Endothelial barrier function is known to be regulated by a number of molecular mechanisms; however, the role of biomechanical signals associated with blood flow is comparatively less explored. Biomimetic microfluidic models comprised of vessel analogues that are lined with endothelial cells (ECs) have been developed to help answer several fundamental questions in endothelial mechanobiology. However, previously described microfluidic models have been primarily restricted to single straight or two parallel vessel analogues, which do not model the bifurcating vessel networks typically present in physiology. Therefore, the effects of hemodynamic stresses that arise due to bifurcating vessel geometries on ECs are not well understood. Here, we introduce and characterize a microfluidic model that mimics both the flow conditions and the endothelial/extracellular matrix (ECM) architecture of bifurcating blood vessels to systematically monitor changes in endothelial permeability mediated by the local flow dynamics at specific locations along the bifurcating vessel structure. We show that bifurcated fluid flow (BFF) that arises only at the base of a vessel bifurcation and is characterized by stagnation pressure of ∼38 dyn cm⁻² and approximately zero shear stress induces significant decrease in EC permeability compared to the static control condition in a nitric oxide (NO)-dependent manner. Similarly, intravascular laminar shear stress (LSS) (3 dyn cm⁻²) oriented tangential to ECs located downstream of the vessel bifurcation also causes a significant decrease in permeability compared to the static control condition via the NO pathway. In contrast, co-application of transvascular flow (TVF) (∼1 μm s⁻¹) with BFF and LSS rescues vessel permeability to the level of the static control condition, which suggests that TVF has a competing role against the stabilization effects of BFF and LSS. These findings introduce BFF at the base of vessel bifurcations as an important regulator of vessel permeability and suggest a mechanism by which local flow dynamics control vascular function in vivo.

Sprouting angiogenesis—the infiltration and extension of endothelial cells from pre-existing blood vessels—helps orchestrate vascular growth and remodeling. It is now agreed that fluid forces, such as laminar shear stress due to unidirectional flow in straight vessel segments, are important regulators of angiogenesis. However, regulation of angiogenesis by the different flow dynamics that arise due to vessel branching, such as impinging flow stagnation at the base of a bifurcating vessel, are not well understood. Here we used a recently developed 3-D microfluidic model to investigate the role of the flow conditions that occur due to vessel bifurcations on endothelial sprouting. We observed that bifurcating fluid flow located at the vessel bifurcation point suppresses the formation of angiogenic sprouts. Similarly, laminar shear stress at a magnitude of ~3 dyn/cm² applied in the branched vessels downstream of the bifurcation point, inhibited the formation of angiogenic sprouts. In contrast, co-application of ~1 µm/s average transvascular flow across the endothelial monolayer with laminar shear stress induced the formation of angiogenic sprouts. These results suggest that transvascular flow imparts a competing effect against bifurcating fluid flow and laminar shear stress in regulating endothelial sprouting. To our knowledge, these findings are the first report on the stabilizing role of bifurcating fluid flow on endothelial sprouting. These results also demonstrate the importance of local flow dynamics due to branched vessel geometry in determining the location of sprouting angiogenesis.

Previous in vitro studies have reported on the use of direct current electric fields (DC-EFs) to regulate vascular endothelial permeability, which is important for tissue regeneration and wound healing. However, these studies have primarily used static 2D culture models that lack the fluid mechanical forces associated with blood flow experienced by endothelial cells (ECs) in vivo. Hence, the effect of DC-EF on ECs under physiologically relevant fluid forces is yet to be systematically evaluated. Using a 3D microfluidic model of a bifurcating vessel, we report the role of DC-EF on regulating endothelial permeability when co-applied with physiologically relevant fluid forces that arise at the vessel bifurcation. The application of a 70 V m⁻¹ DC-EF simultaneously with 1 μL min⁻¹ low perfusion rate (generating 3.8 dyn cm⁻² stagnation pressure at the bifurcation point and 0.3 dyn cm⁻² laminar shear stress in the branched vessel) increased the endothelial permeability 7-fold compared to the static control condition (i.e., without flow and DC-EF). When the perfusion rate was increased to 10 μL min⁻¹ (generating 38 dyn cm⁻² stagnation pressure at the bifurcation point and 3 dyn cm⁻² laminar shear stress in the branched vessel) while maintaining the same electrical stimulation, a 4-fold increase in endothelial permeability compared to the static control was observed. The lower increase in endothelial permeability for the higher fluid forces but the same DC-EF suggests a competing role between fluid forces and the applied DC-EF. Moreover, the observed increase in endothelial permeability due to combined DC-EF and flow was transient and dependent on the Akt signalling pathway. Collectively, these findings provide significant new insights into how the endothelium serves as an electro-mechanical interface for regulating vessel permeability.

