F-actin-rich contractile endothelial pores prevent vascular leakage during leukocyte diapedesis through local RhoA signalling

During immune surveillance and inflammation, leukocytes exit the vasculature through transient openings in the endothelium without causing plasma leakage. However, the exact mechanisms behind this intriguing phenomenon are still unknown. Here we report that maintenance of endothelial barrier integrity during leukocyte diapedesis requires local endothelial RhoA cycling. Endothelial RhoA depletion in vitro or Rho inhibition in vivo provokes neutrophil-induced vascular leakage that manifests during the physical movement of neutrophils through the endothelial layer. Local RhoA activation initiates the formation of contractile F-actin structures that surround emigrating neutrophils. These structures that surround neutrophil-induced endothelial pores prevent plasma leakage through actomyosin-based pore confinement. Mechanistically, we found that the initiation of RhoA activity involves ICAM-1 and the Rho GEFs Ect2 and LARG. In addition, regulation of actomyosin-based endothelial pore confinement involves ROCK2b, but not ROCK1. Thus, endothelial cells assemble RhoA-controlled contractile F-actin structures around endothelial pores that prevent vascular leakage during leukocyte extravasation

F-actin-rich contractile endothelial pores prevent vascular leakage during leukocyte diapedesis through local rhoA signaling in vivo

During immune surveillance and inflammation, leukocytes exit the vasculature through transient openings in the endothelium without causing plasma leakage. However, the exact mechanisms behind this intriguing phenomenon are still unknown. Here we report that maintenance of endothelial barrier integrity during leukocyte diapedesis requires local endothelial RhoA cycling. Endothelial RhoA depletion in vitro or Rho inhibition in vivo provokes neutrophil-induced vascular leakage that manifests during the physical movement of neutrophils through the endothelial layer. Local RhoA activation initiates the formation of contractile F-actin structures that surround emigrating neutrophils. These structures that surround neutrophil-induced endothelial pores prevent plasma leakage through actomyosin-based pore confinement. Mechanistically, we found that the initiation of RhoA activity involves ICAM-1 and the Rho GEFs Ect2 and LARG. In addition, regulation of actomyosin-based endothelial pore confinement involves ROCK2b, but not ROCK1. Thus, endothelial cells assemble RhoA-controlled contractile F-actin structures around endothelial pores that prevent vascular leakage during leukocyte extravasation

A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells

G-protein coupled receptors (GPCRs) can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gαi1, Gαi2 and Gαi3 activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gαi subunit, and cp173Venus fused to the Gγ2 subunit as acceptor. The Gαi FRET biosensors constructs are expressed together with Gβ1 from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gαi FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gαi FRET sensor in single cells upon stimulation of several GPCRs, including the LPA2, M3 and BK2 receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gαi1, Gαi2 and Gαi3 activation will be valuable for live-cell measurements that probe Gαi activatio

Letter to the Editor

Volume: 73Start Page: 215End Page: 21

Development and characterization of the new Gα_i1-sensor.

<p>(A) Representative image showing the plasma membrane localization of Gα_i1 fused to mTurquoise2-Δ9, expressed in HeLa cells. (B) Schematic overview of the plasmid containing pGβ-2A-YFP-Gγ₂-IRES-Gα_i1-CFP, driven by a CMV promoter. The inset shows the DNA sequence encoding the end of the IRES sequence and the start of the Gα_i1 sequence. The proposed protein translation is shown in the line below the DNA sequence (single letter abbreviations of the amino acids). (C) Confocal images of the localization of Gα_i1-mTurquoise2-Δ9 (<i>top row</i>) and cp173Venus-Gγ₂ (<i>bottom row</i>) in HeLa cells, for variant 1.0 (<i>left column</i>) and variant 2.0 (<i>right column</i>) of the Gα_i1-sensor. (D) Quantitative co-expression analysis of the CFP and YFP channels of the cp173Venus-Gγ₂ and Gα_i1-mTurquoise2-Δ9 transfections in HeLa cells. Single plasmid transfection (<i>left</i>) versus the transfection of the separate plasmids (<i>right</i>). The dots depict the CFP and YFP intensity, quantified from individual single cells. The r² is the coefficient of determination. Width of the individual images in A and C is 143μm.</p

Performance of the Gα_i1-sensor in kinetic measurements.

<p>(A) HEK293 cells transfected with the Gα_i1-sensor and the α₂AR were repeatedly stimulated with 20μM norepinephrine during intervals that are indicated by short horizontal lines. The presented data is representative for at least six different transfections performed on six experimental days. Top panel: YFP emission, center panel: CFP emission, bottom panel: corrected and normalized FRET ratio. (B) HEK293 cells transfected with the Gα_i1-sensor and the Adenosine A1-receptor were stimulated with 30μM adenosine, indicated by the short horizontal line. The presented data is representative for at least six different transfections performed on six experimental days. Top panel: YFP emission, center panel: CFP emission, bottom panel: corrected and normalized FRET ratio. (C) A close-up of the on-kinetics of Gα_i1 activation, showing the normalized FRET ratio during the first stimulation of the experiment in (A), fitted to a one component exponential decay function with tau = 1160ms and amplitude = 0.18 (R = 0.99). (D) Scatter plot showing the average exponential time constants (tau) of pooled data from (n = 10) individual fits of HEK293 cells transfected with the Gα_i1-sensor and the α₂AR stimulated with 100μM norepinephrine or pooled data (n = 14) from individual fits of the Gα_i1-sensor and the Adenosine A1-receptor stimulated with 30μM adenosine, respectively. Error bars indicate 95% CI.</p

Insertion of a fluorescent protein at different positions in Gα13.

