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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 unidirectional laminar flow triggers the rapid spatial polarization of full-length Notch1 heterodimers into downstream membrane microdomains. Unlike canonical transactivation, Notch1 receptors are cis-endocytosed into the receptor-bearing cell within polarized microdomains. We discover that the Notch1 intracellular domain critically orchestrates receptor polarization and proteolytic cleavage in response to flow, but is dispensable for canonical ligand transactivation. Shear stress increases intracellular domain interaction with annexin A2 and caveolar proteins, which control Notch1 cis-endocytosis and proteolytic activation. These findings define a flow-specific Notch1 mechanotransduction mechanism linking receptor polarization and endocytosis with proteolytic activation and illuminate a new pathway by which mechanical forces integrate with Notch receptor activation.

The cytoskeleton provides structural integrity to cells and serves as a key component in mechanotransduction. Tensins are thought to provide a force-bearing linkage between integrins and the actin cytoskeleton; yet, direct evidence of tensin’s role in mechanotransduction is lacking. We here report that local force application to epithelial cells using a micrometer-sized needle leads to rapid accumulation of cten (tensin 4), but not tensin 1, along a fibrous intracellular network. Surprisingly, cten-positive fibers are not actin fibers; instead, these fibers are keratin intermediate filaments. The dissociation of cten from tension-free keratin fibers depends on the duration of cell stretch, demonstrating that the external force favors maturation of cten−keratin network interactions over time and that keratin fibers retain remarkable structural memory of a cell’s force-bearing state. These results establish the keratin network as an integral part of force-sensing elements recruiting distinct proteins like cten and suggest the existence of a mechanotransduction pathway via keratin network.

Germinal matrix hemorrhage (GMH) is a devastating neurodevelopmental condition affecting preterm infants, but why blood vessels in this brain region are vulnerable to rupture remains unknown. Here we show that microglia in prenatal mouse and human brain interact with nascent vasculature in an age-dependent manner and that ablation of these cells in mice reduces angiogenesis in the ganglionic eminences, which correspond to the human germinal matrix. Consistent with these findings, single-cell transcriptomics and flow cytometry show that distinct subsets of CD45 + cells from control preterm infants employ diverse signaling mechanisms to promote vascular network formation. In contrast, CD45 + cells from infants with GMH harbor activated neutrophils and monocytes that produce proinflammatory factors, including azurocidin 1, elastase and CXCL16, to disrupt vascular integrity and cause hemorrhage in ganglionic eminences. These results underscore the brain’s innate immune cells in region-specific angiogenesis and how aberrant activation of these immune cells promotes GMH in preterm infants. Chen et al. show that subtypes of immune cells in prenatal human brain promote angiogenesis in the germinal matrix. Conversely, in preterm infants, proinflammatory immune cells disrupt angiogenesis and promote germinal matrix hemorrhage.

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.

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 ( ) and the double homeobox 4 gene ( ), 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 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.

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.

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.

Clinical divergence between patients harboring -rearrangements is frequently observed. For example, the prototypical fusion associates with soft tissue tumors while 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 , , and 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 fusion binding partner may alter overall fusion oncoprotein activity. Thus, these first generation synthetic tools provide an unprecedented resource to study -family fusions beyond and allow for the dissection of this rare subgroup of cancers.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by excessive synchrony in neuronal firing within the basal ganglia. Coordinated reset stimulation (CRS)therapies aim to alleviate symptoms by disrupting this pathological synchrony. Vibrotactile CRS(vCR), a non-invasive variant of CRS, applies vibratory bursts to the fingertips to induce desynchronization. However, the mechanisms by which tactile afferent responses contribute to this effect remain poorly understood.This study employs TouchSim, a computational model of tactile afferents, to investigate the influence of vCR stimulus parameters and device housing design on afferent population responses. Simulations were conducted using models of the vCR prototype glove, exploring variations in pin arrangements, stimulus amplitudes, and housing pre-indentation. Results reveal that both stimulus configuration and housing parameters significantly affect the spatiotemporal firing patterns of mechanoreceptors, particularly in Pacinian corpuscle (PC) populations. These findings suggest that design features of vCR devices may play a critical role in shaping afferent input to central neural circuits.By elucidating the peripheral encoding of vCR stimuli, this work provides insight into optimizing device design and stimulation protocols for improved therapeutic outcomes in PD.

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.Competing Interest StatementThe authors have declared no competing interest.