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First published online October 22, 2008
doi: 10.1242/10.1242/jcs.018648

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ROCK-mediated contractility, tight junctions and channels contribute to the conversion of a preapical patch into apical surface during isochoric lumen initiation

Aldo Ferrari^1,*,, Alexey Veligodskiy^1,2,, Ulrich Berge^1,2,, Miriam S. Lucas³ and Ruth Kroschewski^1,

¹ Institute of Biochemistry, ETH Zurich, Schafmattstrasse 18, 8093 Zurich, Switzerland
² Molecular Life Science Ph.D. Program, Zurich, Switzerland
³ Electron Microscopy ETH Zurich (EMEZ), Wolfgang-Pauli-Strasse 16, 8093 Zurich, Switzerland

View larger version (69K): [in this window] [in a new window]   Fig. 1. A preapical patch (PAP) containing gp135, gp114, CFTR and free of E-cadherin is formed in solid MDCK aggregates. (A,B) Localization of gp135 (A) or gp114 (B) and E-cadherin. Average frequencies of two-cell aggregates (± s.e.m.) are shown in A. (C,D) Localization of gp114 (C) or gp135 (D) and the CFTR channel in two-cell aggregates without PAP, with PAP, and with a lumen. (D) Gp135-positive vesicles (open arrowheads) and compact structure (closed arrowhead). Middle sections of deconvolved confocal Z-stacks of fixed and immunostained cell aggregates are shown. Corresponding DIC images are shown below the merged images. Scale bars: 5 µm.

View larger version (66K): [in this window] [in a new window]   Fig. 2. Establishment of the preapical patch. (A) Time-sequence of deconvolved optical sections of an aggregate expressing mRFP-gp135 (top row) and E-cadherin-GFP (2nd row). Arrowheads delineate a gap in the E-cadherin surface (60 minutes). X-Y projections of the central region (outlined by a dashed line) are shown in the bottom row. The complete recording is available as supplementary material (Movie 1). Scale bar: 5 µm. (B) Dynamics of the normalized contact (Sc, red circles) and basal (Sb, black squares) surfaces of the aggregate shown in A. Error bars correspond to the calculated systematic segmentation error (see Materials and Methods). (C) Transmission electron micrographs of the PAP. The PAP (zone II) contains interdigitating microvilli (box 4) and is bordered by adhering plasma membranes (zone I). Magnified views of selected regions (box 1-box 5) are above the main image, which contains corresponding red and numbered rectangles. The inset in the main panel is an overview (light grey) of the two aggregate cells and the identified zones. Scale bars: 1 µm (main panel) and 200 nm (numbered boxes).

View larger version (52K): [in this window] [in a new window]   Fig. 3. The gp135-positive compact structure has hallmarks of apical recycling endosomes. Two individual fluorescence channels and the merged image represent maximum intensity projections of only confocal slices in which the compact structure was present. In the fourth column maximum intensity projections of merged fluorescence channels of the entire aggregate are overlaid with corresponding iso-colocalization surfaces (whitish objects) between gp135 (green) and indicated proteins (red) in aggregates with compact structures. The corresponding Pearson's correlation coefficient is shown on the right. Scale bars: 5 µm

View larger version (44K): [in this window] [in a new window]   Fig. 4. Characterization of gp135 redistribution. (A) Pseudocolor pH maps of two-cell aggregates with compact structure (left panel) and a lumen (middle panel) beside their corresponding fluorescent images used to generate the pH maps. The calibration bar (upper left corner) reports the relative pH, which was normalized to the value measured at the basal surface. Dotted white lines encircle regions corresponding to a compact structure (left panel) or a lumen (central panel). White arrowheads indicate some central vesicles (left panel). Scale bars: 5 µm. The boxplot (right panel) reports the average and relative pH values (small square) ± s.e.m. (box) and the 10-90% quantiles (whiskers) measured in the compact structure, PAP, lumen and central vesicles (n=14). Statistical comparisons were performed on distinct data sets. (B) Intensity profiles of gp135 and caveolin 1 (cav1) or gp58, respectively measured from maximum projections along contact surfaces (x-axis) of two-cell aggregates with PAP and with (upper row) or without (lower row) compact structure. The Gaussian fit of the gp135 signal is reported as a blue curve in the graphs. The corresponding fluorescent channels are reported below the graphs. Bars 5 µm. (C) Frequencies of two-cell aggregates with PAP or with lumen in cultures treated with BFA. Average values of three experiments (± s.e.m., n=50) are shown. BFA caused significant changes in lumen- and PAP-containing aggregates (*P<0.001).

View larger version (110K): [in this window] [in a new window]   Fig. 5. An initial lumen is formed within tens of minutes in the absence of cell division or apoptosis but during aggregate rotation. Middle sections of Z-stacks collected between 90 minutes before lumen appearance (–90 minutes) and 120 minutes after lumen appearance (+120 minutes) are shown. Cells of a three-cell aggregate are identified as cell 1, cell 2 and cell 3 (labels shown in the pp-YFP channel). The white arrows in the mRFP-gp135 channel (–90 minutes) depict the borders of the PAP before lumen appearance. The white arrowhead points to the first resolvable lumen (0 minutes). Scale bar: 10 µm. The complete recording is available as supplementary material (supplementary material Movie 2).

