Fig 1: The HMHA1 GAP domain induces loss of focal adhesions.The effects of myc-tagged HMHA1 (and deletion constructs) on focal adhesion distribution was studied by Confocal Laser Scanning Microscopy following expression in HeLa cells. Similar to the effects on F-Actin distribution, cells expressing full-length HMHA1 (FL), N-term, or GAP (first, second and fifth rows) constructs show normal focal adhesion distribution as detected using Paxillin immunostainings. Expression of HMHA1 C1-GAP, C1-GAPtail, or GAPtail (marked with asteriks) induces loss of focal adhesions. In the merged images, HMHA1 constructs appear in red, paxillin in green and nuclei in blue. Higher magnification images of the boxed area are included. Scale bars, 10 µm. (B) Mean +/− SD of the average-per-cell paxillin staining intensity (10–20 cells per condition), quantified following background subtraction, is indicated. Statistical differences compared to the Full-Length control are indicated by the respective p-values.
Fig 2: Constitutively active Rac1, but not Cdc42, rescues the altered cell morphology induced by HMHA1 C1-GAPtail.(A,B) Rescue experiments with constitutively active Rac1 Q61L and G12V (A) or Cdc42 G12V (B), co-expressed with the HMHA1 C1-GAPtail protein were performed in HeLa cells and analyzed by Confocal Laser Scanning Microscopy. Ectopically expressed proteins were visualized in combination with F-Actin. (A) Constitutively active Rac1 Q61L (middle panels) and G12V (bottom panels) were able to (partially) rescue the phenotype induced by C1-GAPtail. As a control, mCherry empty vector (EV; upper panels) was unable to rescue the phenotype. (B) Ectopic expression of constitutively active Cdc42 G12V did not rescue the phenotype induced by C1-GAPtail. Scale bars, 10 µm.
Fig 3: ARHGAP45 deficiency stabilizes the polarization and decreases the speed of naïve T cells A, BRepresentative kymographs of WT (A) and Arghap45 −/− (B) naive CD4+ T cells on 2D surface coated with ICAM‐1 and CCL21.C, DTop, representative sequences of cell contours during migration for 466 s of WT (C) and Arghap45 −/− (D) naive T cells on 2D surface coated with ICAM‐1 and CCL21. The color scale highlights each time‐resolved shape. Rectangle length, 112.6 μm (WT) and 51.6 μm (Arghap45 −/−). Bar, 10 µm. Bottom, three representative time‐resolved shapes and their corresponding calculated contours are shown. Bar, 10 µm.E, FHistograms of instant eccentricities (left) and 2D plots of adherent surface versus speed (right) of WT naive T cells on 2D surface coated with ICAM‐1 and CCL21. In the 2D plot, the 3 populations discussed in the results have been highlighted: a, hardly motile (instant speed < 5 µm/min) cell population with low (projected adherent area < 20%) adhesion, b, hardly motile (instant speed < 5 µm/min) cell population with high (projected adherent area > 40%) adhesion, and c, highly adherent (projected adherent area > 20%) cell population with elevated speed (ranging between 15 and 30 µm/min).G, HHistograms of instant eccentricities (left) and 2D plots of adherent surface versus speed (right) of Arghap45 −/− naive T cells on 2D surface coated with ICAM‐1 and CCL21. In the 2D plot, the single adherent (projected adherent area > 25%) and slow (instant speed < 15 µm/min) cell population is highlighted. Data information: In (E) and (F), histograms of instant eccentricities correspond to > 60 cells and > 2,000 contours, and fitted by a double Gaussian for WT naive T cells (means = 0.54, 0.8 and SD = 0.18, 0.06) and a single Gaussian for Arghap45 −/− naïve T cells (Mean = 0.58, SD = 0.18). 2D plots correspond to > 60 cells and > 2,000 events. In (F) and (H), events which combined null motility and adherence over the recorded time have been excluded.
Fig 4: Numbers of B and T cells in the specified organs of WT mice and of mice deficient in ARHGAP45 (Arhgap45 −/−), or expressing a loxP‐flanked Arhgap45 allele prior to (Arhgap45 fl/fl) or after (Arhgap45∆T/∆T) crossing with CD4‐Cre transgenic mice Cellularity of thymus from the specified mice.Numbers of T cells found in the blood of the specified mice.Numbers of B cells found in the blood of the specified mice.Numbers of T cells found in the LNs of the specified mice.Numbers of B cells found in the LNs of the specified mice.Numbers of T cells found in the spleen of the specified mice.Numbers of B cells found in the spleen of the specified mice. Data information: Each dot corresponds to a mouse and the mean and SD are indicated. Data are representative of three independent experiments involving each a total of 8–16 mice. A one‐way Anova test was used to compare each mouse model against WT mouse controls. The resulting probability is indicated above each model. ns, non‐significant, **P ≤ 0.002, ****P ≤ 0.0001.
Fig 5: Analysis of the projected adhesion area of WT and Arghap45 −/− naive T cells on 2D surface coated with ICAM‐1 CCL21 A–FProcessing of images from RICM microscopy to infer projected adhesion area. The projected area of cells is extracted from bright field images (A), which are binarized (B) to extract the contour in red (C). The area of adhesion fingerprint is assessed from RICM images (D) that are inverted and binarized (E) to extract the area of the contact zone in green (F). To illustrate image processing, the final image of the migration sequence shown in Fig 6C has been used. Scale bar: 10 µm.G, HHistograms of instant projected adherent area for WT (G) and ARHGAP45‐/‐ (H) naive T cells on 2D surface coated with ICAM‐1 and CCL21.
Supplier Page from MilliporeSigma for Anti-ARHGAP45 antibody produced in rabbit