Research ArticleCell Biology

Mitotic cell responses to substrate topological cues are independent of the molecular nature of adhesion

See allHide authors and affiliations

Science Signaling  25 Feb 2020:
Vol. 13, Issue 620, eaax9940
DOI: 10.1126/scisignal.aax9940
  • Fig. 1 Cadherin- and integrin-based adhesions elicit divisions parallel to the substrate plane.

    (A and B) Confocal images of interphase HeLa cells on coverslips coated with fibronectin (FN) or N-cadherin Fc (N-Fc). Cells were stained for β-catenin (which colocalizes with cadherins), active integrin β1, and either FAK [N = 220 (A)] or paxillin [N = 256 (B)]. N, number of interphase cells across all conditions from three independent experiments. (C) Representative Z-stacks at the plane of the spindle poles and side projections (xz) of metaphase HeLa cells on FN and N-Fc substrates. Cells were stained for α-tubulin and β-catenin. N = 180 total number of metaphase cells across all conditions from four independent experiments. (D) Distribution of spindle-to-substrate angles in (C). Means ± SEM: HeLa cells on FN, 7.235° ± 0.4565° (N = 60); HeLa cells on N-Fc, 6.428° ± 0.5170° (N = 60); HeLa on PLL, 32.87° ± 1.961° (N = 60). Data were analyzed by the Mann-Whitney test (****P < 0.0001). N, number of metaphase cells, from each condition, from three independent experiments. (E) Z-stacks at the plane of the spindle poles and side projections of metaphase U2OS cells on FN and N-Fc substrates. Cells were stained for α-tubulin and β-catenin. N = 100 total number of metaphase cells across all conditions from four independent experiments. (F) Distribution of spindle-to-substrate angles in (E). Means ± SEM: U2OS cells on FN, 6.566° ± 0.4883° (N = 50); U2OS cells on N-Fc, 6.702° ± 0.4522° (N = 50). Data were analyzed by the Mann-Whitney test. N, number of metaphase cells, from each condition, from three independent experiments. Scale bars, 10 μm. n.s., not significant.

  • Fig. 2 Cadherin engagement is necessary for correct spindle orientation on cadherin substrates.

    (A) Optical sections and side projections (xz) of control and EGTA-treated metaphase HeLa cells on FN or N-Fc substrates. Cells were stained for β-catenin and α-tubulin, and images were acquired at the plane of the spindle poles. N = 240 total number of metaphase cells across all conditions from three independent experiments. (B) Distribution of spindle-to-substrate angles in (A). Means ± SEM: control on FN, 7.235° ± 0.4565° (N = 60); EGTA-treated on FN, 5.210° ± 0.5494° (N = 60); control on N-Fc, 6.428° ± 0.5170° (N = 60); EGTA-treated on N-Fc, 24.42° ± 1.514° (N = 60). Data were analyzed by the Mann-Whitney test (****P < 0.0001). N, number of metaphase cells, from each condition, from three independent experiments. (C) Distribution of spindle-to-substrate angles in metaphase control and brefeldin-A–treated HeLa cells on N-Fc. Control on N-Fc, 8.910° ± 0.6321° (N = 80); brefeldin-A–treated on N-Fc, 9.809° ± 0.7211° (N = 80). Data were analyzed by the Mann-Whitney test. N, number of metaphase cells, from each condition, from three independent experiments. n.s., not significant. (D) Representative optical sections and side projections of representative control and brefeldin-A–treated metaphase HeLa cells on N-Fc. Cells were stained for β-catenin and α-tubulin. N = 160 total number of metaphase cells across all conditions from three independent experiments. Scale bars, 10 μm.

  • Fig. 3 The spatial distribution of LGN and NuMA does not depend on the molecular nature of adhesion.

