Long-range mechanical force enables self-assembly of epithelial tubular patterns - PubMed (original) (raw)

Long-range mechanical force enables self-assembly of epithelial tubular patterns

Chin-Lin Guo et al. Proc Natl Acad Sci U S A. 2012.

Abstract

Enabling long-range transport of molecules, tubules are critical for human body homeostasis. One fundamental question in tubule formation is how individual cells coordinate their positioning over long spatial scales, which can be as long as the sizes of tubular organs. Recent studies indicate that type I collagen (COL) is important in the development of epithelial tubules. Nevertheless, how cell-COL interactions contribute to the initiation or the maintenance of long-scale tubular patterns is unclear. Using a two-step process to quantitatively control cell-COL interaction, we show that epithelial cells developed various patterns in response to fine-tuned percentages of COL in ECM. In contrast with conventional thoughts, these patterns were initiated and maintained by traction forces created by cells but not diffusive factors secreted by cells. In particular, COL-dependent transmission of force in the ECM led to long-scale (up to 600 μm) interactions between cells. A mechanical feedback effect was encountered when cells used forces to modify cell positioning and COL distribution and orientations. Such feedback led to a bistability in the formation of linear, tubule-like patterns. Using micro-patterning technique, we further show that the stability of tubule-like patterns depended on the lengths of tubules. Our results suggest a mechanical mechanism that cells can use to initiate and maintain long-scale tubular patterns.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Cells developed direct branching in response to COL. (A) Experimental setup. (B) (i) Represented acinar morphogenesis at [COL] (Top) approximately 0; (Middle) 0.3 mg/mL; and (Bottom) 0.5 mg/mL. ECM2 gelled at t = 00:00. The represented single-cell motions in acini (“a,” “b,” and “c”) are displayed on the right. Directions of acinar branching are indicated by yellow and pink arrows. Red arrowhead indicates branching cells. Time is in hours and minutes. Scale bar: 40 μm. Initial cell density: 30 ± 5 mm-2. (ii) Correlations of cell motions in i. Time windows to compute correlations: t = 20:00 to 50:00 in Top, t = 44:00 to 69:00 in Middle, and t = 15:00 to 61:00 in Bottom. All cells in acini “a” and “b” were paired to compute correlations (N > 200). Data were normalized to the maximum. (iii) The distributions (mean ± SEM) of acini (N = 200) at different patterns (after 24 h of ECM2 gelification) depended on [COL]. Initial cell density: 30 ± 5 mm-2. (iv) Histograms of the initial distances between interacting acini (N = 100) show long-scale interactions. Data were normalized to the maximum. (v) The percentage (mean ± SEM) of acini (N = 200) developing attraction or extension (after 24 h of ECM2 overlay) depended on initial acinus density. [COL] = 0.5 mg/mL.

Fig. 2.

Fig. 2.

COL-dependent direct branching associated with COL reorganization and required cell contractility. (A) Linear ECM deformation (outlined by black arrows in day 1 and 2 after ECM2 overlay) arose between interacting acini. (B) Collagen alignment and condensation (white arrow) occurred around and away from branched cells (after 36 h of ECM2 overlay). Yellow arrows and white lines indicate cell boundaries. Nucleus (Nuc): H2B-CFP. Cytoplasm (Cyto): mCherry. Collagen: FITC-conjugated type I collagen (FITC-COL, 5% in total COL, total [COL] = 0.5 mg/mL). BF, bright field. (C) (i) Inhibiting ROCK (Left) prevented the initiation and (Right) caused the disruption of direct branching. Data are mean ± SEM (N = 50 for each case). (ii) Represented image of branch disruption after ROCK inhibition [by Y27632 (20 μM) at t = 00:00].

Fig. 3.

Fig. 3.

