Hepatocyte growth factor switches orientation of polarity and mode of movement during morphogenesis of multicellular epithelial structures

Abstract

Epithelial cells form monolayers of polarized cells with apical and basolateral surfaces. Madin-Darby canine kidney epithelial cells transiently lose their apico-basolateral polarity and become motile by treatment with hepatocyte growth factor (HGF), which causes the monolayer to remodel into tubules. HGF induces cells to produce basolateral extensions. Cells then migrate out of the monolayer to produce chains of cells, which go on to form tubules. Herein, we have analyzed the molecular mechanisms underlying the production of extensions and chains. We find that cells switch from an apico-basolateral polarization in the extension stage to a migratory cell polarization when in chains. Extension formation requires phosphatidyl-inositol 3-kinase activity, whereas Rho kinase controls their number and length. Microtubule dynamics and cell division are required for the formation of chains, but not for extension formation. Cells in the monolayer divide with their spindle axis parallel to the monolayer. HGF causes the spindle axis to undergo a variable "seesaw" motion, so that a daughter cells can apparently leave the monolayer to initiate a chain. Our results demonstrate the power of direct observation in investigating how individual cell behaviors, such as polarization, movement, and division are coordinated in the very complex process of producing multicellular structures.

Figure 1

Role of PI3K in HGF-induced extension and chain formation. (A–C) Phase contrast images. (A) Untreated cyst. (B) Cyst treated with CM for 16 h to allow formation of extensions. (C) Cyst treated with CM for 16 h in the presence of 2.5 μm/ml anti-rhHGF IgG, which inhibits formation of extensions. (D and E) Confocal fluorescence images of cysts expressing GFP-PH-Akt. (D) Untreated cyst. (E) Cyst expressing GFP-PH-Akt stimulated with CM for 16 h to allow formation of extensions. (F–H) Phase contrast images. (F) Cyst treated with CM for 16 h. (G) Cyst treated with CM for 16 h in the presence of 20 μM LY294002. (H) Cyst treated with CM and 20 μM LY294002 for 16 h, followed by a washout of LY294002, and subsequent treatment with CM for another 24 h. (I) Fluorescence image of cyst expressing GFP-PH-Akt, stimulated with CM for 40 h to allow formation of chains. (J) Cyst stimulated with CM for 40 h to produce a chain. Fluorescent phalloidin in green, blue is nuclei. (K) Cyst stimulated with CM for 16 h and then treated with CM and 20 μM LY294002 for an additional 24 h. Staining as in J. Bars, 50 μm in A, B, C, F, G, and H and 10 μm in D, E, I, J, and K.

Figure 2

Orientation of the Golgi in extensions and chains and effect of nocodazole on extension and chain formation. (A) Cyst treated with CM for 16 h to produce extension. Golgi marker (GM130) is red, fluorescent phalloidin is green, and nuclei are blue. One cell has formed an extension and has a concentration of actin at the tip of this extension, near the right edge of the panel. This cell still has a small apical surface, which is at the base of a deep indentation of the cyst lumen. Note that the Golgi in this cell is still underneath the apical surface (arrow). (B) Cyst treated with CM for 40 h to form a short chain. The cell in the chain has left the monolayer. Note that the nucleus is close to the nucleus of a cell that remains in the monolayer. Arrow indicates the Golgi in this chain cell, which is oriented toward the leading edge of the cell, away from the cyst. (C and C′) Disassembly of the Golgi in nocodazole-treated extension. Cyst was preincubated for 1 h at 4°C and then treated for 16 h with CM and 200 ng/ml nocodazole. (C) Staining as in A. C′ emphasizes fragmented Golgi by GM130 staining alone. (D) Cyst was preincubated for 1 h at 4°C and then treated for 16 h with CM and 200 ng/ml nocodazole. Green is fluorescent phalloidin, blue is nuclei, and red is microtubules. Fairly normal extensions are seen. (E) As in D, except treatment was for 40 h. Note many cells arrested in mitosis (red spindles) in and around the cyst. Bar, 10 μm.