    (A) Representative optical sections and side projections (xz) of HeLa cells seeded on FN and N-Fc. Images were acquired at the plane of the spindle poles. Cells were stained for α-tubulin, LGN, and NuMA as indicated. N = 215 total number of metaphase cells across all conditions from three independent experiments. (B) Representative confocal image of interphase HeLa cells expressing LGN-GFP on N-Fc linear micropatterns and labeled for LGN, β-catenin, and N-Fc. Images were acquired at the plane of attachment. N = 112 total number across all conditions of interphase cells from three independent experiments. (C) Distribution of spindle-to-substrate angles in metaphase control and E-cadherin–GFP–expressing HeLa cells on FN or E-cadherin Fc (E-Fc). Means ± SEM: control on FN, 9.769° ± 0.4638° (N = 74); control on E-cadherin, 34.17° ± 1.305° (N = 71); E-cadherin–GFP on FN, 9.315° ± 0.4771° (N = 65); E-cadherin–GFP on E-cadherin Fc, 9.184° ± 0.4588° (N = 67). Data were analyzed by the Mann-Whitney test (****P < 0.0001). N, number of metaphase cells from three independent experiments. (D) Representative optical sections and side projections of HeLa cells expressing E-cadherin–GFP on E-cadherin Fc substrate. Cells were stained for α-tubulin, LGN, and NuMA as indicated. Images were acquired at the plane of the spindle poles. N = 277 total number of metaphase cells across all conditions from three independent experiments. (E) Distribution of spindle-to-substrate angles in control and LGN-C′ mCherry–expressing metaphase HeLa cells on FN or N-Fc. Means ± SEM: LGN-C′ mCherry on FN, 3.248° ± 0.8769° (N = 74); LGN-C′ mCherry on N-Fc, 7.509° ± 0.3213° (N = 71); control on FN, 3.189° ± 0.8956° (N = 65); control on N-Fc, 6.878° ± 0.2764° (N = 67). Data were analyzed by the Mann-Whitney test (****P < 0.0001). N, number of metaphase cells, from each condition, from three independent experiments. Scale bars, 10 μm.

  • Fig. 4 Spindle responses to adhesion geometry are independent of the molecular nature of adhesion.

    (A) Optical sections and side projections (xz) of mitotic HeLa cells on FN or N-Fc. Cells were stained for active integrin β1 and β-catenin. Images were acquired at the plane of spindle poles. N = 197 total number of metaphase cells across all conditions from three independent experiments. (B) Representative images of interphase HeLa cells on FN or N-Fc L-shaped micropatterned substrates. Cells were stained for active integrin β1, β-catenin, β-tubulin, FN, and N-Fc as indicated. Images were acquired at the plane of attachment. N = 152 total number of interphase cells across all conditions from three independent experiments. (C) Metaphase HeLa cells on FN or N-Fc L-shaped micropatterned surfaces. Cells were stained for paxillin, β-catenin, β-tubulin, FN, and N-Fc as indicated. The images were acquired at the plane of mitotic spindle. N = 82 total number of metaphase cells across all conditions from three independent experiments. (D) Distribution of XY spindle angles in metaphase HeLa cells on FN or N-Fc L-shaped and linear micropatterns. Means ± SEM: FN bar patterns, 6.399° ± 0.4586° (N = 44); FN L-shaped patterns, 5.829° ± 0.4129° (N = 31); N-Fc linear patterns, 6.235° ± 0.2674° (N = 85); N-Fc L-shaped patterns, 6.402° ± 0.2679° (N = 82). Data were analyzed by the Mann-Whitney test. N, number of metaphase cells, from each condition, from three independent experiments. Scale bars, 10 μm. n.s., not significant.

  • Fig. 5 Integrin activation is required for spindle responses to planar cadherin substrates.

