Integrin-mediated adhesion orients the spindle parallel to the substratum in an EB1- and myosin X-dependent manner - PubMed (original) (raw)

Integrin-mediated cell–substrate adhesion is required for the spindle orientation parallel to the substrate surface. (A) Synchronized HeLa cells were plated on the fibronectin-coated coverslips immediately after the release from a double-thymidine block with dilution by 10 (Sparse) or without dilution (semiconfluent), incubated for 10 h, and subjected to the spindle orientation analysis (right, histogram; _n_=50, inset; mean±s.d.; _n_=50). The images of the cells stained with anti-α-tubulin antibody (red), anti-γ-tubulin antibody (green), and Hoechst (blue) are shown in the left. In sparse culture, the spindle angles were measured in cells that had no cell–cell contacts. (B) Synchronized HeLa cells were diluted by 10, and plated on the coverslips coated with fibronectin, collagen, or poly-

L

-lysine, and subjected to the spindle orientation analysis (left, histogram; _n_=50, inset; mean±s.d.; _n_=50). *P<0.025, as compared with fibronectin, analyzed by _F_-test. Spindle angles were plotted as a function of basal area (right bottom, _n_=50). The merge images of phase contrast, α-tubulin (green), and Hoechst (blue) of a metaphase cell on each substrate are shown in the right upper. (C) Synchronized HeLa cells were plated on the fibronectin-coated coverslip without dilution in the presence of the GRGDNP peptide (RGD) or the GRADSP peptide (RAD), and subjected to the spindle orientation analysis (left, histogram; _n_=50, inset; mean±s.d.; _n_=50). *P<0.005 as compared with controls RAD, analyzed by _F_-test. Spindle angles were plotted as a function of basal area (middle, _n_=50). The average cell width at midsection of metaphase cells was measured (right, mean±s.d.; _n_=50). (D) Synchronized HeLa cells were plated on the fibronectin-coated coverslip without dilution in the presence of mouse IgG, or adhesion-blocking antibodies against β1 integrin, β3 integrin, or αVβ6 integrin, and subjected to the spindle orientation analysis (histogram; _n_=50, inset; mean±s.d.; _n_=50). *P<0.001, _**P_=0.59, and _***P_=0.46 as compared with control IgG, analyzed by _F_-test. (E) Synchronized HeLa cells on fibronectin were transfected with or without β1-integrin siRNA, and subjected to the spindle orientation analysis (right, histogram; _n_=50, inset; mean±s.d.; _n_=50). *P<0.001 as compared with mock, analyzed by _F_-test. Cell lysates were prepared and analyzed by Western blotting with anti-β1 integrin (left, upper) and anti-α-tubulin (left, bottom) antibodies. (F) Synchronized NRK cells were plated on the fibronectin-coated coverslips immediately after the release from an aphidicolin block with dilution by 10 (Sparse) or without dilution (semiconfluent), incubated for 8 h, and subjected to the spindle orientation analysis (histogram; _n_=50, inset; mean±s.d.; _n_=50). In sparse culture, the spindle angles were measured in cells that had no cell–cell contacts. (G) Synchronized NRK cells were plated on the fibronectin-coated coverslip without dilution in the presence of the GRGDNP peptide (RGD) or the GRADSP peptide (RAD), and subjected to the spindle orientation analysis (histogram; _n_=50, inset; mean±s.d.; _n_=50). *P<0.005, as compared with control RAD, analyzed by _F_-test.