Figure 3

Involvement of ROCK in extension and chain formation. Confocal micrographs of cysts. Phalloidin staining in green, nuclei in blue. (A) Untreated cyst. (B, C, and D) Cysts treated with CM for 24, 40, or 72 h, respectively. (E) Cyst treated with 30 μM Y27632 for 24 h. (F–H) Cysts treated with CM and Y27632 for 24, 40, or 72 h, respectively. Bars, 10 μm in A and E; 50 μm in all other panels.

Figure 4

Localization of P-MLC and GFP-MLC. (A, A′, and A") Confocal micrograph of untreated cyst. P-MLC (A, red in merge A"). Arrowheads indicate P-MLC in region of TJ. GFP-MLC (A′, green in merge) was mainly localized at basal plasma membrane, nuclei in blue. (B, D, and F) P-MLC in extension (B, 16 h CM), chain (D, 40 h CM), and extension after Y27632 treatement (F, 16 h CM + 30 μM Y27632), respectively. (B) Solid arrows indicate P-MLC associated with basolateral plasma membrane in extension. (D) Open arrowheads indicate P-MLC in cytoplasm of cell in chain. Left open arrow is in leading region of the cell, whereas right open arrow is in trailing region of the cell. (F) Asterisks indicate elongated, narrow extension. (C, E, and G) GFP-MLC in extension (C, 16 h CM), chain (D, 40 h CM), and extension after Y27632 treatment (G, 16 h CM + 30 μM Y27632), respectively. GFP-MLC is primarily located at the basolateral membrane, but some staining is observed apically, near tight junctions (arrowheads). (H) CM and Y27632 do not affect MCL expression levels. Confluent MDCK cells on 24-mm wells remained untreated or were treated for 16 h with CM in absence or presence of 30 μM Y27632, or with Y27632 alone. MLC in whole cell lysates was detected by Western blotting by using an anti-MLC antibody.

Figure 5

Effect of mitomycin C on extension and chain formation. Cysts were stained with fluorescent phalloidin. (A) Cyst treated with CM for 16 h. (B) Cyst treated with CM and 0.5 μg/ml mitomycin C for 16 h. Note apparently normal extensions. (C) Cyst treated with CM for 40 h to produce chains. (D) Cyst treated with CM and 0.5 μg/ml mitomycin C for 40 h. Note absence of chains.

Figure 6

Location of cell division. (A, B, and C) Three serial confocal sections, 4 μm apart, of a complex branching structure in a cyst stimulated with CM for 72 h, followed by 24 h with CM and 200 ng/ml nocodazole (to accumulate cells in mitosis). Red is immunofluorescence staining for α-tubulin, green is fluorescent phalloidin, and blue is nuclei. Individual mitotic cells are indicated by lowercase letters; some of these cells are visible in more than one section.

Figure 7

Time-lapse images of orientation of mitotic spindle. All images are of cysts expressing GFP-α-tubulin, taken by time-lapse confocal microscopy. (A) Untreated cyst. The mitotic spindle is oriented in the apical-basal axis at 0 min (dotted line), rotates 90^o to the plane of the monolayer by 15 min, and remains in this orientation through the completion of mitosis. (B) Cyst treated with CM. The mitotic spindle is oriented in the apical-basal axis at 0 min (dotted line) and rotates to the plane of the monolayer by 9 min. In this case, the spindle axis remains in this orientation through the completion of mitosis and the cell does not leave the monolayer. (C) Cyst treated with CM. This spindle exhibits little rotation and remains parallel to the apical-basal axis. (D) Cyst treated with CM. This cyst exhibits seesawing motion of spindle. The spindle is oriented in the apical-basal axis at 0 min (solid line). It first rotates counterclockwise to 70^o at 7 min (dotted line). The spindle then rotates clockwise, passing through the apical-basal axis at 17 min and reaches 45^o in the opposite direction at 25 min. Eventually, the spindle rotates out of the plane of focus.

Figure 8

Effect of inhibition of ROCK on spindle orientation of cyst. Images are of live cysts expressing GFP-α-tubulin, taken by time lapse confocal microscopy. (A) In cysts treated with 30 μM Y27632 for 24 h, the spindle axis rotates 90°, from perpendicular to apical-basal axis (at 0′) to parallel to apical-basal axis at 25′). Cell division was not completed after 86′. (B) This spindle exhibits little rotation in the apical-basal axis and finally divides in the apical-basal axis.