The GTPases Rac1, RhoA and Cdc42 act in concert to control cytoskeleton dynamics1-3. Recent biosensor studies have shown that all three GTPases are activated at the front of migrating cells4-7 and biochemical evidence suggests that they may regulate one another: Cdc42 can activate Rac18, and Rac1 and RhoA are mutually inhibitory9-12. However, their spatiotemporal coordination, at the seconds and single micron dimensions typical of individual protrusion events, remains unknown. Here, we examine GTPase coordination both through simultaneous visualization of two GTPase biosensors and using a “computational multiplexing” approach capable of defining the relationships between multiple protein activities visualized in separate experiments. We found that RhoA is activated at the cell edge synchronous with edge advancement, whereas Cdc42 and Rac1 are activated 2 μm behind the edge with a delay of 40 sec. This indicates that Rac1 and RhoA operate antagonistically through spatial separation and precise timing, and that RhoA plays a role in the initial events of protrusion, while Rac1 and Cdc42 activate pathways implicated in reinforcement and stabilization of newly expanded protrusions.

Inhibitors of Na+/H+ exchange proteins block macropinocytosis by lowering the pH near the plasma membrane, which in turn inhibits actin remodeling by Rho family GTPases.

Figure 2. (B) Top: schematic of the structure of membrane-targeted SEpHluorin/mCherry chimaera used to measure pH sm . Bottom: confocal images of SEpHluorin (left) and mCherry fluorescence (right) in A431 cells. Bar, 10 µm.

Summary Background Mechanical forces regulate cell behavior and function during development, differentiation, and tissue morphogenesis. In the vascular system, forces produced by blood flow are critical determinants not only of morphogenesis and function, but also pathological states such as atherosclerosis. Endothelial cells (ECs) have numerous mechanotransducers, including platelet endothelial cell adhesion molecule-1 (PECAM-1) at cell-cell junctions and integrins at cell-matrix adhesions. However, the processes by which forces are transduced to biochemical signals and subsequently translated into downstream effects are poorly understood. Results Here, we examine mechanochemical signaling in response to direct force application on PECAM-1. We demonstrate that localized tensional forces on PECAM-1 result in, surprisingly, global signaling responses. Specifically, force-dependent activation of phosphatidylinositol 3-kinase (PI3K) downstream of PECAM-1 promotes cell-wide activation of integrins and the small GTPase RhoA. These signaling events facilitate changes in cytoskeletal architecture, including growth of focal adhesions and adaptive cytoskeletal stiffening. Conclusions Taken together, our work provides the first evidence of a global signaling event in response to a localized mechanical stress. In addition, these data provide a possible mechanism for the differential stiffness of vessels exposed to distinct hemodynamic force patterns in vivo.

Cerebral cavernous malformations (CCM)3 are clusters of leaky, dilated capillaries in the central nervous system that occur in ϳ0.5% of the general population and up to 1.5% of the Hispanic population (1). CCM frequently lead to clinical sequelae such as hemorrhage, epilepsy, and neurological deficits (1). The disease is associated with both germline and somatic mutations in one of three genes, ccm1, -2, or -3 (2). The three CCM proteins form a common complex, indicating that they function coordinately (3, 4); each lacks defined catalytic domains, indicating that they are scaffold-or adaptor-like proteins for organization of protein complexes (3-5). The identical disease phenotype produced upon loss of any one of the three CCM proteins suggests that they coordinately regulate a common mechanism required for vascular integrity (3,4,6,7).It was recently shown that loss of endothelial cell expression of CCM2 resulted in activation of the GTPase RhoA (7,8). Crose et al. (8) demonstrated that CCM2 knockdown in brain microvascular endothelial cells resulted in defective RhoA degradation because of the dysregulation of Smurf1, a CCM2 binding partner, and an ubiquitin-protein isopeptide ligase (E3) that controls RhoA degradation. RhoA overabundance induced by loss of CCM2 was shown to increase cytoskeletal stability, inhibit vessel-like tube formation, and increase endothelial cell monolayer permeability (7,8). Herein, we show that loss of CCM1, -2, or -3 expression results in a common phenotype associated with RhoA overexpression and activation. We define ROCK as a critical RhoA effector whose increased activation dysregulates endothelial cell function. ROCK is activated by RhoA and phosphorylates several substrates, including myosin light chain, myosin light chain phosphatase, and LIM kinase for the regulation of actin cytoskeletal dynamics (9). ROCK has also been shown to regulate vascular permeability and has been a drug discovery target for regulation of vascular bed diseases (10). Our findings show that ROCK inhibition rescues extracellular matrix invasion and vessel-like tube formation, two endothelial cell functions disrupted by loss of CCM protein expression. EXPERIMENTAL PROCEDURESEstablishment of Knockdown Cell Lines-Lentiviral genespecific shRNAs in pLKO.1 were obtained from the University of North Carolina -Chapel Hill Lenti-shRNA Core Facility.RhoA Biosensor-Imaging and image processing were performed as described (11).Tube Formation Assay and Live Cell Imaging-7.0 ϫ 10 Ϫ4 cells were incubated for 15 h on Matrigel (BD Biosciences) and stained with rhodamine phalloidin as described previously (8).Imaging was performed on either a Pathway (BD Biosciences) or a Cellomics ArrayScan (Thermo Scientific). For live cell imaging, six fields of cells were imaged via transmitted light every 10 min for 15 h. Cellomics ArrayScan software was used to quantitate mean tube area. Statistical Significance-Where indicated, statistical significance was calculated using the two-tailed Student's t test. See supplemental mater...

Preface Cellular signal transduction occurs in complex and redundant interaction networks that are best examined at the level of single cells by simultaneously monitoring the activation dynamics of multiple components. Recent advances in biosensor technology have made it possible to visualize and quantify the activation of multiple network nodes in the same living cell. The precision and scope of this approach has been greatly extended by novel computational approaches to determine the relationships between different networks, studied in separate cells.

We present here the phasor approach to biosensor FRET detection by fluorescence lifetime imaging microscopy (FLIM) and show that this method of data representation is robust towards biosensor design as well as the fluorescence artifacts inherent to the cellular environment. We demonstrate this property on a series of dual and single chain biosensors which report the localization of Rac1 and RhoA activity, whilst performing concomitant ratiometric FRET analysis on the acquired FLIM data by the generalized polarization (GP) approach. We then evaluate and compare the ability of these two methods to quantitatively image biosensor FRET signal as a function of time and space. We find that with lifetime analysis in the phasor plot each molecular species is transformed into a two dimensional coordinate system where independent mixtures of fluorophores can be distinguished from changes in lifetime due to FRET. This enables the fractional contribution of the free and bound state of a dual chain biosensor or the low and high FRET species of a single chain biosensor to be quantified in each pixel of an image. The physical properties intrinsic to each biosensor design are also accurately characterized by the phasor analysis; thus this method could be used to inform biosensor optimization at the developmental stage. We believe that as biosensors become more sophisticated and are multiplexed with other fluorescent molecular tools, biosensor FRET detection by the phasor approach to FLIM will not only become imperative to their use but also their advancement.

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