Geometry sensing by dendritic cells dictates spatial organization and PGE2-induced dissolution of podosomes

Assembly and disassembly of adhesion structures such as focal adhesions (FAs) and podosomes regulate cell adhesion and differentiation. On antigen-presenting dendritic cells (DCs), acquisition of a migratory and immunostimulatory phenotype depends on podosome dissolution by prostaglandin E2 (PGE2). Whereas the effects of physico-chemical and topographical cues have been extensively studied on FAs, little is known about how podosomes respond to these signals. Here, we show that, unlike for FAs, podosome formation is not controlled by substrate physico-chemical properties. We demonstrate that cell adhesion is the only prerequisite for podosome formation and that substrate availability dictates podosome density. Interestingly, we show that DCs sense 3-dimensional (3-D) geometry by aligning podosomes along the edges of 3-D micropatterned surfaces. Finally, whereas on a 2-dimensional (2-D) surface PGE2 causes a rapid increase in activated RhoA levels leading to fast podosome dissolution, 3-D geometric cues prevent PGE2-mediated RhoA activation resulting in impaired podosome dissolution even after prolonged stimulation. Our findings indicate that 2-D and 3-D geometric cues control the spatial organization of podosomes. More importantly, our studies demonstrate the importance of substrate dimensionality in regulating podosome dissolution and suggest that substrate dimensionality plays an important role in controlling DC activation, a key process in initiating immune responses

Podosome regulation by Rho GTPases in myeloid cells

Myeloid cells form a first tine of defense against infections. They migrate from the circulation to the infected tissues by adhering to and subsequently crossing the vascular wall. This process requires precise control and proper regulation of these interactions with the environment is therefore crucial. Podosomes are the most prominent adhesion structures in myeloid cells. Podosomes control both the adhesive and migratory properties of myeloid cells and the regulation of podosomes is key to the proper functioning of these cells. Here we discuss the regulation of podosomes by Rho GTPases, well known regulators of adhesion and migration, focusing on myeloid cells. In addition, the regulation of podosomes by GTPase regulators such as GEFs and GAPs, as well as the effects of some Rho GTPase effector pathways, will be discussed. (C) 2010 Elsevier GmbH. All rights reserve

Rho GTPase Expression in Human Myeloid Cells

Myeloid cells are critical for innate immunity and the initiation of adaptive immunity. Strict regulation of the adhesive and migratory behavior is essential for proper functioning of these cells. Rho GTPases are important regulators of adhesion and migration; however, it is unknown which Rho GTPases are expressed in different myeloid cells. Here, we use a qPCR-based approach to investigate Rho GTPase expression in myeloid cells. We found that the mRNAs encoding Cdc42, RhoQ, Rac1, Rac2, RhoA and RhoC are the most abundant. In addition, RhoG, RhoB, RhoF and RhoV are expressed at low levels or only in specific cell types. More differentiated cells along the monocyte-lineage display lower levels of Cdc42 and RhoV, while RhoC mRNA is more abundant. In addition, the Rho GTPase expression profile changes during dendritic cell maturation with Rac1 being upregulated and Rac2 downregulated. Finally, GM-CSF stimulation, during macrophage and osteoclast differentiation, leads to high expression of Rac2, while M-CSF induces high levels of RhoA, showing that these cytokines induce a distinct pattern. Our data uncover cell type specific modulation of the Rho GTPase expression profile in hematopoietic stem cells and in more differentiated cells of the myeloid lineag

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

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

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