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Pak1 regulates the orientation of apical polarization and lumen formation by distinct pathways.

deLeon O, Puglise JM, Liu F, Smits J, ter Beest MB, Zegers MM - PLoS ONE (2012)

Bottom Line: Here, we investigated whether the Rac1 effector Pak1 is a downstream effector in this pathway.Expression of constitutive active Pak1 phenocopies the effect of β1 integrin inhibition in that it misorients the apical surface and induces a multilumen phenotype.Therefore, Pak1 likely regulates apical polarization and lumen formation by two distinct pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, University of Chicago, Chicago, Illinois, United States of America.

ABSTRACT
The development of the basic architecture of branching tubules enclosing a central lumen that characterizes most epithelial organs crucially depends on the apico-basolateral polarization of epithelial cells. Signals from the extracellular matrix control the orientation of the apical surface, so that it faces the lumen interior, opposite to cell-matrix adhesion sites. This orientation of the apical surface is thought to be intrinsically linked to the formation of single lumens. We previously demonstrated in three-dimensional cyst cultures of Madin-Darby canine kidney (MDCK) cells that signaling by β1 integrins regulates the orientation of the apical surface, via a mechanism that depends on the activity of the small GTPase Rac1. Here, we investigated whether the Rac1 effector Pak1 is a downstream effector in this pathway. Expression of constitutive active Pak1 phenocopies the effect of β1 integrin inhibition in that it misorients the apical surface and induces a multilumen phenotype. The misorientation of apical surfaces depends on the interaction of active Pak1 with PIX proteins and is linked to defects in basement membrane assembly. In contrast, the multilumen phenotype was independent of PIX and the basement membrane. Therefore, Pak1 likely regulates apical polarization and lumen formation by two distinct pathways.

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CA-Pak1 inhibits laminin deposition.A–D: Controls (+ dox, A,C) and cells expressing CA-Pak1 (B) or CA-Pak1ΔPIX (D) were grown on Transwell filters for 6 days. Cells were fixed and stained for laminin underneath the basal surface. E–H: Controls (+ dox, E,G) and cells expressing CA-Pak1 (F) or CA-Pak1ΔPIX (H) were grown in 3D collagen I for 5 days. Cells were fixed and stained for laminin (green). Nuclei, stained with DAPI, are blue. Scale bars in A–H are 10 µm. I, Western blot of total laminin levels of 2D lysates from control (+dox) and CA-Pak1 expressing cells (-dox). Myc shows expressing levels of CA-Pak1, β-tubulin is loading control. J, Quantification of total laminin in two different Pak-L107F clones. Data represent mean ± SEM. n = 3 for #7 and n = 5 for #83. **p<0.02, *p<0.05.
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pone-0041039-g007: CA-Pak1 inhibits laminin deposition.A–D: Controls (+ dox, A,C) and cells expressing CA-Pak1 (B) or CA-Pak1ΔPIX (D) were grown on Transwell filters for 6 days. Cells were fixed and stained for laminin underneath the basal surface. E–H: Controls (+ dox, E,G) and cells expressing CA-Pak1 (F) or CA-Pak1ΔPIX (H) were grown in 3D collagen I for 5 days. Cells were fixed and stained for laminin (green). Nuclei, stained with DAPI, are blue. Scale bars in A–H are 10 µm. I, Western blot of total laminin levels of 2D lysates from control (+dox) and CA-Pak1 expressing cells (-dox). Myc shows expressing levels of CA-Pak1, β-tubulin is loading control. J, Quantification of total laminin in two different Pak-L107F clones. Data represent mean ± SEM. n = 3 for #7 and n = 5 for #83. **p<0.02, *p<0.05.

Mentions: Cell-matrix interactions are sufficient to orient the apical pole of epithelial cells in 2D culture [1]. The deposition and organization of heterotrimeric laminin molecules into a basement membrane-like structure is required for proper orientation of the apical domain and lumen formation in embryoid bodies [7] and in 3D cultures of MDCK and mammary epithelial cells [8], [15], [40]. Cell-matrix adhesion receptors regulate laminin organization [41], and a loss of focal adhesions may thus inhibit this process. To detect extracellular deposited laminin we stained non-permeabilized CA-Pak1-expressing cells grown on Transwell filters or in 3D culture for laminin. For this, we used polyclonal antibodies against laminin-111, which stain the β1 and γ1 chains of laminin-511 in MDCK cells [8], [15]. Co-staining cells for the intracellular protein p120catenin did not yield any signal, thus showing that only extracellular proteins were detected with our fixation and staining procedure. CA-Pak1, but not CA-Pak1ΔPIX, caused a dramatic decrease in deposited laminin in both 2D (Figure 7A–D) and 3D (Figure 7E–H) culture. This was at least partially due to decreased synthesis or stabilization, as Western blot analysis of two different CA-Pak1-expressing clones showed a modest but consistent reduction in laminin levels in 6-day old cultures (Figure 7I,J).


