gfp rhoa biosensor  (Addgene inc)

Journal: Science Advances

Article Title: Mechano-gradients drive morphogen-noise correction to ensure robust patterning

doi: 10.1126/sciadv.adp2357

Figure Lengend Snippet: ( A ) Wnt/β-catenin activity gradient formation. ( B ) Membrane β-catenin, E-cadherin, F-actin, and p-MLC protein levels. Optical sagittal cross sections (dorsal side) of 8-hpf embryos. Scale bar, 50 μm. Right: Means ± SD; n = 10 embryos; across anterior-posterior (AP) axis. Relative fluorescent intensity in the anterior (A), central middle (M), and posterior (P) regions shown in the images on the left was quantified. ( C ) Wnt signaling and cortical actomyosin contraction underneath adherence junction. ( D ) RhoA activity gradients. Tg (HS:GFP-βcatCA) or Tg (HS:dkk1b-GFP) and E-cadherin–mRNA–injected zebrafish embryos and sibling embryos at 9 hpf. Scale bar, 50 μm. Right: Means ± SD; n = 10 embryos. Relative fluorescent intensity in the anterior (A), central middle (M), and posterior (P) regions was quantified. n.s., not significant. ( E ) Left: Laser dissection method. Bottom: Embryos injected with Ruby-Lifeact mRNA (magenta) before and after laser dissection. White arrows: Maximum recoil distances in (right) anterior, central middle, and posterior tissues. Scale bar, 20 μm. EVL, epithelial envelope layer (orange); deep cells (purple). Means ± SD ( n = 31, 29, and 28 experiments). ( F ) d2EGFP in Wnt/β-catenin reporter (OTM:d2EGFP)–transgenic embryos (dorsal view) treated with DMSO (control) or 2.5 μM blebbistatin or injected with bcl-2 mRNA. Red arrowheads: Abnormally high or low Wnt/β-catenin activity. Scale bar, 200 μm. ( G ) otx2 and cdx4 markers in embryos treated with DMSO (control) or 2.5 μM blebbistatin. Scale bar, 200 μm. Left: AP tissue marker expression. ( H ) Percentage of embryos with normal or abnormal expression patterns ( n = 100). Two-tailed one-way analysis of variance (ANOVA). ( I ) Zebrafish larvae (32 hpf) treated with DMSO (control) or 2.5 μM blebbistatin from 6 to 9 hpf. Red arrowheads: Abnormal cell proliferation. Scale bar, 500 μm. Right: Percentages of embryos with normal or abnormal morphology. The total number of embryos analyzed are shown above the graph (Fisher’s exact test). ( J ) Naturally generated “noise” cells are eliminated via E-cadherin gradient formation.

Article Snippet: For visualizing RhoA activity, GFP-RhoA biosensor (a gift from M. Glotzer; Addgene, catalog no. 68026) ( ) was used.

Techniques: Activity Assay, Membrane, Injection, Dissection, Transgenic Assay, Control, Marker, Expressing, Two Tailed Test, Generated

Journal: Science Advances

Article Title: Mechano-gradients drive morphogen-noise correction to ensure robust patterning

doi: 10.1126/sciadv.adp2357

Figure Lengend Snippet: ( A ) Introduction of Wnt/β-catenin–unfit cells mosaically in embryos. ( B ) Active caspase-3 (green) in mosaic embryos expressing mKO2 alone (control) or with βcatCA or Axin1 (magenta) and treated with DMSO (control) or 2.5 μM blebbistatin. White arrowheads: Caspase-3–active cells. Scale bar, 50 μm. Right: Means ± SD ( n = 20 embryos; three independent experiments) of GFP + caspase-3–active cell frequencies; unpaired two-tailed t test. ( C ) Embryos injected with RhoA-biosensor mRNA (RhoA-bio, green) and mosaically introduced with mKO2 + alone (control) or with βcatCA + or Axin1 + cells (magenta). Right: RhoA-bio fluorescence intensities (means ± SD; n = 50 cells); two-tailed one-way ANOVA. Scale bars, 20 μm. ( D ) Left: Active caspase-3 (green) in mosaic embryos expressing either mKO2 + RhoACA + or mKO2 + RhoADN + (magenta). White arrowheads: Caspase-3–active cells. Scale bar, 50 μm. Right graphs: Means ± SD of caspase-3–active cell frequencies within the divided range along the AP axis of embryos mosaically expressing mKO2 + RhoACA + or mKO2 + RhoADN + ( n = 5 embryos). ( E ) Quantification of angle and distance of neighboring cell movements. ( F and G ) Directions and distances of neighboring cell movements relative to βcatCA + cells in the anterior region (F) or GSK3β + cells in the posterior region (G) were quantified (top). Centroid of neighboring cell approaching unfit cells: negative direction ( −D ); centroid of cell that moved away: positive direction ( +D ). Bottom left: mKO2 + βcatCA + (F) or mKO2 + GSK3β + (G) cells (magenta). Plasma membrane (green). Lines: Distance between centroids of mKO2 + (yellow spot) and neighboring cells (white spot) at time points t (white lines) and t + 1 (red lines). Images were acquired in the anterior region (mKO2 + βcatCA + ) or posterior region (mKO2 + GSK3β + ). Scale bar, 20 μm. Bottom right: Means ± SD ( n = 102 cells; three independent experiments) of change in distance between the centroid of mKO2 + cells and neighboring cells during 10 s, separated according to the movement direction; unpaired two-tailed t test. ( H ) Actomyosin contractile force changes around cells with abnormal Wnt/β-catenin signaling activity.

Article Snippet: For visualizing RhoA activity, GFP-RhoA biosensor (a gift from M. Glotzer; Addgene, catalog no. 68026) ( ) was used.

Techniques: Expressing, Control, Two Tailed Test, Injection, Fluorescence, Clinical Proteomics, Membrane, Activity Assay

Journal: Nature immunology

Article Title: Dynamic encounters with red blood cells trigger splenic marginal zone B cell retention and function.

doi: 10.1038/s41590-023-01690-z

Figure Lengend Snippet: Fig. 6 | CD97 promotes cell membrane retraction through activation of RhoA. a, HEK293T cells were transfected with CD97 (WT)–GFP, CD97 (T419G)–GFP or CTF–GFP fusion constructs. Longest diameters of individual WT- (n = 62), T419G- (n = 62) and CTF-transfected (n = 63) cells are shown. Each symbol represents one cell. One of three independent experiments with similar results is shown. b, Schematic diagram of an optically trapped ligand-coated bead applying pulling force while in contact with the cell membrane. The cell is also indicated to contain an anillin–GFP RhoA biosensor. c,d, HEK293T cells were cotransduced with anillin–GFP RhoA biosensor and CD97 (WT)–Scarlet, CD97 (T419G)–Scarlet or CD97 (PBM)–Scarlet fusion lentivirus and sorted. c, Cell trap forces of individual WT- (n = 70), T419G- (n = 64) and PBM-transduced (n = 62) cells. d, Normalized active RhoA biosensor fluorescence intensity of WT- (n = 27),

Article Snippet: Anillin–GFP RhoA biosensor was cloned from pEGFP-RhoA Biosensor (a gift from M. Glotzer, University of Chicago, Addgene plasmid 68026) and inserted into the lentiviral vector.

Techniques: Membrane, Activation Assay, Transfection, Construct, Fluorescence