow labeled sarcomere components. Here, we generated the mCherry knock-in at titins Z-disk to study skeletal muscle development and remodeling. We find titins integration into the sarcomere tightly regulated and its unexpected mobility facilitating a homogenous distribution of titin after cell fusion - an integral part of syncytium formation and maturation of skeletal muscle. In adult mCherry-titin mice, treatment of muscle injury by implantation of titin-eGFP myoblasts reveals how myocytes integrate, fuse and contribute to the continuous myofilament system across cell boundaries. Unlike in immature primary cells, titin proteins are retained at the proximal nucleus and do not diffuse across the whole syncytium with implications for future cell-based therapies of skeletal muscle disease.

cell biology↗

bioRxiv2023

Unravelling the metastasis-preventing effect of miR-200c in vitro and in vivo

Advanced breast cancer as well as insufficient treatment can lead to the dissemination of malignant cells from the primary tumor to distant organs. Recent research has shown that miR-200c can hamper certain steps of the invasion-metastasis cascade. However, it is still unclear, whether sole miR-200c expression is sufficient to prevent breast cancer cells from metastasis formation. Hence, we performed a xenograft mouse experiment with inducible miR-200c expression in MDA-MB 231 cells. The ex vivo analysis of metastatic sites in a multitude of organs including lung, liver, brain, and spleen has revealed a dramatically reduced metastatic burden of mice with miR-200c expressing tumors. A fundamental prerequisite for metastasis formation is the motility of cancer cells and, therefore, their migration. Consequently, we analyzed the effect of miR-200c on collective and single cell migration in vitro, utilizing MDA-MB 231 and MCF7 cell systems with genetically modified miR-200c expression. Analysis of collective cell migration has resulted in confluence dependent motility of cells with altered miR-200c expression. Additionally, scratch assays have shown enhanced predisposition of miR-200c negative cells to leave cell clusters. The in-between stage of collective and single cell migration was validated using transwell assays, which have displayed reduced migration of miR-200c positive cells. Finally, to measure migration on single cell level, a novel assay on dumbbell shaped micropatterns was performed, which revealed that miR-200c critically determines confined cell motility. All of these results demonstrate that exclusive expression of miR-200c impedes metastasis formation in vivo and migration in vitro and highlight miR-200c as metastatic suppressor in breast cancer.

cancer biology↗

bioRxiv2022

DNA-PKcs is required for cGAS/STING-dependent viral DNA sensing in human cells

To mount an efficient interferon response to virus infection, intracellular pattern recognition receptors (PRRs) sense viral nucleic acids and activate anti-viral gene transcription. The mechanisms by which intracellular DNA and DNA viruses are sensed are relevant not only to antiviral innate immunity, but also to autoinflammation and anti-tumour immunity through initiation of sterile inflammation by self-DNA recognition. The PRRs that directly sense and respond to viral or damaged self-DNA function by signalling to activate interferon regulatory factor (IRF)-dependent type one interferon (IFN-I) transcription. We and others have previously defined DNA-dependent protein kinase (DNA-PK) as an essential component of the DNA-dependent antiviral innate immune system. Here, we show that DNA-PK is essential for STING-dependent IFN-I responses in human cells during stimulation with exogenous DNA and infection with DNA viruses.

immunology↗