HMHA1 colocalizes with RhoGTPases.

<p>(A-D) Colocalization of myc-tagged HMHA1 with endogenous Rac1 (A), Rac1 Q61L (B), Cdc42 G12V (C) and RhoA V14 (D) was studied by Confocal Laser Scanning Microscopy. Myc-tagged HMHA1 and HMHA-tagged Cdc42 and RhoA were detected by immunostaining in combination with detection of F-Actin. HMHA1 colocalized with endogenous Rac1 (A) and Rac1 Q61L (B) in membrane ruffles (arrows). A partial colocalization of HMHA1 with Cdc42 G12V (C) and RhoA V14 (D) was observed (arrows) although less clear than for Rac1. Higher magnification images of the boxed areas are included. Scale bars, 10 µm.</p

Visualization and flow cytometry analysis of endogenous HMHA1 using ImageStream.

<p>(A) Jurkat T-cells were fixed and immunostained for endogenous HMHA1 and Rac1 and stained for F-actin and DNA. Left panel shows three examples of the distribution of HMHA1, Rac1 and F-actin revealing colocalization of HMHA1 and Rac1 in F-actin rich areas. The nucleus (DNA) and cell morphology (phase image) are included to show the integrity of the cell. Right panel shows intensity distribution of Rac1 (Y-axis) and HMHA1 (X-axis) signals, underscoring the fact that most cells are double positive. (B) Jurkat cells were stimulated for the indicated time-points with 100 ng/ml CXCL12 and analyzed as in A. Two examples of each condition are shown in the left panels. Changes in F-actin distribution in response to CXCL12 can be observed, in particular after 1 and 5 minutes. Right panels show the extent of colocalization (AU, arbitrary units) quantified by the image stream software. Ave, average colocalization, n, number of cells.</p

The HMHA1 GAP domain negatively affects cell spreading.

<p>Cell spreading was measured by Electrical Cell-substrate Impedance Sensing (ECIS) following seeding of 100.000 cells on fibronectin-coated electrodes. Left panel: A significant decrease in electrical resistance, as a measure of cell spreading, was observed in HeLa cells expressing HMHA1 C1-GAPtail (black), C1-GAP (light green), and GAPtail (grey) compared to control cells (red). Ectopic expression of HMHA1 full-length (blue), N-term (dark green), and GAP (magenta) did not affect cell spreading. Right panel: Relative cell spreading at 60 minutes post-seeding. Data are mean values of three independent experiments. Error bars indicate SEM. ns, not significant, ** p<0.01, *** p<0.001.</p

HMHA1 is a RhoGAP <i>in vitro</i>.

<p>(A) Sequence alignment of HMHA1 with the typical RhoGAP, p50RhoGAP, and the structurally-related BAR-GAPs, GRAF1 and OPHN1. Green indicates two matching amino acids. Pink indicates three matching amino acids. Purple indicates four matching amino acids. The arginine finger region is indicated with a black bar. (B) 3D model of the protein-protein complex between RhoA and the HMHA1 RhoGAP domain highlighting the catalytic residues (in sticks, colour coding as indicated; P-loop-Switch I-Switch II of RhoA in light green). The homology model for the GAP domain of human HMHA1 is based on the structure of the human p50RhoGAP domain (PDB ID: 1tx4), using Phyre. The position of the HMHA1 GAP domain in the complex with human RhoA (from RhoA⋅GDP⋅AlFx⋅p50RhoGAP; PDB ID: 1tx4) was obtained through its overlay on the p50RhoGAP domain. The RhoGAP domain of GRAF1 from <i>Gallus gallus</i> (PDB ID: 1f7c) was superimposed onto the model of the HMHA1 GAP domain. (C) HMHA1 C1-GAPtail has <i>in vitro</i> GAP activity towards Rac1, Cdc42, and RhoA but not towards Ras (purple bars). p50RhoGAP was used as a positive control (red bars). GTPases or HMHA1 only were used as a control and as a measure for intrinsic nucleotide hydrolysis (yellow bars). Data are mean values of two independent experiments. Error bars indicate SD. (D) HMHA1 GAP activity is inhibited by the N-terminal BAR domain as full-length HMHA1 has no GAP activity while C1-GAPtail, lacking the N-terminal region, shows GAP activity (purple bars). GTPases or HMHA1 only were used as a control and as a measure for intrinsic hydrolysis (yellow bars). Data are mean values of two independent experiments. Error bars indicate SD.</p

Localization and effects on F-actin of HMHA1 and selected mutants.

<p>Intracellular localization of myc-tagged HMHA1 (and mutant constructs) was studied by Confocal Laser Scanning Microscopy following expression in HeLa cells. Myc-tagged HMHA1 was detected by immunostaining for the myc epitope in combination with detection of F-Actin with phalloidin. Full-length HMHA1 (FL) as well as HMHA1 N-term are partially localized at membrane ruffles as well as in the cytoplasm. For HMHA1 N-term localization at vesiculo-tubular structures is occasionally observed (arrows). Cells expressing FL or the N-term constructs are morphologically similar to control cells and no effects are seen on F-Actin (upper two rows). HMHA1 constructs lacking the C-terminal tail (GAP and C1-GAP) are partly mislocalized into protein aggregates. In cells expressing HMHA1 C1-GAP, C1-GAPtail, and GAPtail (marked with asteriks), F-Actin distribution is altered and cell morphology is dramatically changed. Higher magnification images of the boxed area are included. Scale bars, 10 µm.</p

The HMHA1 GAP domain induces loss of focal adhesions.

<p>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 <i>p</i>-values.</p

HMHA1 regulates RhoGTPase activity <i>in vivo</i>.

<p>(A) HeLa cells were transfected as indicated. After 24 hours, a CRIB pull-down assay was performed to measure levels of Rac1GTP. HMHA1 full-length (blue bar) and N-term (purple bar) do not significantly decrease Rac1GTP loading. The N-terminal BAR domain auto-inhibits GAP function as HMHA1 C1-GAP (green bar) and C1-GAPtail (black bar), which lack the N-terminal BAR domain, show a significant decrease in Rac1GTP loading compared to control cells (red bar). Data are mean values of five independent experiments. Error bars indicate SEM. ns, not significant. * p<0.05, ** p<0.01. (B) HeLa cells were transfected as indicated. After 24 hours, a RhoA G-LISA (Cytoskeleton) was used to measure levels of RhoA-GTP. HMHA1 full-length (red bar) and N-term (purple bar) did not significantly decrease RhoA-GTP loading. In contrast, HMHA1 C1-GAPtail (pink bar), which lacks the N-terminal BAR domain, showed a significant decrease in RhoA-GTP loading compared to control cells (red bar). Data are mean values of three independent experiments. Error bars indicate SEM. ns, not significant, * <0.05, *** p<0.001. (C) Jurkat cells were transduced with control (shC) or HMHA1 (shHMHA1 #2 or #3) short hairpin RNAs. After 72 hours, a CRIB pull-down assay was performed to measure levels of Rac1GTP. Knock-down of HMHA1 significantly increases Rac1GTP loading compared to control cells. Data are mean values of two (for shHMHA1 #3) or three (for shHMHA1 #2) independent experiments. Error bars indicate SD. * p<0.05. (D) Jurkat cells transduced as indicated were lysed and RhoA-GTP levels were measured using a RhoA G-LISA. No significant differences in levels of RhoA-GTP loading were observed between control cells and cells treated with shRNAs against HMHA1. Data are mean values of four measurements of two independent experiments. Error bars indicate SD.</p