No abstract

A robust nanopillar platform with increased spatial resolution reveals that perinuclear forces, originating from stress fibres spanning the nucleus of fibroblasts, are significantly higher on these nanostructured substrates than the forces acting on peripheral adhesions. Many perinuclear adhesions embrace several nanopillars at once, pulling them into β1-integrin- and zyxin-rich clusters, which are able to translocate in the direction of cell motion without losing their tensile strength. The high perinuclear forces are greatly reduced upon inhibition of cell contractility or actin polymerization and disruption of the actin cap by KASH dominant-negative mutant expression. LMNA null fibroblasts have higher peripheral versus perinuclear forces, impaired perinuclear β-integrin recruitment, as well as YAP nuclear translocation, functional alterations that can be rescued by lamin A expression. These highly tensed actin-cap fibres are required for YAP nuclear signalling and thus play far more important roles in sensing nanotopographies and mechanochemical signal conversion than previously thought.

Nogo-A is an important axonal growth inhibitor in the adult and developing CNS. In vitro, Nogo-A has been shown to inhibit migration and cell spreading of neuronal and nonneuronal cell types. Here, we studied in vivo and in vitro effects of Nogo-A on vascular endothelial cells during angiogenesis of the early postnatal brain and retina in which Nogo-A is expressed by many types of neurons. Genetic ablation or virus-mediated knock down of Nogo-A or neutralization of Nogo-A with an antibody caused a marked increase in the blood vessel density in vivo. In culture, Nogo-A inhibited spreading, migration, and sprouting of primary brain microvascular endothelial cells (MVECs) in a dose-dependent manner and induced the retraction of MVEC lamellipodia and filopodia. Mechanistically, we show that only the Nogo-A–specific Delta 20 domain exerts inhibitory effects on MVECs, but the Nogo-66 fragment, an inhibitory domain common to Nogo-A, -B, and -C, does not. Furthermore, the action of Nogo-A Delta 20 on MVECs required the intracellular activation of the Ras homolog gene family, member A (Rho-A)-associated, coiled-coil containing protein kinase (ROCK)-Myosin II pathway. The inhibitory effects of early postnatal brain membranes or cultured neurons on MVECs were relieved significantly by anti–Nogo-A antibodies. These findings identify Nogo-A as an important negative regulator of developmental angiogenesis in the CNS. They may have important implications in CNS pathologies involving angiogenesis such as stroke, brain tumors, and retinopathies.

Thermotropic properties were characterized by differential scanning calorimetry (Perkin-Elmer DSC-7) in conjunction with hot-stage polarizing optical microscopy (DMLM, Leica; FP90 central processor and F82 hot stage, Mettler Toledo). Optically flat glass substrates used in this study included CaF 2 (Almaz Optics, 1 inch diameter (1 inch » 2.5 cm) and 0.125 inch thick, transparent down to 200 nm), and Schott optical glass FK5 (n = 1.4828 at 780 nm, transparent above 320 nm) and SF57 (n = 1.8258 at 780 nm, transparent above 400 nm), both 1 inch diameter and 0.177 inch thick with nearly matched thermal expansion coefficients, 10.0 10 ±6 and 9.2 10 ±6 K ±1 from 20 to 300 C for FK5 and SF57, respectively. All three types of glass substrate were coated with a polyimide layer and then uniaxially buffed for the preparation of glassy nematic films with their thickness controlled by glass fiber spacers (Bangs Laboratory). The light absorption and linear dichroism were characterized using a UV-vis-NIR spectrophotometer (Lambda 900, Perkin-Elmer), and infrared dichroism using a FTIR spectrometer (Nicolet 20 SXC). An AbbØ refractometer (Bellingham and Stanley, Model 60/HR) was used for the measurement of n e and n o with a 780 nm laser (LiCONiX Diolite 800, 6.0±8.0 mW). A solid sample of II was heated to 250 C and then cooled at a rate of ±20 C/min to room temperature for X-ray diffraction using a Rigaku Bragg-Brentano diffractometer equipped with a copper rotating anode and a position sensitive detector. The optics for this instrument were set up to look for a low-angle peak resulting in a narrow X-ray beam irradiating the sample, particularly at higher 2h angles.