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. 2010 Jul;299(1):C21-32.
doi: 10.1152/ajpcell.00543.2009. Epub 2010 Mar 24.

Pak1 regulates branching morphogenesis in 3D MDCK cell culture by a PIX and beta1-integrin-dependent mechanism

Michael P Hunter  1 , Mirjam M Zegers
Affiliations

Pak1 regulates branching morphogenesis in 3D MDCK cell culture by a PIX and beta1-integrin-dependent mechanism

Michael P Hunter et al. Am J Physiol Cell Physiol. 2010 Jul.

Abstract

Branching morphogenesis is a fundamental process in the development of the kidney. This process gives rise to a network of ducts, which form the collecting system. Defective branching can lead to a multitude of kidney disorders including agenesis and reduced nephron number. The formation of branching tubules involves changes in cell shape, cell motility, and reorganization of the cytoskeleton. However, the exact intracellular mechanisms involved are far from understood. We have used the three-dimensional (3D) Madin-Darby canine kidney (MDCK) cell culture system to study how p21-activated kinase 1 (Pak1), which is an important regulator of the cytoskeleton, modulates branching. Our data reveal that Pak1 plays a crucial role in regulating branching morphogenesis. Expression of a dominant-negative Pak1 mutant (DN-Pak1) in MDCK cysts resulted in the spontaneous formation of extensions and branching tubules. Cellular contractility and levels of phosphorylated myosin light chain (pMLC) were increased in DN-Pak1 cells in collagen. Expression of a DN-Pak1 mutant that does not bind to PIX (DN-Pak1-DeltaPIX) failed to form extensions in collagen and did not have increased contractility. This shows that the DN-Pak1 mutant requires PIX binding to generate extensions and increased contractility in 3D culture. Furthermore, a beta1-integrin function-blocking antibody (AIIB2) inhibited the formation of branches and blocked the increased contractility in DN-Pak1 cysts. Taken together, our work shows that DN-Pak1-induced branching morphogenesis requires PIX binding and beta1-integrin signaling.

