p114RhoGEF governs cell motility and lumen formation during tubulogenesis through a ROCK-myosin-II pathway

Abstract

Tubulogenesis is fundamental to the development of many epithelial organs. Although lumen formation in cysts has received considerable attention, less is known about lumenogenesis in tubes. Here, we utilized tubulogenesis induced by hepatocyte growth factor (HGF) in MDCK cells, which form tubes enclosing a single lumen. We report the mechanism that controls tubular lumenogenesis and limits each tube to a single lumen. Knockdown of p114RhoGEF (also known as ARHGEF18), a guanine nucleotide exchange factor for RhoA, did not perturb the early stages of tubulogenesis induced by HGF. However, this knockdown impaired later stages of tubulogenesis, resulting in multiple lumens in a tube. Inhibition of Rho kinase (ROCK) or myosin IIA, which are downstream of RhoA, led to formation of multiple lumens. We studied lumen formation by live-cell imaging, which revealed that inhibition of this pathway blocked cell movement, suggesting that cell movement is necessary for consolidating multiple lumens into a single lumen. Lumen formation in tubules is mechanistically quite different from lumenogenesis in cysts. Thus, we demonstrate a new pathway that regulates directed cell migration and formation of a single lumen during epithelial tube morphogenesis.

Keywords: Epithelia; Hepatocyte growth factor; Madin–Darby canine kidney cells; Migration; Tube.

Fig. 1.

A multicellular cystic structure undergoes dynamic remodeling in the presence of HGF, and ROCK regulates tubular lumen formation in a 3D matrix. (A) Selected stills of a phase-contrast movie of HGF-induced tubulogenesis for 72 h. Outlines of the apical region, marked by a dashed line, indicate lumen opening. The arrow indicates a small lumen. Insets show higher magnification. Scale bar: 10 μm. (B) MDCK cysts were pre-treated with HGF for 24 h and stimulated with solvent control (DMSO) or Y-27632. Representative images of F-actin (red) and nuclei (blue) are shown. Arrows indicate multiple lumens and the dashed line outlines the lumen. Scale bars: 10 μm. (C) Quantification of tubules with multiple lumens in DMSO or Y-27632 treated cysts. Results are mean±s.d. (n=3). *P<0.05 (Student's t-test).

Fig. 2.

ROCK regulates oriented cell motility in a 3D matrix. (A) Selected stills of time-lapse sequences of a control (left panels; Movie 1) and Y-27632-treated 3D structures expressing an apical membrane marker, GFP fused to podocalyxin (GFP–PODXL), and a nuclear marker, monomeric RFP fused to histone 2B (mRFP–H2B, right panels; Movie 2). Time is indicated in min:seconds. Scale bars: 10 μm. Arrows indicate multiple lumens and the dashed line outlines the lumen. (B) Migration tracks of cells are displayed as displacement plots. For each group, the trajectories of 15 to 30 cells at 30 min intervals over 45 h are presented. The origin of each track was superimposed at position (0, 0).

Fig. 3.

Depletion of ROCK1 impairs tubular lumen formation. (A) Representative images of HGF-induced tubules stably expressing control (Scramble) or ROCK1 (ROCKI KD) shRNA. Tubular structures were stained for F-actin (green), E-cadherin (white) and nuclei (blue). Arrows indicate multiple lumens and the dashed line outlines the lumen. Scale bar: 10 μm. (B) Quantification of tubules with multiple lumens in control or ROCK1-depleted cysts. Results are mean±s.d. (n=3). *P<0.01 (Student's t-test). (C) Selected stills of movies of tubulogenesis in control (Movie 4) or ROCK1-depleted cysts (Movie 5). Arrows indicate multiple lumens and the dashed line outlines the lumen. Scale bars: 10 μm. (D) Migration tracks of cells are displayed as displacement plots. For each group, the trajectories of 15 to 30 cells at 30 min intervals over 24 h are presented.

Fig. 4.

ROCK inhibition has no effect on apico-basal polarity. (A) Representative images of ZO-1 (green), E-cadherin (red) and F-actin (white) in DMSO-, Y-27632- or blebbistatin-treated tubules. Arrows indicate multiple lumens and arrowheads indicate tight junctions. Scale bars: 10 μm.

Fig. 5.

ROCK inhibition attenuates the localization of myosin IIA at the basal surface of tubule. (A) Myosin IIA [green, shown separately in top panels; merged with phalloidin (white) in bottom panels] at the periphery of tubular structure is substaintally reduced upon Y-27632 treatment. Arrows indicate multiple lumens. Scale bars: 10 μm. (B) Myosin IIA (green) and phalloidin (red) fluorescence intensity ratios were quantified along the cross-section indicated in red in A (right panels). All measurements were carried out on confocal images and the line-scan analysis for pixel intensity was performed using a Zeiss LSM510 microscope. (C) Representative images of myosin IIA (left) and merged with E-cadherin (red), and nuclei (blue) (right) in E17.5 mouse kidney. Scale bars: 10 μm.

Fig. 6.

Inhibition of myosin IIA induces multiple lumens in tubes. (A) Representative images of cysts and tubules stably expressing control (Scramble), myosin IIA or myosin IIB shRNA, and stained for myosin IIA (green), F-actin (red) and E-cadherin (white). Arrows indicate multiple lumens. Scale bars: 10 μm. (B) Immunoblot of myosin IIA or myosin IIB depletion. (C) Quantification of tubules with multiple lumens in control-, myosin-IIA- or myosin-IIB-depleted cells. Results are mean±s.d. (n=3). *P<0.01, **P<0.05 (Student's t-test).

Fig. 7.

Inhibition of myosin IIA impairs directed tubular cell migration during tubulogenesis. (A) Stills of movies of tubulogenesis in control- (Scramble) (Movie 6) or myosin-IIA-depleted cysts (Movie 7). Arrows indicate multiple lumens and the dashed line outlines the lumen. Scale bars: 10 μm. (B) Migration tracks of cells are displayed as displacement plots. For each group, the trajectories of 30 to 40 cells at 30 min intervals over 24 h are presented.

Fig. 8.

p114RhoGEF is necessary for tubular lumen formation. (A) Representative images of tubules stably expressing control (Scramble) and p114RhoGEF shRNA. Tubular structures were stained for F-actin (red), E-cadherin (white) and nuclei (blue). Arrows indicate multiple lumens. Scale bars: 10 μm. (B) Quantification of tubules with multiple lumens in control or p114RhoGEF-depleted cysts. Results are mean±s.d. (n=3). *P<0.01 (Student's t-test). (C) Selected stills of time-lapse sequences of tubulogenesis in control- (Movie 8) or p114RhoGEF-depleted (Movie 9). Arrows indicate multiple lumens and the dashed line outlines the lumen. Scale bars: 10 μm. (D) Migration tracks of cells are displayed as displacement plots. For each group, the trajectories of 15 to 30 cells at 30 min intervals over 24 h are presented.