Substrate rigidity-dependent positive feedback regulation between YAP and ROCK2

<p>Extracellular matrix (ECM) stiffness influences gene expression, leading to modulation of various cellular functions. While ROCK2 regulates actomyosin activity as well as cell migration and proliferation, expression of ROCK2 is increased in response to stiffening ECM. However, the mechanism underlying rigidity-dependent <i>ROCK2</i> expression remains elusive. Here, we show that YAP, a mechanically regulated transcription coactivator, upregulates <i>ROCK2</i> expression in an ECM rigidity-dependent manner. YAP interacted with the <i>ROCK2</i> promoter region in an actomyosin activity-dependent manner. Knockdown of <i>YAP</i> decreased <i>ROCK2</i> expression while activity of the <i>ROCK2</i> promoter was upregulated by expressing constitutively active YAP. Furthermore, we found that <i>ROCK2</i> expression promotes transcriptional activation by YAP. Our results reveal a novel positive feedback loop between YAP and ROCK2, which is modulated by ECM stiffness.</p

Identification of a novel strain of green algae, <i>Parachlorella</i> sp binos (Binos). a

<p>, Microscopic image of <i>P.</i> sp <i>binos</i> (Binos) (x2000). <b>b</b>, India ink staining of <i>P.</i> sp <i>binos</i> (Binos) (x400). India ink was diluted with water to make a 10% solution. <b>c, d</b>, Phylogenetic analysis of members of the phylum Chlorophyta based on (<b>c</b>) 18S rRNA gene and (<b>d</b>) actin amino acid sequences. <b>e, f</b>, Transmission electron micrographs of (<b>e</b>) <i>P.</i> sp <i>binos</i> (Binos) and (<b>f</b>) <i>Chlorella</i> sp. <b>g</b>, Infrared spectra of the extracellular matrix of <i>P.</i> sp <i>binos</i> (red line) and purified sodium alginate (standard; black line) analyzed by Fourier transform infrared spectroscopy from 400 to 4000 cm⁻¹.</p

Binding of radioisotopes of cesium with <i>P.</i> sp <i>binos.</i> a

<p>, 10, 30, and 100 mg/ml of wet algal cells were incubated with 1.92 kBq of ¹³⁷CsCl at room temperature for 10 min. Radioactivity accumulated in algal cells were measured, and data are shown as % of total radioactivity (mean ± SD). *, <i>P</i><0.05, **, <i>P</i><0.01 (n = 4). <b>b</b>, After 10 min incubation, the radioactivities of pellets and supernatant were measured and the bound/free ratios were calculated (n = 4). <b>c</b>, Ten mg/ml of wet algal cells were incubated with nonradioactive CsCl (solid circles) or NaCl (open circles) and 1.92 kBq of ¹³⁷CsCl at room temperature for 10 min. Radioactivity accumulated in algal cells were measured, and data are shown as % of control uptake without nonradioactive CsCl. Non-linear curve fitting to the model of two binding sites with high and low affinities was performed using the CsCl and NaCl competition data (n = 4).</p

Radioiodine uptake by <i>P.</i> sp <i>binos</i> (Binos). a

<p>, Ten mg/ml of <i>P.</i> sp <i>binos</i> was incubated with 1.48 kBq of Na¹²⁵I at room temperature under illumination at 2000 lux (open circles) or in the dark (solid circles) (n = 4). Radioactivity accumulated in algal cells were measured, and data are shown as maximal uptake. <b>b</b>, Algal cells incubated with Na¹²⁵I at room temperature under illumination for 24 h. Radioactivity accumulated in algal cells were measured, and data are shown as % of total radioactivity. *, <i>P</i><0.05, **, <i>P</i><0.01 (n = 4). <b>c</b>, After 24 h incubation, pellet and supernatant radioactivities were measured and the bound/free ratios of ¹²⁵I were calculated (n = 4). <b>d</b>, Ten mg/ml of wet algal cells were incubated with nonradioactive NaI and 1.48 kBq of Na¹²⁵I (n = 4). Radioactivity accumulated in algal cells were measured, and data are shown as % of control uptake without nonradioactive NaI <b>e</b>, Algal cells were incubated with or without 10 µM of hydrogen peroxide (H₂O₂) for 24 h (n = 3). <b>f, g</b>, After incubation of algal cells with 1 µM of sodium iodide for 24 h, elemental distribution of ¹²C¹⁴N (f) and ¹²⁷I (g) on the section of algal cells were visualized by NanoSIMS. <b>h</b>, Marged image of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044200#pone-0044200-g002" target="_blank">Figure 2f and 2g</a>.</p

Binding of radionuclides in contaminated water and soil samples from Fukushima. a

<p>, Soil and water soil samples were collected from Namie-machi, approximately 20 km from the Fukushima Daiichi Nuclear Plant. The upper photograph shows a signboard prohibiting entry to the 20 km exclusion zone. Gross radioactivity of ground samples measured using a Geiger-Müller counter was approximately 60 kcpm (lower photograph). Water samples (<b>b</b>) and soil-water extracts (<b>c</b>) contaminated by radioactive fallout were incubated with algal cells under illumination at 2000 lux for 8 h. Gross β radioactivity (closed bars) and γ radioactivity (open bars) from ¹³⁷Cs were measured by liquid scintillation and γ counters, respectively.</p