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Figures

Fig. 1.
Fig. 1.
Expression of DN-Pak1 in Madin-Darby canine kidney (MDCK) cells in three-dimensional (3D) culture results in a spontaneous branching phenotype. A: in the presence of doxycycline (+Dox), dominant-negative Pak1 mutant (DN-Pak1) clones 5 and 3 form cysts with a normal spherical morphology and central lumen (left) at 8 days in culture. In the absence of doxycycline (−Dox), clones 5 and 3 form cysts with many narrow (arrowhead) and broad (arrow) branches. B: expression of the hemagglutin (HA)-tagged DN-Pak1 mutant protein in cysts was verified by Western blot. The HA-tagged DN-Pak1 protein was expressed at high levels only in the −Dox treated cysts in both clone 5 and 3. GAPDH was used as a loading control. C: quantitation of morphology of cysts. n = 3. Error bars represent SD. D: DN-Pak1 (−Dox) branching cyst at day 7 (top), arrow marks a single branch arising from this cyst. Bottom: branch has retracted in this cyst after the addition of Dox from day 7 to day 10. E: analysis of DN-Pak1 (−Dox) cyst morphology from days 7 to 10 in culture. Five representative branching cysts are shown. Arrowheads mark branches that broaden and/or elongate from day 7 to day 8. Arrows mark the formation of new branches at day 9. The line in images for cyst 4 marks a grid line at the bottom of the dish. Scale bars in A, D, and E are 30 μm.
Fig. 2.
Fig. 2.
DN-Pak1 does not induce loss of apical-basal polarity or junctional integrity. A: control cyst showing podocalyxin (green) is localized to the apical surface (arrowhead). B: DN-Pak1 (−Dox) cyst showing localization of podocalyxin to the apical surface (arrowhead). Arrow shows that cells initiating branching maintain contact with the lumen. Inset in B shows a branch in a DN-Pak1 cyst that forms a tubule and contains a lumen (arrow), which is continuous with the cyst lumen. C and D: podocalyxin staining (green) in two additional examples of DN-Pak1 cysts. Arrows show that cells that initiate branching maintain contact with the lumen. E: low magnification of control cyst stained for E-cadherin (green). F: high magnification of cross section of control cyst wall showing E-cadherin (green) is localized to the basal membrane (double arrowhead) and lateral membrane (arrow). Podocalyxin is shown in red (arrowhead) at the apical surface. G: low magnification of DN-Pak1 cyst (partially through the cyst lumen) stained for E-cadherin (green). H: high magnification of DN-Pak1 cyst and cells initiating branching with E-cadherin (green) at the basal (double arrowhead) and lateral (arrow) membranes, and podocalyxin (red) at the apical membrane (arrowhead). Scale bars are 20 μm in E and G, and 5 μm in F and H. I: control cyst showing localization of ZO-1 (green) to the tight junctions near the apical surface (arrowhead), and β-catenin (red) to the lateral membrane (arrow). J–L: localization of β-catenin (red) and ZO-1 (green) to the basal membrane (arrow) and tight junctions (arrowhead) in DN-Pak1 nonbranching (J) and branching cells (K and L). β-Catenin and ZO-1 colocalization is shown in yellow. Scale bars are 5 μm. M and N: laminin (green) is deposited at the basal surface (arrows) of both control (M) and DN-Pak1 (N) cysts. Insets show projection images of serial confocal sections of the basal surface of cysts. Scale bars are 20 μm. O and P: F-actin staining (green) strongly labels the apical microvilli in control and DN-Pak1 cysts (arrows). Q: staining for F-actin in a DN-Pak1 cyst extension showing F-actin-rich stress fibers (arrowhead). Scale bar is 10 μm. Nuclei in A–Q were stained with DAPI in blue.
Fig. 3.
Fig. 3.
Expression of DN-Pak1 in cysts results in increased phosphorylated myosin light chain (pMLC) levels. A: Western blot showing levels of pMLC in control (+Dox) and DN-Pak1 (−Dox) cysts. This anti-pMLC antibody recognizes a doublet in MDCK cells (59), which probably represents multiple isoforms of pMLC. GAPDH is used as a loading control. B: quantitation of pMLC levels in control and DN-Pak1 cysts. pMLC levels were normalized to GAPDH. n = 4. Error bar represents SD. P < 0.05.
Fig. 4.
Fig. 4.
DN-Pak1 cells have increased contractility in collagen gel. A: images of floating collagen I gels containing control and DN-Pak1 cells treated with DMSO (vehicle) or blebbistatin (50 μM) at 0, 12, and 16 h of incubation. Scale bar is 1 mm. B: measurements of gel diameters (in mm) for collagen gels at 0, 12, and 16 h. n = 3. Error bars represent SD. *P < 0.05. C: Western blot analysis for HA-tagged DN-PAK1 in control and DN-Pak1 expressing cells in collagen gels at 16 h. GAPDH was used as a loading control.
Fig. 5.
Fig. 5.
Cells expressing a DN-Pak1 protein that cannot bind PIX (DN-Pak1-ΔPIX) do not form branching cysts or have increased contractility in collagen. A: control (+Dox) and DN-Pak1-ΔPIX expressing (−Dox) cysts both have a normal spherical morphology. Scale bar is 30 μm. B: quantitation of control and DN-Pak1-ΔPIX cyst morphology. Data are represented as the mean of four independent experiments. Error bars represent SD. C: images of control and DN-Pak1-ΔPIX cysts at 0 and 16 h of incubation. Scale bar is 1 mm. D: measurement of gel diameters (in mm) at 0 and 16 h of incubation. n = 3. Error bars represent SD. E: Western blot analysis was used to confirm the expression of the myc-tagged DN-Pak1-ΔPIX protein in DN-Pak1-ΔPIX cells in collagen. GAPDH was used as a loading control.
Fig. 6.
Fig. 6.
DN-PAK1 mutant protein requires β1-integrin signaling to generate branches and increase cellular contractility in 3D culture. A: control and DN-Pak1 cysts in BME. Scale bar is 30 μm. B: control and DN-Pak1 control cysts (no AIIB2) or cysts treated with AIIB2 from days 5 to 8 in culture. Scale bar is 30 μm. C: quantitation of branching cysts in control and DN-Pak1 no AIIB2-treated and AIIB2-treated cysts. n = 3. Error bars represent SD. *P < 0.05. D: Western blot for the HA-tagged DN-Pak1 protein in control (no AIIB2) and AIIB2-treated cysts. GAPDH was used as a loading control. E: images of collagen gels containing control and DN-Pak1 cells no AIIB2-treated or AIIB2-treated for 0 and 16 h of incubation. Scale bar is 1 mm. F: measurement of gel diameters (in mm) at 0 and 16 h. n = 3. Error bars represent SD. *P < 0.05. G: Western blot for HA-tagged DN-Pak1 protein in control and DN-Pak1 cells in collagen gels at 16 h incubation. GAPDH was used as a loading control.
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