eNOS actin interaction.

<p>Effect of high [K⁺]_e and CD on endothelial NO release. Cells loaded with the fluorescent NO indicator DAF-FM DA were incubated in control buffer, high [K⁺]_e or CD in the presence of control peptide or P326TAT, respectively (N = 5, n = 30). DAF-FM fluorescence intensity under control conditions was averaged and set to 100%. Respective values are given as % of control ± SEM. High [K⁺]_e and CD significantly increase NO release relative to control. In all approaches P326TAT inhibits the eNOS/actin interaction and thereby the NO release.</p

Proof of principle.

<p>a) Fluorescence images of a Lifeact-eGFP transfected cell (grown in a confluent monolayer of non-transfected cells) before and after treatment with 1 µM CD. Cellular as well as cortical F-actin depolymerizes upon treatment with CD. Highlighted details illustrate the time course of the CD effect. Cortical F-actin (#) as well as F-actin stress fibres (*) are destabilized. b) Lifeact-eGFP transfected cell (grown in a confluent monolayer of untransfected cells) treated with 50 nM CD. Application of low doses of CD destabilizes cortical F-actin (#) and spares bulk stress fibres (*). c) Simultaneous analysis of cortical F-actin ( = peripheral Lifeact-eGFP fluorescence intensity, black circles), cortical stiffness (К_cortex, red circles) and bulk stiffness (К_bulk, red triangles). Application of 50 nM cytochalasin D (CD) decreases cortical F-actin and К_cortex significantly (after 4 minutes, p<0.05) whereas К_bulk is unaffected (n = 7). d) Correlation of CD-induced changes of К_cortex and cortical F-actin. The correlation coefficient, R² = 0.85, indicates a linear correlation. e) Analysis of the correlation between К_bulk and cortical F-actin indicates no correlation (R² = 0.1).</p

Exclusion of volume artefacts.

<p>a) Effect of low [Cl^-]_e on cortical F-actin (black circles) and К_cortex (red circles) of endothelial cells are detected simultaneously. Changing to low [Cl^-]_e decreases stiffness and F-actin in the cell cortex. b) Correlation of low [Cl^-]_e-induced changes between cortical F-actin and К_cortex. The correlation coefficient of R² = 0.76 indicates a linear dependence (n = 8). c) Effect of low [Cl^-]_e on К_bulk (triangles) and cortical F-actin (black circles). While cortical F-actin depolymerizes (see also d), К_bulk increases simultaneously.</p

Cortical Actin Nanodynamics Determines Nitric Oxide Release in Vascular Endothelium

<div><p>The release of the main vasodilator nitric oxide (NO) by the endothelial NO synthase (eNOS) is a hallmark of endothelial function. We aim at elucidating the underlying mechanism how eNOS activity depends on cortical stiffness (К_cortex) of living endothelial cells. It is hypothesized that cortical actin dynamics determines К_cortex and directly influences eNOS activity. By combined atomic force microscopy and fluorescence imaging we generated mechanical and optical sections of single living cells. This approach allows the discrimination between К_cortex and bulk cell stiffness (К_bulk) and, additionally, the simultaneous analysis of submembranous actin web dynamics. We show that К_cortex softens when cortical F-actin depolymerizes and that this shift from a gel-like stiff cortex to a soft G-actin rich layer, triggers the stiffness-sensitive eNOS activity. The results implicate that stiffness changes in the ∼100 nm phase of the submembranous actin web, without affecting К_bulk, regulate NO release and thus determines endothelial function.</p> </div

Physiological relevance.

<p>a) Simultaneous measurement of cortical F-actin intensity (black circles), cortical stiffness (К_cortex, red circles) and bulk stiffness (К_bulk, red triangles). After an increase of [K⁺]_e from 5 to 12 mM cortical F-actin and К_cortex decrease. The effect is significant after 6 minutes (n = 12). К_bulk is not affected by an increased [K⁺]_e. b) Changes in К_cortex and Lifeact-eGFP intensity show an almost linear correlation (R² = 0.79), c) whereas the correlation of K⁺-induced changes in cortical F-actin and К_bulk do not correlate (R² = 0.06).</p

Lifeact verification.

<p>Double staining of F-actin in GM7373 bovine aortic endothelial cells using Lifeact-eGFP and TRITC-phalloidin. DAPI is used to stain nuclei. The merge image verifies that Lifeact-eGFP and TRITC-phalloidin are equally applicable to detect F-actin in endothelial cells.</p

Proposed model of cortical stiffness-mediated eNOS activity.

<p>A stiff cortex implies a high rate of actin polymerization. eNOS, associated to actin filaments, exhibits a low activity. Actin depolymerisation causes cortical softening. Free G-actin, instead of F-actin, binds to eNOS stimulating its activity.</p

Cortical fluorescence in the cell periphery.

<p>a) 3D topography map of a confluent endothelial monolayer generated by an AFM surface scan. Cell height is illustrated by the colour code, shown at the right. The dashed line indicates the section for the height profile (scale bar = 20 µm). b) Height profile of the transfected endothelial cell (see also a). c) Height analysis of cell periphery. Four representative surface scans of endothelial cells are shown. Values indicate mean heights of the respective cell periphery. Quantification of all analysed cells (n = 33) is displayed. d) Proposed model of Lifeact-eGFP binding to actin filaments. Actin depolymerisation of cortical filaments leads to a dissociation of actin and Lifeact which results in a diffusion of fluorophores into the cell centre and a decrease of peripheral fluorescence intensity. e) Epifluorescence of the Lifeact-eGFP transfected cell (see also e). The white frames indicate the region of interest used for analysis of peripheral fluorescence intensity.</p

Sodium permeability <i>P_Na+</i> of human vascular endothelium (EA.hy 926 cell monolayer).

<p>Statistics (Mann Whitney U-Test with Bonferonni correction): <i>P_Na+(total)</i> is significantly smaller than <i>P_Na+(cell layer)</i> of control group (<i>p</i><0.05) and WS 1442 group (<i>p</i><0.01). <i>P_Na+(total)</i> of the WS 1442 group is significantly smaller in comparison to <i>P_Na+(total)</i> of the control group(<i>p</i><0.05). There is no significant difference in <i>P_Na+(cell layer)</i> between control group and WS 1442 group.</p

Impact of WS 1442 on the ESL-stiffness, the ESL-height (n = 36, 50 force-distance curves per n respectively) and the amount of heparan sulfates (n = 12).

<p>After administration of 10 µg/ml WS 1442 for 30 min the ESL becomes softer (A) and thicker (B), whereas the amount of heparan sulfates remains constant (C). Shown were median, IQR (box) and SD (whiskers).</p