Research ArticleCELL MODELS

Curvature and Rho activation differentially control the alignment of cells and stress fibers

See allHide authors and affiliations

Science Advances  06 Sep 2017:
Vol. 3, no. 9, e1700150
DOI: 10.1126/sciadv.1700150
  • Fig. 1 Fibroblasts in confluent monolayers sense weaker curvature fields than isolated cells.

    (A) Three-dimensional reconstruction of MEFs stained with phalloidin-TRITC (red) on cylinder (Rc = 40 μm) functionalized with Alexa Fluor 488–conjugated fibronectin (green). (B) Projection along the surface normal (premapping; left) and mapped image (postmapping; right) of surface in (A) and corresponding cross sections. (C) AIs of isolated and confluent monolayers of MEFs on cylinders with Rc = 40 and 200 μm. (D) Mapped images of isolated (left) and confluent monolayer (right) of MEFs on cylinders with Rc = 200 μm. Arrows indicate cylinder axis orientation. Scale bars, 100 μm. (E) Analysis of outlines of MEFs on cylinders with Rc = 200 μm. Area of outlined cells (left), ratio of major-to-minor axis length of ellipses fit to cell outlines (middle), and orientation angle of long axes of fit ellipses relative to cylinder long axis (right). Cells on at least seven independent cylinders were analyzed in each condition. Results are mean and SE. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test. ns, not significant.

  • Fig. 2 VSMCs align weakly on cylinders when isolated.

    (A) Isolated hVSMCs have a smaller AI than isolated MEFs on cylinders with Rc = 40 μm; this is due to weaker axial alignment (B) and not a difference in elongation (C), as measured from the dimensions of ellipses fit to cell outlines. At least 16 cells were analyzed in each condition. Representative images of phalloidin-TRITC (gray) and 4′,6-diamidino-2-phenylindole (DAPI; blue) staining of MEFs and hVSMCs on coverslips (D) and corresponding phalloidin-TRITC intensities (E). Scale bars, 50 μm. At least 40 cells of each type in two independent experiments were analyzed. Results are mean and SE. *P < 0.05, ***P < 0.001, Student’s t test.

  • Fig. 3 hVSMCs in confluent monolayers sense weaker curvature fields than isolated cells.

    (A) Representative mapped images of isolated (left) and confluent monolayers (right) of hVSMCs on cylinders with Rc = 200 μm. Arrow indicates cylinder axis orientation. Scale bars, 100 μm. (B) AIs of isolated and confluent hVSMCs on cylinders with Rc = 200 μm. (C) Spread area of isolated hVSMCs and hVSMCs in confluent monolayers on cylinders with Rc = 200 μm. Aspect ratio (D) and orientation angle relative to cylinder axis (E) of ellipses fit to cell outlines on cylinders with Rc = 200 μm. Cells on at least eight independent cylinders were analyzed in each condition. Results are mean and SE. *P < 0.05, ***P < 0.001, Student’s t test.

  • Fig. 4 Apical and basal SFs align in distinct patterns in response to curvature.

    (A) Apical SFs sit above the nucleus, and basal SFs sit below the nucleus relative to the cylinder surface shown in cross section. (B) Representative images of an hVSMC on a cylinder with Rc = 40 μm stained with phalloidin-TRITC showing apical and basal SFs from a single confocal stack. Arrow indicates cylinder axis orientation. Scale bars, 30 μm. (C and D) Angle distribution plots showing the directions in which basal and apical SFs are oriented (top) on small and large cylinders. The bottom plots show the longest 30% (Long; 22.3 μm ≤ L40μm ≤ 67.4 μm; 18.6 μm ≤ L200μm ≤ 47.3 μm), shortest 30% (Short; L40μm ≤ 12.1 μm; L200μm ≤ 11.3 μm), and the remaining 40% (Mid) of SFs, where Embedded Image denotes the length of SFs on cylinders with radius Rc. Axial and circumferential orientations are given by orientation angles of 0° and 90°, respectively. (E) Angle between the mean orientation of basal SFs and the mean orientation of apical SFs in cells on each substrate. At least 272 SFs in 10 cells were analyzed in each condition. Results are mean and SE. **P < 0.01, Student’s t test.

  • Fig. 5 Activation of Rho alters the sizes of SF populations.

    (A) Representative mapped phalloidin-TRITC images of hVSMCs on cylinders with Rc = 125 μm treated with CN03. Scale bars, 50 μm. (B) Z-projection and orthogonal slices of hVSMC on a cylinder with Rc = 125 μm treated with CN03. Red, phalloidin-TRITC; blue, DRAQ5. Scale bar, 30 μm. Arrows indicate cylinder axis orientation. (C) Angle distribution plots showing the directions in which basal and apical SFs are oriented (top) in control cells and cells treated with CN03. The bottom plots show the longest 30% (Long; 19.6 μm ≤ L0μg/ml ≤ 50.7 μm; 18.1 μm ≤ L5μg/ml ≤ 38.4 μm), shortest 30% (Short; L0μg/ml ≤ 12.3 μm; L5μg/ml ≤ 12.0 μm), and the remaining 40% (Mid) of SFs, where L[CN03] denotes the length of SFs in cells treated with a concentration of CN03 equal to [CN03]. Axial and circumferential orientations are given by orientation angles of 0° and 90°, respectively. (D) Ratio of the number of apical-to-basal SFs per cell in isolated hVSMCs treated with CN03 on cylinders with Rc = 125 μm. At least 259 SFs in 11 cells were analyzed in each condition. Results are mean and SE. *P < 0.05, Student’s t test.

  • Fig. 6 Rho activation establishes F-actin alignment observed in vivo.

    hVSMCs on cylinders with Rc = 125 μm were treated with CN03 [0 μg/ml (A) or 5 μg/ml (B)]. Arrow indicates cylinder axis orientation. Scale bars, 100 μm. (C) AIs of CN03-treated hVSMCs in confluent monolayers on cylinders with Rc = 125 μm. Cells on at least 13 independent cylinders were analyzed in each condition. Results are mean and SE. ***P < 0.001, Student’s t test.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/9/e1700150/DC1

    fig. S1. MEFs and hVSMCs coalign on planar surfaces.

    fig. S2. Apical and basal SFs align in distinct patterns in MEFs in response to curvature.

    fig. S3. Lengths of apical and basal SFs in hVSMCs on small and large cylinders.

    fig. S4. Phalloidin intensity of isolated hVSMCs on coverslips treated with CN03.

    fig. S5. F-actin organization is not recovered after CN03 washout.

    fig. S6. Basal SFs remain after inhibition of ROCK.

    fig. S7. Assembly of cylinder substrates.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. MEFs and hVSMCs coalign on planar surfaces.
    • fig. S2. Apical and basal SFs align in distinct patterns in MEFs in response to curvature.
    • fig. S3. Lengths of apical and basal SFs in hVSMCs on small and large cylinders.
    • fig. S4. Phalloidin intensity of isolated hVSMCs on coverslips treated with CN03.
    • fig. S5. F-actin organization is not recovered after CN03 washout.
    • fig. S6. Basal SFs remain after inhibition of ROCK.
    • fig. S7. Assembly of cylinder substrates.

    Download PDF

    Files in this Data Supplement: