Rho GTPase expression compared in progenitor cells and differentiated myeloid cells.

<p>(A) Expression pattern of RhoC and RhoV in the monocyte-lineage. The expression of RhoC and RhoV are depicted as a percentage of total Rho GTPase expression. The expression of RhoC is lowest in CD34⁺ cells, low in monocytes and increases during differentiation of monocytes, while RhoV displays an inverse expression pattern. CD34; CD34⁺ cells, iDCs; immature DCs, mDCs (LPS); LPS-matured DCs, mDCs (PGE2); PGE₂-matured DCs. (B) Rho GTPase expression in CD34⁺ cells, neutrophils and monocytes. The percentage of total Rho GTPase expression is depicted for each cell type in a pie chart. Rho GTPase subfamilies and individual Rho GTPases are color coded (for example Rho subfamily is green and RhoA is dark green). (C) The 2^−ΔΔCt values of the Rho GTPases in CD34⁺ cells, neutrophils and monocytes. The 2^−ΔΔCt values of the individual data points for each cell type are depicted. Donormix 1 and 2 are derived from 9 and 3 donors, resp. Donormix CD14 is derived from the same donors as donormix 2, but monocytes were obtained by elutriation followed by CD14 MACS isolation.</p

Primer sequences.

<p>Accessions are derived from <a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>.</p

General expression pattern of Rho GTPases in myeloid cells.

<p>The mRNA expression of the Rho GTPases is depicted as the average 2^−ΔΔCt value per Rho GTPases for the individual cell types and averaged for myeloid cells (upper panel) and for the control cell types, i.e. PBLs, HeLa and neuroblastoma cells (second panel). CD34; CD34⁺ cells, iDCs; immature DCs, mDCs (LPS); LPS-matured DCs, mDCs (PGE2); PGE₂-matured DCs. Expression of the Rho GTPases in each cell type was determined in 2 or 3 different donors or donormixes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042563#s4" target="_blank">Materials and Methods</a>). The colors mark the expression level. The lower panel shows a ranking of the Rho GTPase expression in myeloid cells and in the control cell types.</p

Morphology of differentiated myeloid cells on fibronectin.

<p>The cells are seeded on fibronectin-coated coverslips and stained for vinculin (green, second column). Phalloidin Texas Red (red, first column) is used to detect F-actin and Hoechst33258 is used to visualize nuclei (blue, third column). Images were obtained by confocal microscopy using a Zeiss LSM 510-meta microscope with a Plan-Apochromatic 63× 1.4 NA oil immersion objective (Carl Zeiss, Jena, Germany) and analyzed using Zen software (Carl Zeiss). The third column shows the merged image and the fourth column shows a zoom of a part of the merge image. Podosomes can be seen as actin dots surrounded by vinculin rings. In the osteoclasts podosome rings (arrows) can be observed. iDCs; immature DCs, mDCs (LPS); LPS-matured DCs, mDCs (PGE2); PGE₂-matured DCs. Representative images are shown. Scale bar; 20 µm.</p

Overview of most abundant Rho GTPases per cell type.

<p>The different cells investigated in this study are depicted with the three most highly expressed Rho GTPases next to them. CD34+; CD34⁺ cell, MΦ (GM-CSF); macrophage differentiated with GM-CSF, MΦ (M-CSF); macrophage differentiated with M-CSF, iDC; immature DC, osteo (mono); osteoclast differentiated from monocyte, mDC (LPS); LPS-matured DC, mDC (PGE2); PGE₂-matured DC, osteo (DC); osteoclast differentiated from DC. An arrow indicates that a cell type is differentiated from the cell type at the start of the arrow. Two arrows between CD34⁺ and monocyte or neutrophil indicate that the monocytes and neutrophils are not directly differentiated from CD34⁺ cells, but that there are intermediate cells in between.</p

The family of Rho GTPases.

<p>Rho GTPases can be classified as classical or atypical. Classical Rho GTPases cycle between the GDP- and GTP-bound form, while atypical Rho GTPases are almost always in the active form. GeneIDs were derived from <a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>.</p

Rho GTPase expression in macrophages and osteoclasts.

<p>(A) Rho GTPase expression in macrophages generated with M-CSF or GM-CSF. The percentage of total Rho GTPase expression is depicted for each macrophage type in a pie chart. Rho GTPase subfamilies and individual Rho GTPases are color coded. (B) Rho GTPase expression in osteoclasts generated from monocytes or DCs. The percentage of total Rho GTPase expression is depicted for each osteoclast type in a pie diagram. Rho GTPase subfamilies and individual Rho GTPases are color coded. (C) The 2^−ΔΔCt values of the Rho GTPases in macrophages and osteoclasts. The 2^−ΔΔCt values of the individual data points for each cell type are depicted. Donormix 1 and 2 are derived from 9 and 3 donors, resp. Donormix CD14 is derived from the same donors as donormix 2, but monocytes were obtained by elutriation followed by CD14 MACS isolation and differentiated to macrophages.</p

Rho GTPase expression during DC maturation.

<p>(A) Rho GTPase expression in DCs during maturation with LPS or PGE₂. The percentage of total Rho GTPase expression is depicted for each DC in a pie chart. Rho GTPase subfamilies and individual Rho GTPases are color coded. iDCs; immature DCs, mDCs (LPS); LPS-matured DCs, mDCs (PGE2); PGE₂-matured DCs. (B) The 2^−ΔΔCt values of the Rho GTPases in immature and mature DCs. The 2^−ΔΔCt values of the individual data points for each cell type are depicted. Donormix 1 and 2 are derived from 9 and 3 donors, resp. Donormix CD14 is derived from the same donors as donormix 2, but monocytes were obtained by elutriation followed by CD14 MACS isolation and differentiated to DCs. iDCs; immature DCs, mDCs (LPS); LPS-matured DCs, mDCs (PGE2); PGE₂-matured DCs.</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

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