Biology subjects

Jacobs, K. A.

Publications and source records attributed to Jacobs, K. A..

16 recordsLinked to original sources

bioRxiv2026

Macrophage-instructed GSDME couples glioblastoma cell-state plasticity with inflammatory cell death

Glioblastoma (GBM) cell states reflect spatial microenvironmental interactions. Here, using COMET spatial proteomics and RNAscope across multiregional human GBM tissue, spanning tumor cores with pseudopalisading regions, infiltrative margins and peripheral regions, together with multiplex spatial profiling of 202 specimens from 33 patients with matched primary and recurrent tumors, we identify full-length gasdermin E (GSDME-FL) as a macrophage-instructed regulator of malignant cell plasticity. Integration with single-cell transcriptomics and functional perturbation shows that GSDME-high tumor cells localize to macrophage-rich perivascular niches and are associated with delayed recurrence and longer patient survival. Mechanistically, macrophage-derived S100A4 activates EGFR-Sp1 signaling to induce GSDME-FL in neighboring GBM cells. GSDME-FL restrains hypoxia-associated mesenchymal plasticity and shapes macrophage-induced tumor state transitions independently of caspase activation. During immunogenic cell death (ICD), cleaved GSDME promotes pre-lytic swelling, early ATP efflux, and the release of canonical ICD-associated cytokines and chemokines. NLRP3 signaling further supports ATP release and licenses macrophage phagocytosis of dying GBM cells, whereas NINJ1-dependent membrane rupture enables terminal HMGB1 release. Vaccination with ICD-treated glioma cells elicits tumor rejection in a prophylactic intracranial challenge model. Notably, co-expression of GSDME and NINJ1 in macrophage-rich perivascular niches provides a spatial correlate of pathway convergence in patient tumors. Thus, our findings define how a spatial macrophage niche induces GSDME-FL, thereby coupling malignant cell state plasticity to the inflammatory properties of GBM cell death.

cancer biology↗

bioRxiv2026

FAM83H couples keratin organization to Notch signaling during epidermal morphogenesis

Keratin intermediate filaments are essential for epidermal integrity, yet how keratin organization is coupled to the cell fate signaling that coordinates keratinocyte differentiation during epidermal morphogenesis remains poorly understood. Here, we identify FAM83H as a previously unrecognized regulator that links keratin cytoskeletal organization to epidermal cell fate decisions. Using mouse models and a human 3D microphysiological epidermis model, we show that loss of FAM83H disrupts epidermal architecture by impairing basal keratinocyte differentiation, organization, and cell-cell adhesion. Single-cell transcriptomic analysis of 3D epidermal tissues identified Notch signaling as prominently associated with cell populations lost upon FAM83H depletion. Mechanistically, FAM83H localizes to cell-cell junctions, where it organizes keratin filaments and desmosome integrity. Loss of FAM83H disrupts a desmoplakin-keratin-Notch1 complex at junctions, impairing Notch1 proteolytic activation and proper keratinocyte differentiation. Together, our work identifies FAM83H as a key regulator of epidermal morphogenesis that couples keratin cytoskeletal architecture to Notch1 signaling, and positions keratin-associated proteins as active participants in the epithelial fate decisions that govern epidermal homeostasis.

cell biology↗

bioRxiv2026

JCAD couples tight junction condensates to actin and RhoA to maintain the endothelial barrier

How endothelial cell-cell junctions integrate cytoskeletal, adhesive, and local signaling networks to maintain vascular barrier integrity remains incompletely defined. Here, we identify junctional cadherin 5-associated protein (JCAD) as a modular scaffold that organizes endothelial tight junction architecture by coupling junctional condensates to actin and RhoA signaling. Genetic deletion of Jcad in mice does not affect baseline vascular permeability but causes inflammation-dependent barrier hyperpermeability. JCAD depletion in primary human endothelial cells disrupts tight junction continuity and increases paracellular permeability. Mechanistically, JCAD localizes to ZO-1-positive tight junctions independently of VE-cadherin, directly binds filamentous actin, and forms dynamic actin-associated condensates at cell-cell contacts. Structure-function analysis reveals separable domains mediating tight junction targeting and actin binding, establishing a bipartite architecture that distinctly coordinates junctional signaling and cytoskeletal coupling. Together, these findings identify JCAD as a cell-cell adhesion scaffold that integrates the phase-separated tight junction plaque with actin and RhoA-dependent mechanics, enabling endothelial barrier adaptation to inflammatory stress.