    (A) Optical sections of interphase HeLa cells on FN or N-Fc. Cells were stained for β-catenin and active integrin β1, and images were acquired at the cell-substrate plane. N = 357 total number of interphase cells across all conditions from three independent experiments. (B) Optical sections and side projections (xz) of control HeLa cells on FN or N-Fc at the plane of the spindle pole. Cells were stained for β-tubulin and active integrin β1. N = 205 total number of metaphase cells across all conditions from three independent experiments. (C) Optical sections and side projections of control and AIIB2-treated metaphase HeLa cells on FN or N-Fc. Cells were stained for β-tubulin and β-catenin. Images were acquired at the plane of each spindle pole. N = 342 total number of metaphase cells across all conditions from three independent experiments. (D) Distribution of spindle-to-substrate angles in control and AIIB2-treated metaphase HeLa cells on FN or N-Fc. Means ± SEM: Control on FN, 7.358° ± 0.3919° (N = 85); AIIB2 treated on FN, 31.63° ± 1.110° (N = 85); control on N-Fc, 6.665° ± 0.3849° (N = 86); AIIB2 treated on N-Fc, 30.82° ± 1.104° (N = 86). Data were analyzed by the Mann-Whitney test (****P < 0.0001). N, number of metaphase cells, from each condition, from three independent experiments. (E) Distribution of the cell spread of control and AIIB2-treated interphase HeLa cells on FN or N-Fc. Means ± SEM: Control on FN, 19.89 ± 0.5394 μm (N = 180); AIIB2 treated on FN, 12.88 ± 0.3790 μm (N = 181); control on N-Fc, 21.67 ± 0.4622 μm (N = 176); AIIB2 treated on N-Fc, 19.56 ± 0.5394 μm (N = 181). Data were analyzed by the Mann-Whitney test (****P < 0.0001). N, number of interphase cell diameter, from each condition, from four independent experiments. Scale bars, 10 μm.

  • Fig. 6 The CMC proteins p130Cas and Src are required for spindle responses to planar cadherin substrates.

    (A) Optical sections of representative metaphase HeLa cells on N-Fc) at the spindle plane, costained for β-tubulin plus FAK, p130Cas, or Src. (B) Distribution of spindle-to-substrate angles in cells in p130Cas−/− and p130Cas-reconstituted metaphase cells seeded on FN and N-Fc. Means ± SEM: p130Cas−/− on FN, 33.03° ± 2.280° (N = 60); p130Cas−/− on N-Fc, 32.01° ± 2.167° (N = 60); p130Cas−/− + WTp130Cas on FN, 8.756° ± 0.5179° (N = 60); p130Cas−/− + WTp130Cas on N-Fc, 8.462° ± 0.5809° (N = 60). Data were analyzed by the Mann-Whitney test (****P < 0.0001). N, number of metaphase cells, from each condition, from three independent experiments. (C) Representative optical sections and side projections (xz) of p130Cas−/− and p130Cas-reconstituted metaphase cells seeded on FN and N-Fc. Cells were stained for β-tubulin and β-catenin, and the images were acquired at the spindle poles plane. N = 240 total number of metaphase cells across all conditions from three independent experiments. (D) Distribution of spindle angles in control and PP2-treated metaphase HeLa cells on FN and N-Fc. Means ± SEM: control on FN, 7.402° ± 0.5001° (N = 60); control on N-Fc, 6.428° ± 0.5170° (N = 60); PP2 treated on FN, 16.075° ± 1.304° (N = 60); PP2 treated on N-Fc, 13.84° ± 0.9846° (N = 60). Data were analyzed by the Mann-Whitney test (****P < 0.0001). N, number of metaphase cells, from each condition, from three independent experiments. (E) Representative optical sections at the spindle poles and side projections of control and PP2-treated metaphase HeLa cells on FN and N-Fc acquired at the pane of spindle poles. N = 240 total number of metaphase cells across all conditions from three independent experiments. Scale bars, 10 μm.

Supplementary Materials

  • This PDF file includes:

    • Fig. S1. Planar cadherin substrates drive cell divisions parallel to the plane of adhesion.
    • Fig. S2. Integrin activation is necessary for mitotic cell responses to planar substrates.

    [Download PDF]

Stay Connected to Science Signaling

Navigate This Article