Dlg1 is required to orient the spindle in dissociated cells cultured on adhesive micropatterns. (A, left) Cortical localization of Dlg1 in HeLa cells cultured on nonpatterned coverslips. Dlg1 staining was lost upon siRNA treatment. (right) Distribution of the mitotic spindle angles relative to the coverslip in control and Dlg1 RNAi cells (αz, means ± SEM, n > 70 cells). **, P ≤ 0.01. (B) Cortical localization of Dlg1 during prometaphase and metaphase in cells cultured on L shape fibronectin micropatterns, as schematized on the left. (C) Control and Dlg1 siRNA–treated H2B-Cherry–expressing HeLa cells were cultured on L shape patterns and recorded by time-lapse microscopy. (left) Representative examples of time-lapse sequences of control or siDlg1 cells. A scheme of L shape micropattern and orientation of the mitotic spindle at anaphase onset is provided. (right) Distribution of mitotic spindle angles relative to pattern orientation (αxy) at anaphase onset (means ± SD, n > 750 cells from three independent experiments). The gray box highlights the 15° bin centered around 45°. (D) Evolution of the mitotic spindle αxy orientation during mitosis plotted for a dozen cells from C. (E, top left) Dlg1–LGN–NuMA interacting domains. Yellow star and triangle represent amino acids necessary for Dlg1–LGN interaction: P769 in Dlg1 and S401 in LGN, respectively. (bottom left) Confocal slices of nonpatterned control and Dlg1 siRNA–transfected metaphase cells stained for LGN and NuMA. (right) Graph showing mean LGN and NuMA cortical intensity profiles for control and Dlg1 siRNA–treated cells. Cortical coordinates along the plot correspond to the blue and red circles depicted in the LGN images. a.u., arbitrary unit; Ctrl, control; TPR, tetratricopeptide repeat; PDZ, PSD95/Dlg/ZO-1 domain. Bars: (A, B, and E) 10 µm; (C) 5 µm.