Pak1 regulates the orientation of apical polarization and lumen formation by distinct pathways.

deLeon O, Puglise JM, Liu F, Smits J, ter Beest MB, Zegers MM - PLoS ONE (2012)

CA-Pak1 inhibits laminin deposition.A–D: Controls (+ dox, A,C) and cells expressing CA-Pak1 (B) or CA-Pak1ΔPIX (D) were grown on Transwell filters for 6 days. Cells were fixed and stained for laminin underneath the basal surface. E–H: Controls (+ dox, E,G) and cells expressing CA-Pak1 (F) or CA-Pak1ΔPIX (H) were grown in 3D collagen I for 5 days. Cells were fixed and stained for laminin (green). Nuclei, stained with DAPI, are blue. Scale bars in A–H are 10 µm. I, Western blot of total laminin levels of 2D lysates from control (+dox) and CA-Pak1 expressing cells (-dox). Myc shows expressing levels of CA-Pak1, β-tubulin is loading control. J, Quantification of total laminin in two different Pak-L107F clones. Data represent mean ± SEM. n = 3 for #7 and n = 5 for #83. **p<0.02, *p<0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3399788&req=5

pone-0041039-g007: CA-Pak1 inhibits laminin deposition.A–D: Controls (+ dox, A,C) and cells expressing CA-Pak1 (B) or CA-Pak1ΔPIX (D) were grown on Transwell filters for 6 days. Cells were fixed and stained for laminin underneath the basal surface. E–H: Controls (+ dox, E,G) and cells expressing CA-Pak1 (F) or CA-Pak1ΔPIX (H) were grown in 3D collagen I for 5 days. Cells were fixed and stained for laminin (green). Nuclei, stained with DAPI, are blue. Scale bars in A–H are 10 µm. I, Western blot of total laminin levels of 2D lysates from control (+dox) and CA-Pak1 expressing cells (-dox). Myc shows expressing levels of CA-Pak1, β-tubulin is loading control. J, Quantification of total laminin in two different Pak-L107F clones. Data represent mean ± SEM. n = 3 for #7 and n = 5 for #83. **p<0.02, *p<0.05.
Mentions: Cell-matrix interactions are sufficient to orient the apical pole of epithelial cells in 2D culture [1]. The deposition and organization of heterotrimeric laminin molecules into a basement membrane-like structure is required for proper orientation of the apical domain and lumen formation in embryoid bodies [7] and in 3D cultures of MDCK and mammary epithelial cells [8], [15], [40]. Cell-matrix adhesion receptors regulate laminin organization [41], and a loss of focal adhesions may thus inhibit this process. To detect extracellular deposited laminin we stained non-permeabilized CA-Pak1-expressing cells grown on Transwell filters or in 3D culture for laminin. For this, we used polyclonal antibodies against laminin-111, which stain the β1 and γ1 chains of laminin-511 in MDCK cells [8], [15]. Co-staining cells for the intracellular protein p120catenin did not yield any signal, thus showing that only extracellular proteins were detected with our fixation and staining procedure. CA-Pak1, but not CA-Pak1ΔPIX, caused a dramatic decrease in deposited laminin in both 2D (Figure 7A–D) and 3D (Figure 7E–H) culture. This was at least partially due to decreased synthesis or stabilization, as Western blot analysis of two different CA-Pak1-expressing clones showed a modest but consistent reduction in laminin levels in 6-day old cultures (Figure 7I,J).

Bottom Line: Here, we investigated whether the Rac1 effector Pak1 is a downstream effector in this pathway.Expression of constitutive active Pak1 phenocopies the effect of β1 integrin inhibition in that it misorients the apical surface and induces a multilumen phenotype.Therefore, Pak1 likely regulates apical polarization and lumen formation by two distinct pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, University of Chicago, Chicago, Illinois, United States of America.

ABSTRACT
The development of the basic architecture of branching tubules enclosing a central lumen that characterizes most epithelial organs crucially depends on the apico-basolateral polarization of epithelial cells. Signals from the extracellular matrix control the orientation of the apical surface, so that it faces the lumen interior, opposite to cell-matrix adhesion sites. This orientation of the apical surface is thought to be intrinsically linked to the formation of single lumens. We previously demonstrated in three-dimensional cyst cultures of Madin-Darby canine kidney (MDCK) cells that signaling by β1 integrins regulates the orientation of the apical surface, via a mechanism that depends on the activity of the small GTPase Rac1. Here, we investigated whether the Rac1 effector Pak1 is a downstream effector in this pathway. Expression of constitutive active Pak1 phenocopies the effect of β1 integrin inhibition in that it misorients the apical surface and induces a multilumen phenotype. The misorientation of apical surfaces depends on the interaction of active Pak1 with PIX proteins and is linked to defects in basement membrane assembly. In contrast, the multilumen phenotype was independent of PIX and the basement membrane. Therefore, Pak1 likely regulates apical polarization and lumen formation by two distinct pathways.

Show MeSH
Related in: MedlinePlus