Linker histone H1 is an essential chromatin architectural protein that compacts chromatin into transcriptionally silent regions by interacting with nucleosomal and linker DNA, while rapidly exchanging in vivo . How H1 targets nucleosomes while being dynamic remains unanswered. Using a single-molecule strategy, we investigated human H1.0 interactions with DNA and nucleosomes. H1.0 directly binds nucleosomes and naked DNA with a preference toward nucleosomes. DNA-bound H1.0 exhibited a range of bound lifetimes with both mobile and immobile states, where the mobile H1.0 did not load onto nucleosomes. The histone chaperone Nap1 facilitated H1.0-nucleosome loading by enabling H1.0 loading through DNA sliding, reducing DNA resident times without impacting nucleosome resident times, increasing mobility along DNA, and targeting H1.0 loading onto the nucleosome dyad. These findings reveal linker histones load onto nucleosomes through multiple distinct mechanisms that are facilitated by chaperones to regulate chromatin accessibility.

Sequence-specific DNA-binding proteins must efficiently locate their target sequences within large, structurally heterogeneous genomes while avoiding nonproductive off-target interactions. Here, we show that an autoinhibitory conformational state of the site-specific recombinase Cre lowers nonspecific DNA-binding affinity to promote one-dimensional DNA scanning while suppressing off-target assembly of recombinogenic complexes. Recent studies indicate that the C-terminal segment of Cre transiently adopts a sequestered conformation that occludes the catalytic domain's DNA-binding surface. Using NMR spectroscopy, single-molecule fluorescence microscopy, and optical trapping, we investigated how this conformational equilibrium regulates target search. NMR measurements reveal that the sequestered state predominates in the absence of DNA and persists at noncognate sites, whereas binding to cognate loxP sequences relieves autoinhibition. Single-molecule tracking demonstrates that full-length Cre undergoes processive one-dimensional diffusion and transitions to stable binding upon cognate-site recognition, whereas C-terminal truncation impairs scanning efficiency by arresting diffusion. These findings demonstrate how transient motif sequestration coordinates target search with site-specific complex assembly, suggesting a general mechanism by which DNA-modifying enzymes balance search efficiency and target specificity.

The interactions of cells with signaling molecules present in their local microenvironment maintain cell proliferation, differentiation, and spatial organization, and mediate progression of diseases such as metabolic disorders and cancer. Real-time monitoring of the interactions between cells and their extracellular ligands in a 3-D microenvironment can inform detection and understanding of cell processes and development of effective therapeutic agents. DNA-origami technology allows for the design and fabrication of biocompatible and 3-D functional nanodevices via molecular self-assembly for various applications including molecular sensing. Here, we report a robust method to monitor live cell interactions with molecules in their surrounding environment in a 3-D tissue model using a microfluidic device. We used a DNA origami Cell Sensing Platform (CSP) to detect two specific nucleic acid sequences on the membrane of B cells and dendritic cells. We further demonstrated real-time detection of biomolecules with the DNA sensing platform on the surface of dendritic cells in a 3-D microfluidic tissue model. Our results establish the integration of live cells with membranes engineered with DNA nanodevices into microfluidic chips as a highly capable biosensor approach to investigate subcellular interactions in physiologically relevant 3-D environments under controlled biomolecular transport.