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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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β1 integrin localization, synthesis and transport in Pak1-L107F expressing cells.A–B’: Control (A) or CA-Pak1-expressing cells (B,B’) were grown in 3D collagen for 4 days and stained for F-actin (red) and β1-integrins (green). Scale bar is 10 µm. C: Control (+dox) and CA-Pak1-expressing cells (-dox) were grown on Transwell filters for 6 days and extracellular proteins were removed by mild trypsinisation. Western blots show untreated, total (PBS) and intracellular (trypsin) levels of β1 integrin as determined by a trypsin protection assay. D: Control (+dox) and CA-Pak1-expressing cells (-dox) were grown on Transwell filters for 6 days. Levels of apical (Ap) and basolateral (Bl) β1 integrin were determined by cell surface biotinylation, followed by Western blotting as shown in top panel (IP). Arrowhead shows mature β1 integrin. Levels of β1 integrin in total lysates (TL) are shown in bottom panel.
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pone-0041039-g002: β1 integrin localization, synthesis and transport in Pak1-L107F expressing cells.A–B’: Control (A) or CA-Pak1-expressing cells (B,B’) were grown in 3D collagen for 4 days and stained for F-actin (red) and β1-integrins (green). Scale bar is 10 µm. C: Control (+dox) and CA-Pak1-expressing cells (-dox) were grown on Transwell filters for 6 days and extracellular proteins were removed by mild trypsinisation. Western blots show untreated, total (PBS) and intracellular (trypsin) levels of β1 integrin as determined by a trypsin protection assay. D: Control (+dox) and CA-Pak1-expressing cells (-dox) were grown on Transwell filters for 6 days. Levels of apical (Ap) and basolateral (Bl) β1 integrin were determined by cell surface biotinylation, followed by Western blotting as shown in top panel (IP). Arrowhead shows mature β1 integrin. Levels of β1 integrin in total lysates (TL) are shown in bottom panel.

Mentions: Since the phenotypes of spheroids expressing CA-Pak1 resembled that of spheroids in which β1 integrins were inhibited, we next tested whether CA-Pak1 affected the localization of β1 integrins. Control and CA-Pak1 expressing spheroids exhibited prominent staining of β1 integrins at the basal surface (Figure 2A,B,B′; β1 integrin in green), but the protruding (apical) surfaces in the CA-Pak1 spheroids were mostly devoid of β1 integrins. To investigate if the β1 integrins were correctly trafficked to the plasma membrane, we analyzed the levels of intracellular β1 integrins in filter-grown cells, using mild trypsinization to digest extracellular β1 integrins. This treatment protected intracellular proteins from digestion, as shown by unaltered levels of β-tubulin, but removed all detectable β1 integrins in both control and induced cells. This confirms that most β1 integrins were located at the plasma membrane under both conditions (Figure 2C). MDCK cells were reported to express a small pool of β1 integrins at their apical surface at levels that are too low to be detected by immunofluorescence [11]. To test if CA-Pak1 increased this pool by missorting β1 integrins, we biotinylated proteins at either the apical and basolateral surface, and analyzed apical and basolateral integrins as described in the Methods. Consistent with a previously report [11], we detected an apical pool of β1 integrins, but this pool was not affected by expression of CA-Pak1 (Figure 2D). Finally, Western blot analysis showed that the total protein expression of α3 (data not shown) and β1 integrins (Figure 2C,D) was not altered upon expression of CA-Pak1. Taken together, our data indicate that the CA-Pak1-induced phenotype is not due to a gross defect in transport of β1 integrins to the plasma membrane.


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)

β1 integrin localization, synthesis and transport in Pak1-L107F expressing cells.A–B’: Control (A) or CA-Pak1-expressing cells (B,B’) were grown in 3D collagen for 4 days and stained for F-actin (red) and β1-integrins (green). Scale bar is 10 µm. C: Control (+dox) and CA-Pak1-expressing cells (-dox) were grown on Transwell filters for 6 days and extracellular proteins were removed by mild trypsinisation. Western blots show untreated, total (PBS) and intracellular (trypsin) levels of β1 integrin as determined by a trypsin protection assay. D: Control (+dox) and CA-Pak1-expressing cells (-dox) were grown on Transwell filters for 6 days. Levels of apical (Ap) and basolateral (Bl) β1 integrin were determined by cell surface biotinylation, followed by Western blotting as shown in top panel (IP). Arrowhead shows mature β1 integrin. Levels of β1 integrin in total lysates (TL) are shown in bottom panel.
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Related In: Results  -  Collection

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pone-0041039-g002: β1 integrin localization, synthesis and transport in Pak1-L107F expressing cells.A–B’: Control (A) or CA-Pak1-expressing cells (B,B’) were grown in 3D collagen for 4 days and stained for F-actin (red) and β1-integrins (green). Scale bar is 10 µm. C: Control (+dox) and CA-Pak1-expressing cells (-dox) were grown on Transwell filters for 6 days and extracellular proteins were removed by mild trypsinisation. Western blots show untreated, total (PBS) and intracellular (trypsin) levels of β1 integrin as determined by a trypsin protection assay. D: Control (+dox) and CA-Pak1-expressing cells (-dox) were grown on Transwell filters for 6 days. Levels of apical (Ap) and basolateral (Bl) β1 integrin were determined by cell surface biotinylation, followed by Western blotting as shown in top panel (IP). Arrowhead shows mature β1 integrin. Levels of β1 integrin in total lysates (TL) are shown in bottom panel.
Mentions: Since the phenotypes of spheroids expressing CA-Pak1 resembled that of spheroids in which β1 integrins were inhibited, we next tested whether CA-Pak1 affected the localization of β1 integrins. Control and CA-Pak1 expressing spheroids exhibited prominent staining of β1 integrins at the basal surface (Figure 2A,B,B′; β1 integrin in green), but the protruding (apical) surfaces in the CA-Pak1 spheroids were mostly devoid of β1 integrins. To investigate if the β1 integrins were correctly trafficked to the plasma membrane, we analyzed the levels of intracellular β1 integrins in filter-grown cells, using mild trypsinization to digest extracellular β1 integrins. This treatment protected intracellular proteins from digestion, as shown by unaltered levels of β-tubulin, but removed all detectable β1 integrins in both control and induced cells. This confirms that most β1 integrins were located at the plasma membrane under both conditions (Figure 2C). MDCK cells were reported to express a small pool of β1 integrins at their apical surface at levels that are too low to be detected by immunofluorescence [11]. To test if CA-Pak1 increased this pool by missorting β1 integrins, we biotinylated proteins at either the apical and basolateral surface, and analyzed apical and basolateral integrins as described in the Methods. Consistent with a previously report [11], we detected an apical pool of β1 integrins, but this pool was not affected by expression of CA-Pak1 (Figure 2D). Finally, Western blot analysis showed that the total protein expression of α3 (data not shown) and β1 integrins (Figure 2C,D) was not altered upon expression of CA-Pak1. Taken together, our data indicate that the CA-Pak1-induced phenotype is not due to a gross defect in transport of β1 integrins to the plasma membrane.

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