View larger version (67K): [in this window] [in a new window]   Fig. 6. Morphometric analysis of lumen formation. (A-D) Average normalized aggregate volume (A) does not change (P=0.06), whereas the average normalized cell volume (B) is reduced (P=0.02) in the window 60 minutes before and after lumen appearance. The average normalized sum of the apical and contact surfaces of individual cells (C) and normalized aggregates sphericities (D) remained constant (P=0.18 or 0.57, respectively). Individual data points (± s.e.m.) were normalized to the values at time 0 (only cells facing the lumen were measured in seven-cell aggregates; n=18). The indicated P-values were calculated by linear regression analysis (see Materials and Methods) (see also supplementary material Fig. S4D-H). (E) Luminal volume increases steadily after lumen initiation (P=6x10–5). Coloured areas in the background of (A-E) define the morphogenetic stages shown in (A) (n=7). (F) Development of a luminal space (shaded 3D object) reconstructed from 4D images (aggregate of Fig. 5). Scale bar, 5 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 7. Inhibition of ROCK1/2 or myosin II promotes both PAP formation and lumen initiation. (A) Normalized probabilities of PAP formation (x) and lumen initiation (y) in 24 hour 3D cultures treated as indicated. Values were normalized to medium (control, Y-27632) or DMSO (DMSO, ML-7, blebbistatin) values. Values (± s.e.m.) that are significantly different from control (medium or DMSO; P<0.05) are indicated with asterisks. The values were calculated from frequencies of aggregates with PAP or lumen (see Materials and Methods) (supplementary material Fig. S5A,B). (B) Schematic representation of lumen formation as a linear sequence of events. The probabilities (see Materials and Methods for calculation) of PAP formation (x) and lumen initiation (y) in cultures treated with ML-7, Y-27632, or blebbistatin relative to control values (medium or DMSO) are shown in A. (C) Western blot analysis of myosin light chain phosphorylation after ML-7 and Y-27632 treatment. Depicted are levels of pMLC and ppMLC in monolayer cultures treated for 24 hours as indicated; β-actin levels as input control (see also supplementary material Fig. S5C). (D) Frequencies of aggregates with lumen in 96-hour cultures. The cells were treated with ML-7, Y-27632, or blebbistatin during the final 24 hours of culture, or were left untreated (control). The average frequency values are shown (± s.e.m.; n=50, three experiments; see also Materials and Methods).

View larger version (53K): [in this window] [in a new window]   Fig. 8. Tight junction (TJ) formation around PAP triggers lumen initiation. (A) Localization of occludin-1 and gp135 in two-cell aggregates with a PAP or with a lumen. In the merged images, grey lines indicate the aggregate outline. Frequencies of aggregates (± s.e.m.) with cytoplasmic (w/o TJ) or ring-like occludin localization surrounding PAP (with TJ; ring in X-Y view or dots in X-Z view; arrowheads) relative to all two-cell aggregates with PAP. Scale bars: 5 µm. (B) Maximum projections of 5-20 central sections of deconvolved confocal images of MDCK two-cell aggregates transiently expressing mRFP-gp135 stained without permeabilization with anti-gp135 antibodies (gp135 ab). Scale bars: 5 µm. (C) Frequencies of aggregates containing PAP and lumen in dependence of mSnail overexpression. The frequencies of control Tet-Off MDCK cells (TO) and two different clones overexpressing mSnail (#1 and #10, ±dox; with/without doxycyclin) in a group of randomly picked two-cell aggregates (n>45) are represented (± s.e.m.; three experiments). Significantly decreased frequencies upon overexpression of mSnail (P<0.05) are indicated with an asterisk. Key for column colours is shown in D. (D) Frequencies of PAP- and lumen-containing aggregates in cultures after discontinuation of mSnail expression. mSnail overexpression was stopped after 40 hours by addition of dox (0 h no mSnail). The mSnail #1 cultures were grown for an additional 4 or 8 hours and quantified as in (C) (4 h and 8 h no mSnail; n>50; three replicates). After 4 hours of release, the cells were exposed for an additional 4 hours to 20°C (20°C) or 0.1% ethanol in medium (EtOH), BFA, bumetanide, or silver nitrate (Ag+). In the negative control `–dox', the cells were grown in dox-free medium for a total of 48 hours; in the positive control `+dox', cells were grown in presence of dox for 48 hours. The sequence of treatments is indicated with a vertical arrow. The categories of aggregates with a statistically significant difference in frequencies in different conditions are connected to the corresponding controls with white bars. The significance is shown with asterisks (*P<0.01; **P<0.001).

View larger version (14K): [in this window] [in a new window]   Fig. 9. Model of sequential steps during lumen formation. A PAP is established in the contact surface of neighbouring cells via insertion of gp135-positive membranes originating from a compact ARE-like structure (bigger red spots) (I). PAP maturation occurs through formation of tight junctions around the PAP and the enrichment of it with water and ion channels (II). It triggers lumen initiation (III), which is followed by lumen enlargement (IV). The enlargement phase is subdivided into a phase without cell volume increase but with continued luminal growth (Phase a) and a phase with both cell and luminal growth (Phase b). Only two-cell aggregates are shown for simplicity and the object sizes represent the physical trends. The relative changes in aggregate volume, individual cell volume and lumen volume are represented below the scheme as bars of variable height indicating constancy, increase or decrease over time.

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