Biology subjects

Falk, M.

Publications and source records attributed to Falk, M..

6 recordsLinked to original sources

bioRxiv2026

Tumor-to-endothelium mitochondrial transfer licenses endothelial cells for CD8+ T cell recognition via mitochondrial neoantigen presentation

Renal cell carcinoma (RCC) frequently exhibits resistance to immune checkpoint blockade, highlighting the need for strategies that enhance tumor-specific T cell priming and improve immune access to the tumor microenvironment. Here we show that vaccination targeting tumor-associated mitochondrial antigens (TAMAs), derived from tumor-specific mitochondrial DNA (mtDNA) missense mutations, synergizes with PD-1/PD-L1 blockade to overcome checkpoint refractoriness in the RENCA RCC model. TAMAs vaccination elicits antigen-specific T cell responses, increases intratumoral CD8+ T cell infiltration, and reduces immunosuppressive myeloid populations, resulting in delayed tumor progression and improved survival when combined with checkpoint inhibition. In parallel, TAMAs + checkpoint blockade induces vascular remodeling characterized by increased pericyte coverage, reduced vascular leakage, improved perfusion and reduced hypoxia. Mechanistically, vascular remodeling is driven by CD8+ T cell-dependent, IFN{gamma}-associated immune activity and is associated with endothelial apoptosis and diminished intratumoral CD31 signal. We further identify tumor-to-endothelium mitochondrial transfer as a mechanism linking mitochondrial neoantigens to the tumor vascular compartment: tumor-derived mitochondria enter human and mouse endothelial cells in vitro and in vivo, and tumor-associated mtDNA mutations are detectable in endothelial fractions from murine tumors and human RCC specimens. Human endothelial cells can present mitochondrial neoantigens via MHC class I and become targets of TAMAs-specific CD8+ T cell cytotoxicity, including following mitochondrial acquisition from tumor cells. Together, these findings establish mitochondrial neoantigen immunity as a tractable approach to enhance checkpoint responses and reveal mitochondrial transfer as an antigenic bridge that expands immune targeting to the tumor vasculature.

cancer biology↗

bioRxiv2025

Endogenous recovery of hippocampal function following global cerebral ischemia in juvenile female mice is influenced by neuroinflammation and circulating sex hormones.

Cardiac arrest-induced global cerebral ischemia (GCI) in childhood often results in learning and memory deficits. We previously demonstrated in a murine cardiac arrest and cardiopulmonary resuscitation (CA/CPR) mouse model that a cellular mechanism of learning and memory, long-term potentiation (LTP), is acutely impaired in the hippocampus of juvenile males, correlating with deficits in memory tasks. However, little is known regarding plasticity impairments in juvenile females. We performed CA/CPR in juvenile (P21-25) female mice and used slice electrophysiology and hippocampal dependent behavior to assess hippocampal function. LTP was and contextual fear were impaired 7-days after GCI and endogenously recovered by 30-days. LTP remained impaired at 30 days in ovariectomized females, suggesting the surge in gonadal sex hormones during puberty mediates endogenous recovery. Unlike juvenile males, recovery of LTP in juvenile females was not associated with BDNF expression. NanoString transcriptional analysis revealed a potential role of neuroinflammatory processes, and specifically Cd68 pathways, in LTP impairment and hormone-dependent recovery. We were able to restore LTP in ovariectomized females with chronic and acute PPT administration, implicating estrogen receptor alpha in recovery mechanisms. This study supports a mechanism of endogenous LTP recovery after GCI in juvenile female mice which differs mechanistically from juvenile males and does not occur in adults of either sex.

neuroscience↗

bioRxiv2024

Structural Basis for the Activation of Proteinase-Activated Receptors (PARs) by Endogenous Ligands

The proteinase-activated receptor (PAR) subfamily of G protein-coupled receptors (GPCRs) include four members, PAR1-PAR4, that play critical roles in hemostasis, thrombosis, embryonic development, wound healing, inflammation, and cancer progression. The PARs share a unique activation mechanism driven by proteinase cleavage at a specific site within the extracellular amino-terminus, exposing a tethered ligand that self-activates the receptor. Subsequent activation allows PAR family members to initiate complex intracellular signaling networks via traditional G protein-mediated pathways and beta-arrestin signaling and, in this way, the PARs link extracellular protease signaling molecules to cellular functions. Despite a primary reliance on biochemical studies for understanding tethered ligand recognition, direct structural visualization of these ligand-receptor complexes has been elusive. Here, we present structural snapshots of activated PAR1 and PAR2 bound to their endogenous tethered ligands, revealing, for the first time, shallow and constricted orthosteric binding pockets and highlighting critical residues involved in ligand recognition and receptor activation. Surprisingly, comparisons with antagonist-bound structures show minimal conformational changes in the TM6 helix, a typical signature of GPCR activation, with large movements of TM7 observed upon activation. These insights lead to the identification of a common mechanism for PAR1 and PAR2 activation and provide a structural template for designing novel antagonists targeting the orthosteric binding site, potentially opening new avenues for therapeutic interventions.

