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Sodium channels are the crucial electrical elements of neuronal excitation. As a step toward hybrid neuron-semiconductor devices, we studied the activation of recombinant NaV1.4 sodium channels in human embryonic kidney (HEK293) cells by stimulation from an electrolyte/oxide/silicon (EOS) capacitor. HfO2 was used as an insulator to attain a high capacitance. An effective activation was achieved by decaying voltage ramps at constant intracellular voltage at a depleted NaCl concentration in the bath to enhance the resistance of the cell-chip contact. We were also able to open sodium channels at a NaCl concentration close to physiological conditions. This experiment provides a basis for noninvasive capacitive stimulation of nerve cells with semiconductor chips. [ABSTRACT FROM AUTHOR]

The extracellular matrix plays a critical role in modulating cell behaviour in the developing and adult central nervous system influencing neural cell morphology, function and growth. Neurons and astrocytes, play vital roles in neural signalling and support respectively and respond to cues from the surrounding matrix environment. However, a better understanding of the impact of specific individual extracellular matrix proteins on both neurons and astrocytes is critical for advancing the development of matrix‐based scaffolds for neural repair applications. This study aimed to provide an in‐depth analysis of how different commonly used extracellular matrix proteins— laminin‐1, Fn, collagen IV, and collagen I—affect the morphology and growth of trophic induced pluripotent stem cell (iPSC)‐derived astrocyte progenitors and mouse motor neuron‐like cells. Following a 7‐day culture period, morphological assessments revealed that laminin‐1, fibronectin, and collagen‐IV, but not collagen I, promoted increased process extension and a stellate morphology in astrocytes, with collagen‐IV yielding the greatest increases. Subsequent analysis of neurons grown on the different extracellular matrix proteins revealed a similar pattern with laminin‐1, fibronectin, and collagen‐IV supporting robust neurite outgrowth. fibronectin promoted the greatest increase in neurite extension, while collagen‐I did not enhance neurite growth compared to poly‐L‐lysine controls. Super‐resolution microscopy highlighted extracellular matrix‐specific nanoscale changes in cytoskeletal organization, with distinct patterns of actin filament distribution where the three basement membrane‐associated proteins (laminin‐1, fibronectin, and collagen‐IV) promoted the extension of fine cellular processes. Overall, this study demonstrates the potent effect of laminin‐1, fibronectin and collagen‐IV to promote both iPSC‐derived astrocyte progenitor and neuronal growth, yielding detailed insights into the effect of extracellular matrix proteins on neural cell morphology at both the whole cell and nanoscale levels. The ability of laminin‐1, collagen‐IV and fibronectin to elicit strong growth‐promoting effects highlight their suitability as optimal extracellular matrix proteins to incorporate into neurotrophic biomaterial scaffolds for the delivery of cell cargoes for neural repair. [ABSTRACT FROM AUTHOR]

Platelets interact with multiple adhesion proteins during thrombogenesis, yet little is known about their ability to assemble fibronectin matrix. In vitro three-dimensional superresolution microscopy complemented by biophysical and biochemical methods revealed fundamental insights into how platelet contractility drives fibronectin fibrillogenesis. Platelets adhering to thrombus proteins (fibronectin and fibrin) versus basement membrane components (laminin and collagen IV) pull fibronectin fibrils along their apical membrane versus underneath their basal membrane, respectively. In contrast to other cell types, platelets assemble fibronectin nanofibrils using αIIbβ3 rather than α5β1 integrins. Apical fibrillogenesis correlated with a stronger activation of integrin-linked kinase, higher platelet traction forces, and a larger tension in fibrillar-like adhesions compared to basal fibrillogenesis. Our findings have potential implications for how mechanical thrombus integrity might be maintained during remodeling and vascular repair.

Haemostatic platelet function is intimately linked to cellular mechanics and cytoskeletal morphology. How cytoskeletal reorganizations give rise to a highly contractile phenotype that is necessary for clot contraction remains poorly understood. To elucidate this process in vitro, we developed a morphometric screen to quantify the spatial organization of actin fibres and vinculin adhesion sites in single spread platelets. Platelets from healthy donors predominantly adopted a bipolar morphology on fibrinogen and fibronectin, whereas distinguishable, more isotropic phenotypes on collagen type I or laminin. Specific integrin α IIb β 3 inhibitors induced an isotropic cytoskeletal organization in a dose-dependent manner. The same trend was observed with decreasing matrix stiffness. Circular F-actin arrangements in platelets from a patient with type II Glanzmann thrombasthenia (GT) were consistent with the residual activity of a small number of α IIb β 3 integrins. Cytoskeletal morphologies in vitro thus inform about platelet adhesion receptor identity and functionality, and integrin α IIb β 3 mechanotransduction fundamentally determines the adoption of a bipolar phenotype associated with contraction. Super-resolution microscopy and electron microscopies further confirmed the stress fibre-like contractile actin architecture. For the first time, our assay allows the unbiased and quantitative assessment of platelet morphologies and could help to identify defective platelet behaviour contributing to elusive bleeding phenotypes.

Super-resolution fluorescence microscopy methods are increasingly applied to study the structure of biological molecules within their natural context or at biomaterial interfaces. We here provide a protocol for Single-molecule High-Resolution Imaging with Photobleaching (SHRImP) that can be used to obtain information about the conformation of large proteins or other macromolecules at the single-molecule level. This procedure requires site-specific protein labeling with fluorescent dyes, immobilization and sample preparation, optimization of imaging buffer composition and microscope settings, and acquisition of short time-lapse movies that capture the stepwise bleaching behavior of individual molecules. We then describe a method for reliably determining the relative positions of labels from bleaching movies using the free image processing package Fiji (ImageJ) with the help of auxiliary macros that are provided as Supplementary Material. The presented approach allows for measuring intramolecular distance distributions in the range of a few to hundreds of nanometers and can be applied to a wide variety of biological systems.