cell biology↗

bioRxiv2026

StrataChip: a microphysiological system capturing dynamic keratinocyte fate and mechanical transitions during human epidermal morphogenesis

Epidermal development and homeostasis require precise coordination between keratinocyte differentiation and mechanics. Still, the mechanisms integrating these processes remain poorly understood in part due to limitations of existing experimental systems. Here, we introduce StrataChip, a tractable microphysiological system that enables dynamic, multimodal interrogation of human epidermal morphogenesis. The platform integrates a media perfused dermal tissue with human epidermal keratinocytes within a microfluidic device and supports rapid epidermal stratification following establishment of an air-liquid interface. High-resolution confocal imaging and single-cell RNA-sequencing demonstrate that the StrataChip recapitulates key architectural and molecular features of human epidermis, including distinct basal, spinous, and granular layers defined by canonical differentiation markers and adhesion molecule organization. Single-cell profiling reveals transcriptionally distinct basal and spinous subpopulations, including transitional states associated with suprabasal commitment. Live 3D imaging in situ captures keratinocyte morphodynamics including basal cell delamination and asymmetric division, linking dynamic cellular behaviors to defined differentiation fates and stratification. Altogether, StrataChip provides a robust platform for a dynamic and mechanistic interrogation of how gene regulation and cell mechanics are coupled during epidermal morphogenesis.

bioengineering↗

bioRxiv2025

The Notch1 intracellular domain orchestrates mechanotransduction of fluid shear stress

Hemodynamic shear stress regulates endothelial phenotype through activation of Notch1 signaling, yet the mechanistic basis for this activation is unclear. Here, we establish a fluid shear stress-dependent mechanism of Notch1 activation that is distinct from canonical ligand trans-endocytosis. Application of laminar flow triggers the rapid spatial polarization of full-length Notch1 heterodimers into downstream membrane microdomains. Unlike canonical transactivation, this response occurs independently of ligand redistribution, and Notch1 receptors are cis-endocytosed into the receptor-bearing cell within polarized microdomains prior to proteolytic activation. We discover that the Notch1 intracellular domain (ICD) critically orchestrates receptor polarization and proteolytic activation in response to flow but is dispensable for canonical ligand transactivation. Shear stress increases ICD interaction with annexin A2 and caveolar proteins which control Notch1 endocytosis and proteolytic compartmentalization. These findings define a flow-specific Notch1 mechanotransduction pathway linking receptor polarization and endocytosis with proteolytic activation and establish new mechanisms regulating Notch receptor activation.

cell biology↗

bioRxiv2025

First Generation Tools for the Modeling of Capicua (CIC) - Family Fusion Oncoprotein-Driven Cancers

Clinical divergence between patients harboring CIC-rearrangements is frequently observed. For example, the prototypical CIC::DUX4 fusion associates with soft tissue tumors while CIC::NUTM1 fusions typically localize to the CNS (brain/spinal cord). The basis for these differences is poorly understood due to a lack of molecular tools. To address this need, we generated patient-informed, synthetic coding sequences for CIC::NUTM1, CIC::LEUTX, and ATXN1::DUX4 and validated them in structure-function studies. We found that CIC::NUTM1 drives a transcriptional program distinct from that of CIC::DUX4 due to a C-terminal NUTM1 functional domain, CIC::LEUTX weakly activates CIC target genes through LEUTX transactivation sequences, and ATXN1::DUX4 upregulates CIC target genes via the ATXN1 AXH domain. Our findings indicate that the CIC fusion binding partner may alter overall fusion oncoprotein activity. Thus, these first generation synthetic tools provide an unprecedented resource to study CIC-family fusions beyond CIC::DUX4 and allow for the dissection of this rare subgroup of cancers. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/653825v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1c1cf27org.highwire.dtl.DTLVardef@1073faforg.highwire.dtl.DTLVardef@e61455org.highwire.dtl.DTLVardef@140e952_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Scrib organizes cortical actomyosin clusters to maintain adherens junctions and angiogenic sprouting

