Condensate functionalization with microtubule motors directs their nucleation in space and allows manipulating RNA localization.

The localization of RNAs in cells is critical for many cellular processes. Whereas motor-driven transport of ribonucleoprotein (RNP) condensates plays a prominent role in RNA localization in cells, their study remains limited by the scarcity of available tools allowing to manipulate condensates in a spatial manner. To fill this gap, we reconstitute in cellula a minimal RNP transport system based on bioengineered condensates, which were functionalized with kinesins and dynein-like motors, allowing for their positioning at either the cell periphery or centrosomes. This targeting mostly occurs through the active transport of the condensate scaffolds, which leads to localized nucleation of phase-separated condensates. Then, programming the condensates to recruit specific mRNAs is able to shift the localization of these mRNAs toward the cell periphery or the centrosomes. Our method opens novel perspectives for examining the role of RNA localization as a driver of cellular functions.

RNA at the surface of phase-separated condensates impacts their size and number.

Although it is now recognized that specific RNAs and protein families are critical for the biogenesis of ribonucleoprotein (RNP) condensates, how these molecular constituents determine condensate size and morphology is unknown. To circumvent the biochemical complexity of endogenous RNP condensates, the use of programmable tools to reconstitute condensate formation with minimal constituents can be instrumental. Here we report a methodology to form RNA-containing condensates in living cells programmed to specifically recruit a single RNA species. Our bioengineered condensates are made of ArtiGranule scaffolds composed of an orthogonal protein that can bind to a specific heterologously expressed RNA. These scaffolds undergo liquid-liquid phase separation in cells and can be chemically controlled to prevent condensation or to trigger condensate dissolution. We found that the targeted RNAs localize at the condensate surface, either as isolated RNA molecules or as a homogenous corona of RNA molecules around the condensate. The recruitment of RNA changes the material properties of condensates by hardening the condensate body. Moreover, the condensate size scales with RNA surface density; the higher the RNA density is, the smaller and more frequent the condensates are. These results suggest a mechanism based on physical constraints, provided by RNAs at the condensate surface, that limit condensate growth and coalescence.

PHI-1 induced enhancement of myosin phosphorylation in chicken smooth muscle.

Herein, we provide evidence that in chicken smooth muscle, G-protein stimulation by a Rho-kinase pathway leads to an increase in myosin light chain phosphorylation. Additionally, G-protein stimulation did not increase MYPT1 phosphorylation at Thr695 or Thr850, and CPI-17, was not expressed in chicken smooth muscle. However, PHI-1 was present in chicken smooth muscle tissues. Both agonist and GTP(gamma)S stimulation result in an increase in PHI-1 phosphorylation, which is inhibited by inhibitors to both Rho-kinase (Y-27632) and (PKC) GF109203x. These data suggest that PHI-1 may act as a CPI-17 analog in chicken smooth muscle and inhibit myosin phosphatase activity during G-protein stimulation to produce Ca2+ sensitization.

Hemolysis in sickle cell mice causes pulmonary hypertension due to global impairment in nitric oxide bioavailability.

Pulmonary hypertension is a highly prevalent complication of sickle cell disease and is a strong risk factor for early mortality. However, the pathophysiologic mechanisms leading to pulmonary vasculopathy remain unclear. Transgenic mice provide opportunities for mechanistic studies of vascular pathophysiology in an animal model. By microcardiac catheterization, all mice expressing exclusively human sickle hemoglobin had pulmonary hypertension, profound pulmonary and systemic endothelial dysfunction, and vascular instability characterized by diminished responses to authentic nitric oxide (NO), NO donors, and endothelium-dependent vasodilators and enhanced responses to vasoconstrictors. However, endothelium-independent vasodilation in sickle mice was normal. Mechanisms of vasculopathy in sickle mice involve global dysregulation of the NO axis: impaired constitutive nitric oxide synthase activity (NOS) with loss of endothelial NOS (eNOS) dimerization, increased NO scavenging by plasma hemoglobin and superoxide, increased arginase activity, and depleted intravascular nitrite reserves. Light microscopy and computed tomography revealed no plexogenic arterial remodeling or thrombi/ emboli. Transplanting sickle marrow into wild-type mice conferred the same phenotype, and similar pathobiology was observed in a nonsickle mouse model of acute alloimmune hemolysis. Although the time course is shorter than typical pulmonary hypertension in human sickle cell disease, these results demonstrate that hemolytic anemia is sufficient to produce endothelial dysfunction and global dysregulation of NO.