COL mediated a nondispersed transmission of traction force in direct branching. (A) (Left) Schematics to show motions (red arrows) of cells (formula image) and beads [formula imageat the central region (between cyan lines) and formula image at the peripheral regions (between cyan and pink lines)]. Center and Right ECM deformation induced by cell motions was (Center) nondispersed or (Right) dispersed. (B) (i) Represented images of cell and bead motions (Δ_t_: 13 min). Blue arrows indicate the motions of cells (“a,” “b”). Bead overlay: overlay of fluorescent images of beads at the earlier time (red) and the later time (green) to show bead displacements (white, pink, and yellow arrows). Beads in yellow indicate no movements of beads. Time is in hours and minutes. BF, bright field. (ii) and (iii) Histograms of the correlations between cell motions and bead movements in (Left) the central and (Right) the peripheral regions show distinct behaviors for [COL] = (ii) 0.5 mg/mL and (iii) 0.N = 10 × 10 × 10 (cells × beads × events). Data are normalized to the maximum in each case. Inset in (ii): Frequency (mean ± SEM) of bead motions (≥0.1 μm/ min, see

SI Text

) at central regions in 50 events of cell motions (≥0.1 μm/ min). [COL] = 0.5 mg/mL.

Fig. 4.

Fig. 4.

The formation of long-scale linear patterns requires cell repositioning. (A) Experimental setup. Circular cell traps were created by PDMS stamps with diameter l and separation λ. (B) Potential models to form long-scale linear patterns (see text for details). Long-scale linear structure was defined if the number of branches (N b) at each cluster in the structure is ≤ 2. (C) Represented images of various patterns. (Left) No branching (defined if N b = 0). (Center) Direct branching (red arrows, defined if 0 < _N_ _b_ ≤ 6). (_Right_) Random branching (defined if _N_ _b_ > 6). Time is in hours and minutes. Scale bar: 200 μm. Fluorescence signal was from mCherry expressed in the whole cell. To enhance visualization, background scattering from the argarose gels was removed. (D) (Left) The average number of branches per trap formula image depends on λ and [COL]. (Center) and (Right) The maximal length of linear structures, Λ, and its normalization with λ show that cells could not form linear structures over three traps. Λ = 0 if there is no linear structure. For Center and Right, no significant difference was found in [COL] = 0.3–1 mg/mL. Data are represented in mean ± SEM.

Fig. 5.

Fig. 5.

The stability of linear pattern depended on its length. (A). Experimental setup. Rectangular cell traps were created by PDMS stamps with various aspect ratios (L y/L x). (B) Potential outcomes in a rectangular trap covered by COL in random orientations (light purple lines). Initial cell density (yellow circles) is fixed (3–5 × 108/mL). The outcomes of two COL fibers (green and blue) and associated cells (green and red) are illustrated. (C_–_E) Represented images of (C) and (D) unstable and (E) stable linear structures. The instability was associated with the collapse (red arrows) of linear structures. Time is in hours and minutes. To enhance visualization, background scattering from the argarose gels was removed. (F) The stability of linear structures (mean ± SEM) increased with L y (N = 20 and L x = 200 μm for each case).

Fig. 6.

Fig. 6.

COL-dependent direct branching required PI3K/Rac1 and exhibited evidence of mechano-transduction. (A) The percentage of acini developing branching after 24 h of ECM2 overlay ([COL] = 0.5 mg/mL) in the presence of various growth factors or inhibitors. (B) The percentage of branched acini that lose branches after 24 h treatment of various growth factors or inhibitors. In _A_and B, EGF: 100 ng/mL, TGFβ1: 5 ng/mL, LY294002 (for PI3K): 20 μM; NSC553502 (for Rac1): 70 μM; and PD98095 (for ERK1): 10 μM. Control: serum-free and growth-factor-free. “***” indicates P < 0.001 in student’s _t_-test. (B) Represented images of YAP immuno-staining at (Upper, Left) [COL] = 0, (Upper, Right) 0.5 mg/mL, and (Lower, Left) 1.0 mg/mL. Nucleus (Nuc): H2B-CFP. Cells were exposed to ECM2 containing various [COL] for 24 h and fixed for YAP immuno-staining. Note the nuclear translocation of YAP (white arrows) in [COL] = 0.5 mg/mL. Scale bar: 20 μm. (Lower, Right) Ratios of average fluorescent intensities (mean ± SEM, subtracted by background) of YAP immuno-staining in the nucleus and the cytoplasm depended on [COL] (N = 50 for each case). (C) Proposed mechanical feedback loop for long-scale linear pattern formation. See text for details.

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