Spatiotemporal control of adherens junction fluidity and integrity is critical for angiogenesis, but underlying mechanisms are incompletely understood. To identify unappreciated regulators of endothelial adherens junctions, we performed VE-cadherin proximity ligation mass spectrometry, revealing significant interaction with the multifunctional scaffold Scrib. Utilizing a 3D angiogenesis-on-chip model, we find Scrib-depleted microvessels generate reduced intact sprouts and increased single-cell detachments. This defect was characterized by adherens junction instability and decreased actomyosin in the junctional cortex, yet was not caused by changes in catenin-dependent VE-cadherin coupling to actin. Instead, Scrib controls the formation of cortical actomyosin clusters, which critically organize the architecture and dynamics of the junctional actomyosin cortex to promote adherens junction stability. We further discovered that unconventional myosin-1c is a critical effector linking Scrib cortical dynamics and VE-cadherin to stabilize adherens junctions during angiogenic initiation. Our results demonstrate a new role for Scrib directing cortical actomyosin organization that is critical for precise control of adherens junctions during angiogenesis.

cell biology↗

bioRxiv2024

Arterial endothelial deletion of Alk1 causes epistaxis and cerebral microhemorrhage with aberrant arteries and defective smooth muscle coverage.

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant vascular disorder with manifestations including severe nose bleeding and microhemorrhage in brains. Despite being the second most common inherited bleeding disorder, the pathophysiological mechanism underlying HHT-associated hemorrhage is poorly understood. HHT pathogenesis is thought to follow a Knudsonian two-hit model, requiring a second somatic mutation for lesion formation. Mutations in activin receptor-like kinase 1 (ALK1) gene cause HHT type 2. We hypothesize that somatic mutation of Alk1 in arterial endothelial cells (AECs) leads to arterial defects and hemorrhage. Here, we mutated Alk1 in AECs in postnatal mice using Bmx(PAC)-CreERT2 and found that somatic arterial endothelial mutation of Alk1 was sufficient to induce spontaneous epistaxis and multifocal cerebral microhemorrhage. This bleeding occurred in the presence of tortuous and enlarged blood vessels, loss of arterial molecular marker Efnb2, disorganization of vascular smooth muscle, and impaired vasoregulation. Our data suggest that arterial endothelial deletion of Alk1 leading to reduced arterial identity and disrupted vascular smooth muscle cell coverage is a plausible molecular mechanism for HHT-associated severe epistaxis. This work provides the first evidence that somatic Alk1 mutation in AECs can cause hemorrhagic vascular lesions, offering a novel preclinical model critically needed for studying HHT-associated epistaxis, and delineating an arterial mechanism to HHT pathophysiology.

pathology↗

bioRxiv2024

Quantitative Comparison of Monomeric StayGold Variants Using Protein Nanocages in Living Cells

To standardize comparison of fluorescent proteins and independently determine which monomeric StayGold variant is best for live microscopy, we analyzed fluorescent protein tagged I3-01 peptides that self-assemble into stable sixty subunit dodecahedrons inside live cells. We find mStayGold is 3-fold brighter and 3-fold more photostable compared with EGFP and superior to other monomeric variants in mammalian cytoplasm. In addition, analysis of intracellular nanocage diffusion confirms the monomeric nature of mStayGold.

cell biology↗

bioRxiv2024

The Capicua C1 Domain is Required for Full Activity of the CIC::DUX4 Fusion Oncoprotein