<p>(A) The protein structure of human Gα13 (PDB ID: 1ZCB). The highlighted residues indicate the amino acid preceding the inserted fluorescent protein. Successful sites for inserting mTurquoise2-Δ9 into Gα13 in pink and unsuccessful sites in orange. (B) A partial protein sequence alignment (full alignment see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193705#pone.0193705.s001" target="_blank">S1 Fig</a>) of different Gα classes. The highlighted residues indicate the amino acid preceding the inserted fluorescent protein (or luciferase). In bold, the sites that were previously used to insert Rluc [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193705#pone.0193705.ref026" target="_blank">26</a>]. Insertion of mTurquoise2-Δ9 in Gα13 after residue Q144 (black) was based on homology with previous insertions in Gαq and Gαi (black). Successful sites for inserting mTurquoise2-Δ9 (R128, A129 and R140) in pink and unsuccessful sites (L106 and L143) in orange. The numbers indicated below the alignment correspond with the Gα13 variant numbers, used throughout the manuscript. The colors under the alignment match with the colors of the αHelices shown in (A). (C) Confocal images of the tagged Gα13 variants transiently expressed in HeLa cells. The numbers in the left bottom corner of each picture indicate the number of cells that showed plasma membrane localization out of the total number of cells analyzed. The tagged Gα13 variants also localize to structures inside the cell, which are presumably endomembranes,. The width of the images is 76μm.</p

A FRET-based biosensor for measuring Gα13 activation in single cells

<div><p>Förster Resonance Energy Transfer (FRET) provides a way to directly observe the activation of heterotrimeric G-proteins by G-protein coupled receptors (GPCRs). To this end, FRET based biosensors are made, employing heterotrimeric G-protein subunits tagged with fluorescent proteins. These FRET based biosensors complement existing, indirect, ways to observe GPCR activation. Here we report on the insertion of mTurquoise2 at several sites in the human Gα13 subunit, aiming to develop a FRET-based Gα13 activation biosensor. Three fluorescently tagged Gα13 variants were found to be functional based on i) plasma membrane localization and ii) ability to recruit p115-RhoGEF upon activation of the LPA2 receptor. The tagged Gα13 subunits were used as FRET donor and combined with cp173Venus fused to the Gγ2 subunit, as the acceptor. We constructed Gα13 biosensors by generating a single plasmid that produces Gα13-mTurquoise2, Gβ1 and cp173Venus-Gγ2. The Gα13 activation biosensors showed a rapid and robust response when used in primary human endothelial cells that were exposed to thrombin, triggering endogenous protease activated receptors (PARs). This response was efficiently inhibited by the RGS domain of p115-RhoGEF and from the biosensor data we inferred that this is due to GAP activity. Finally, we demonstrated that the Gα13 sensor can be used to dissect heterotrimeric G-protein coupling efficiency in single living cells. We conclude that the Gα13 biosensor is a valuable tool for live-cell measurements that probe spatiotemporal aspects of Gα13 activation.</p></div

Capacity of the tagged Gα13 variants to recruit p115-RhoGEF.

<p>(A) Confocal images of a representative HeLa cell expressing SYFP1-p115-RhoGEF, Gα13.2-mTurquoise2-Δ9 and LPA2-P2A-mCherry (here only SYFP1-p115-RhoGEF is shown, for the localization of the other constructs see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193705#pone.0193705.s002" target="_blank">S2 Fig</a>) (before (t = 0s) and after (t = 100s) addition of 3μM LPA). The width of the pictures is 67μm. (B) The mean cytoplasmic fluorescence intensity of SYFP1-p115-RhoGEF over time. After 8s, 3μM LPA was added. All cells transiently expressed LPA2 receptor-P2A-mCherry. The number of cells imaged is p115-RhoGEF <i>n</i> = 5, Gα13 untagged + p115-RhoGEF <i>n</i> = 15, Gα13.1 + p115-RhoGEF <i>n</i> = 27, Gα13.2 + p115-RhoGEF <i>n</i> = 28, Gα13.3 + p115-RhoGEF <i>n</i> = 24, Gα13.5 + p115-RhoGEF <i>n</i> = 20. Data have been derived from three independent experiments. (C) Quantification of the fluorescence intensity at t = 50s for each Gα13 variant, relative to t = 0s. The dots indicate individual cells and the error bars show 95% confidence intervals. The numbers of cells analyzed is the same as in (B).</p