Narendra Suryavanshi

Figure S1. Thrombin induces an increase in MLC2 phosphorylation. (A) HUVECs were treated with 1 U/ml thrombin for 10 min, then lysed and analysed by immunoblotting for phospho-MLC2 (pMLC2) and MLC2. GAPDH was used as a loading control. Results for 3 separate experiments (Exp.) are shown. (B) Quantification of pMLC2 levels was performed by individually normalising pMLC2 and MLC2 levels to GAPDH levels. The ratio of the normalised pMLC2 level to the normalised MLC2 level was used as a measure of MLC2 phosphorylation, and is shown for thrombin stimulation as % of control cells (no thrombin). Data are means of 3 independent experiments ± SEM (PDF 166 kb).

Nesprins are large multi-domain proteins that link the nuclear envelope to the cytoskeleton and nucleoskeleton. Here we show that nesprin-1 and nesprin-2 play important roles in regulating cell shape and migration in endothelial cells. Nesprin-1 or nesprin-2 depletion by RNAi increased endothelial cell spread area and the length of cellular protrusions, as well as stimulating stress fibre assembly which correlated with an increase in F-actin levels. Nuclear area was also increased by nesprin depletion, and localization of the inner nuclear membrane protein emerin to the nuclear envelope was reduced. Depletion of nesprin-1 or nesprin-2 reduced migration of endothelial cells into a cell-free area, and decreased loop formation in an in vitro angiogenesis assay. Taken together, our results indicate that nesprin-1 and nesprin-2 both regulate nuclear and cytoplasmic architecture, which we propose leads to their effects on endothelial cell migration and angiogenic loop formation.

The Rho GTPases are members of the Ras superfamily of GTPases that are pivotal regulators of the actin cytoskeleton. They also contribute to other cellular processes such as gene transcription, cell polarity, microtubule dynamics, cell cycle progression and vesicle trafficking. Most Rho GTPases act as molecular switches cycling between an "active" GTP-bound form and an "inactive" GDP-bound form. Hence, to elucidate the mechanisms by which Rho GTPases regulate cellular responses, an important parameter to determine is the GTP-loading of each Rho family member in cells under different conditions. Here we describe a biochemical technique to assess this based on affinity-precipitation of the GTP-bound form from whole cell lysates.