Rearrangements between genes can yield neomorphic fusions that drive oncogenesis. Fusion oncogenes are made up of fractional segments of the partner genes that comprise them, with each partner potentially contributing some of its own function to the nascent fusion oncoprotein. Clinically, fusion oncoproteins driving one diagnostic entity are typically clustered into a single molecular subset and are often treated a similar fashion. However, knowledge of where specific fusion breakpoints occur in partner genes, and the resulting retention of functional domains in the fusion, is an important determinant of fusion oncoprotein activity and may differ between patients. This study investigates this phenomena through the example of CIC::DUX4, a fusion between the transcriptional repressor capicua (CIC) and the double homeobox 4 gene (DUX4), which drives an aggressive subset of undifferentiated round cell sarcoma. Using a harmonized dataset of over 100 patient fusion breakpoints from the literature, we show that most bona fide CIC::DUX4 fusions retain the C1 domain, which is known to contribute to DNA binding by wild type CIC. Mechanistically, deletion or mutation of the C1 domain reduces, but does not eliminate, activation of CIC target genes by CIC::DUX4. We also find that expression of C1-deleted CIC::DUX4 is capable of exerting intermediate transformation-related phenotypes compared with those imparted by full-length CIC::DUX4, but was not sufficient for tumorigenesis in a subcutaneous mouse model. In summary, our results suggest a supercharging role for the C1 domain in the activity of CIC::DUX4. Significance StatementFusion oncogenes are neomorphic entities comprised of fractional coding sequences from two partner genes that have been inappropriately rearranged. The functional domains contributed by the partner genes shape the function of the resulting fusion. We use CIC::DUX4, a transcription factor fusion that drives an ultra-rare soft tissue sarcoma, to explore how preferential retention of a partner gene domain may influence the activity of the overall fusion. Our results indicate that the capicua C1 domain is retained in most CIC::DUX4 transcripts and is required for full activity of the CIC::DUX4 oncoprotein. We demonstrate that knowledge of where breakpoints occur and the resulting impact this has on the retention of functional domains can teach us about fusion behavior.

cancer biology↗

bioRxiv2024

Doublecortin reinforces microtubules to promote growth cone advance in soft environments

Doublecortin (DCX) is a microtubule-associated protein critical for brain development. Although most highly expressed in the developing central nervous system, the molecular function of DCX in neuron morphogenesis remains unknown and controversial. We demonstrate that DCX function is intimately linked to its microtubule-binding activity. By using human induced pluripotent stem cell (hiPSC)- derived cortical i3Neurons genome engineered to express mEmerald-tagged DCX from the endogenous locus, we find that DCX-MT interactions become highly polarized very early during neuron morphogenesis. DCX becomes enriched only on straight microtubules in advancing growth cones with approximately 120 DCX molecules bound per micrometer of growth cone microtubule. At a similar saturation, microtubule-bound DCX molecules begin to impede lysosome transport, and thus can potentially control growth cone organelle entry. In addition, by comparing control, DCX-mEmerald and knockout DCX -/Y i3Neurons, we find that DCX stabilizes microtubules in the growth cone peripheral domain by reducing the microtubule catastrophe frequency and the depolymerization rate. DCX -/Y i3Neuron morphogenesis was inhibited in soft microenvironments that mimic the viscoelasticity of brain tissue and DCX -/Y neurites failed to grow toward brain-derived neurotrophic factor (BDNF) gradients. Together with high resolution traction force microscopy data, we propose a model in which DCX-decorated, rigid growth cone microtubules provide intracellular mechanical resistance to actomyosin generated contractile forces in soft physiological environments in which weak and transient adhesion-mediated forces in the growth cone periphery may be insufficient for productive growth cone advance. These data provide a new mechanistic understanding of how DCX mutations cause lissencephaly-spectrum brain malformations by impacting growth cone dynamics during neuron morphogenesis in physiological environments.

cell biology↗

bioRxiv2023

TUMOR ENDOTHELIAL CELL AUTOPHAGY IS A KEY VASCULAR-IMMUNE CHECKPOINT IN MELANOMA

Tumor endothelial cells (TECs) actively repress inflammatory responses and maintain an immune-excluded tumor phenotype. However, the molecular mechanisms that sustain TEC-mediated immunosuppression remain largely elusive. Here, we show that autophagy ablation in TECs boosts antitumor immunity by supporting infiltration and effector function of T cells, thereby restricting melanoma growth. In melanoma-bearing mice, loss of TEC autophagy leads to the transcriptional expression of an immunostimulatory/inflammatory TEC phenotype driven by heightened NF-kB and STING signaling. In line, single-cell transcriptomic datasets from melanoma patients disclose an enriched InflammatoryHigh/AutophagyLow TEC phenotype in correlation with clinical responses to immunotherapy. Congruently, patients responding to immunotherapy exhibit an increased presence of inflamed vessels, interfacing with infiltrating CD8+ T cells. Mechanistically, STING-dependent immunity in TECs is not critical for the immunomodulatory effects of autophagy ablation, since NF-kB-driven inflammation remains functional in STING/ATG5 double knockout TECs. Hence, autophagy is a principal tumor vascular anti-inflammatory mechanism dampening melanoma antitumor immunity.