biochemistry↗

bioRxiv2022

Cancer-Associated Mutations Enhance The Sensitivity Of The Trupath GαQ/11 System

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and are a common drug target. They can be stabilised in different conformational states by ligands to activate multiple transducers and effectors leading to a variety of cellular responses. The potential of agonists to activate select pathways has important implications for drug discovery. Thus, there is a clear need to profile the initial GPCR signal transduction event, activation of G proteins, to enhance understanding of receptor coupling and guide drug design. The BRET-based biosensor suite, TRUPATH, was recently developed to enable quantification of the activation profiles of all non-visual G proteins (excluding Golf and G14) and has since been utilised in numerous studies. However, it fails to detect Gq/11 activation for a number of GPCRs previously reported to display promiscuous secondary coupling to Gq/11. Here we report modifications to the Gq and G11 biosensors in the switch I region that prevent intrinsic GTPase activity (R183C/Q). Except for the PAC1R, substitution with cancer-associated mutations, Cys or Gln, significantly increased sensitivity to allow detection of robust, reliable, and representative Gq/11 responses to Class B1 GPCRs. We also demonstrate the utility of these modified biosensors for promiscuously coupled class A GPCR that have primary Gs-coupling. Thus, we propose that modification to Gq/11 may also be necessary in other biosensor systems to enable detection of Gq/11 activation.

pharmacology and toxicology↗

bioRxiv2020

DeepFoci: Deep Learning-Based Algorithm for Fast Automatic Analysis of DNA Double Strand Break Ionizing Radiation-Induced Foci

DNA double-strand breaks, marked by Ionizing Radiation-Induced (Repair) Foci (IRIF), are the most serious DNA lesions, dangerous to human health. IRIF quantification based on confocal microscopy represents the most sensitive and gold standard method in radiation biodosimetry and allows research of DSB induction and repair at the molecular and a single cell level. In this study, we introduce DeepFoci - a deep learning-based fully-automatic method for IRIF counting and its morphometric analysis. DeepFoci is designed to work with 3D multichannel data (trained for 53BP1 and {gamma}H2AX) and uses U-Net for the nucleus segmentation and IRIF detection, together with maximally stable extremal region-based IRIF segmentation. The proposed method was trained and tested on challenging datasets consisting of mixtures of non-irradiated and irradiated cells of different types and IRIF characteristics - permanent cell lines (NHDF, U-87) and cell primary cultures prepared from tumors and adjacent normal tissues of head and neck cancer patients. The cells were dosed with 1-4 Gy gamma-rays and fixed at multiple (0-24 h) post-irradiation times. Upon all circumstances, DeepFoci was able to quantify the number of IRIF foci with the highest accuracy among current advanced algorithms. Moreover, while the detection error of DeepFoci remained comparable to the variability between two experienced experts, the software kept its sensitivity and fidelity across dramatically different IRIF counts per nucleus. In addition, information was extracted on IRIF 3D morphometric features and repair protein colocalization within IRIFs. This allowed multiparameter IRIF categorization, thereby refining the analysis of DSB repair processes and classification of patient tumors with a potential to identify specific cell subclones. The developed software improves IRIF quantification for various practical applications (radiotherapy monitoring, biodosimetry, etc.) and opens the door to an advanced DSB focus analysis and, in turn, a better understanding of (radiation) DNA damaging and repair. HighlightsO_LINew method for DSB repair focus (IRIF) detection and multi-parameter analysis C_LIO_LITrainable deep learning-based method C_LIO_LIFully automated analysis of multichannel 3D datasets C_LIO_LITrained and tested on extremely challenging datasets (tumor primary cultures) C_LIO_LIComparable to an expert analysis and superb to available methods C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/321927v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@7a0127org.highwire.dtl.DTLVardef@1e1d3cborg.highwire.dtl.DTLVardef@117e7a7org.highwire.dtl.DTLVardef@69c620_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2018

Heterochromatin drives organization of conventional and inverted nuclei

The mammalian cell nucleus displays a remarkable spatial segregation of active euchromatic from inactive heterochromatic genomic regions. In conventional nuclei, euchromatin is localized in the nuclear interior and heterochromatin at the nuclear periphery. In contrast, rod photoreceptors in nocturnal mammals have inverted nuclei, with a dense heterochromatic core and a thin euchromatic outer shell. This inverted architecture likely converts rod nuclei into microlenses to facilitate nocturnal vision, and may relate to the absence of particular proteins that tether heterochromatin to the lamina. However, both the mechanism of inversion and the role of interactions between different types of chromatin and the lamina in nuclear organization remain unknown. To elucidate this mechanism we performed Hi-C and microscopy on cells with inverted nuclei and their conventional counterparts. Strikingly, despite the inversion evident in microscopy, both types of nuclei display similar Hi-C maps. To resolve this paradox we developed a polymer model of chromosomes and found a universal mechanism that reconciles Hi-C and microscopy for both inverted and conventional nuclei. Based solely on attraction between heterochromatic regions, this mechanism is sufficient to drive phase separation of euchromatin and heterochromatin and faithfully reproduces the 3D organization of inverted nuclei. When interactions between heterochromatin and the lamina are added, the same model recreates the conventional nuclear organization. To further test our models, we eliminated lamina interactions in models of conventional nuclei and found that this triggers a spontaneous process of inversion that qualitatively reproduces the pathway of morphological changes during nuclear inversion in vivo. Together, our experiments and modeling suggest that interactions among heterochromatic regions are central to phase separation of the active and inactive genome in inverted and conventional nuclei, while interactions with the lamina are essential for building the conventional architecture from these segregated phases. Ultimately our data suggest that an inverted organization constitutes the default state of nuclear architecture.

biophysics↗