cancer biology↗

bioRxiv2023

The paracaspase MALT1 controls cholesterol homeostasis in glioblastoma stem-like cells through lysosome proteome shaping

Glioblastoma stem-like cells (GSCs) compose a tumor-initiating and -propagating population, remarkably vulnerable to any variation in the stability and integrity of the endolysosomal compartment. Previous work showed that the expression and activity of the paracaspase MALT1 control GSC viability via lysosomal abundance. However, the underlying mechanisms remain elusive. By combining RNAseq with proteome-wide label-free quantification, we now report that MALT1 repression in patient-derived GSCs alters the cholesterol homeostasis, which aberrantly accumulates in lysosomes. This failure in cholesterol supply culminates in cell death and autophagy defects, which can be partially reverted by providing exogenous membrane-permeable cholesterol to GSCs. From a molecular standpoint, targeted lysosome proteome analysis unraveled that NPC lysosomal cholesterol transporters were exhausted when MALT1 was held in check. Accordingly, we found that hindering NPC1 and NPC2 phenocopies MALT1 inhibition. This supports the notion that GSC fitness relies on lysosomal cholesterol homeostasis.

cancer biology↗

bioRxiv2023

Notch1 cortical signaling regulates epithelial architecture and cell-cell adhesion

Notch receptors control tissue morphogenic processes that involve coordinated changes in cell architecture and gene expression, but how a single receptor can produce these diverse biological outputs is unclear. Here we employ a 3D organotypic model of a ductal epithelium to reveal tissue morphogenic defects result from loss of Notch1, but not Notch1 transcriptional signaling. Instead, defects in duct morphogenesis are driven by dysregulated epithelial cell architecture and mitogenic signaling which result from loss of a transcription-independent Notch1 cortical signaling mechanism that ultimately functions to stabilize adherens junctions and cortical actin. We identify that Notch1 localization and cortical signaling are tied to apical-basal cell restructuring and discover a Notch1-FAM83H interaction underlies stabilization of adherens junctions and cortical actin. Together, these results offer new insights into Notch1 signaling and regulation, and advance a paradigm in which transcriptional and cell adhesive programs might be coordinated by a single receptor.

cell biology↗

bioRxiv2021

The Necroptosis Effector MLKL drives Small Extracellular Vesicle Release and Tumour Growth in Glioblastoma

Extracellular vesicles (EVs) are lipid-based nano-sized particles that convey biological material from donor to recipient cells. They play key roles in tumour progression, notably in glioblastoma in which the subpopulation of Glioblastoma Stem-like Cells (GSCs) might represent a meaningful source of tumour-derived EVs. However, the mechanisms involved in the production and release of EVs by GSCs are still poorly understood. Here, we report the identification of MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of small EVs from GSCs. The targeting of MLKL by genetic, protein depletion or chemical approaches alters endosomal trafficking and EV release and reduces GSC expansion in vitro. This function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL triggers a reduction of both the tumour burden in xenografted mice and of the level of plasmatic EVs. This work reinforces the idea of a non-deadly role for MLKL in endosomal trafficking and suggests that interfering with EV biogenesis is a promising therapeutic option to sensitize glioblastoma cells to death.

cell biology↗

bioRxiv2019

Control of the Endo-Lysosome Homeostasis by the Paracaspase MALT1 regulates Glioma Cell Survival

Glioblastoma is one of the most lethal forms of adult cancer with a median survival of around 15 months. A potential treatment strategy involves targeting glioblastoma stem-like cells (GSC), which constitute a cell autonomous reservoir of aberrant cells able to initiate, maintain, and repopulate the tumor mass. Here, we report that the expression of the paracaspase mucosa-associated lymphoid tissue l (MALT1), a protease previously linked to antigen receptor-mediated NF-{kappa}B activation and B-cell lymphoma survival, inversely correlates with patient probability of survival. The knockdown of MALT1 largely impaired the expansion of patient-derived stem-like cells in vitro, and this could be recapitulated with pharmacological inhibitors, in vitro and in vivo. Blocking MALT1 protease activity increases the endo-lysosome abundance, impaired autophagic flux, and culminates in lysosomal-mediated death, concomitantly with mTOR inactivation and dispersion from lysosomes. These findings place MALT1 as a new druggable target involved in glioblastoma and unveil ways to modulate the homeostasis of endo-lysosomes.

cell biology↗