Extended Data Figure 1. Mud, Pins, Gαi and Gli localization during symmetric epithelial cell division in the Drosophila notum.
Within the Drosophila pupal notum tissue cells divide according to their intephasic cell shape long-axis (a,b), thereby following the 130-year-old Hertwig rule. However, upon entry into mitosis cells round up (the cell shown in a, -15 to -2 min and Fig. 3b).
(a) Time-lapse images of Dlg:GFP in a dividing cell (out of 249 cells quantified in b) in the pupal notum tissue illustrating cell rounding during mitosis (The same cell is shown as inset in Fig. 3b.). Prior to mitosis (-30 min) the cell (marked by asterisk) is clearly elongated and divides according to its interphasic cell shape (5 min). Upon entry into mitosis (-15 min) the cell rounds up and reaches a minimal anisotropy just prior to anaphase (-2 min, see also Fig. 3b).
(b) Rose plot of the difference between the experimental (θdivision) and predicted division orientations by the average (60-30 min prior to mitosis) interphase cell long-axis (θshape). The data are duplicated relative to 0° line (light green). Number of cells (n) analysed is indicated.
(c,d,e) Gαi localization in fixed epithelial dorsal thorax tissue (c), Pins:YFP localization in pins mutant tissue (d) and GFP:Mud localization (e) showing cells in G2 interphase (left) and mitosis (right). Gαi is hardly detected at the cell cortex in G2 phase and Gαi is mostly homogenously distributed around the cortex during mitosis. Pins:YFP is homogenously distributed around the cell cortex in both interphase and mitotic cells. In mitosis Pins:YFP also weakly localizes at the mitotic spindle. GFP:Mud localize at TCJs during interphase and mitosis (see also f). n=24 cells (c, left); n=19 cells (c, right); n=80 cells (d, left); n=12 cells (d, right); n=111 cells (e, left) and 54 cells (e, right).
(f) GFP:Mud time-lapse images from G2 interphase to telophase (n=21 cells). White arrows: GFP:Mud at TCJ (numbered at t=-22min). Red and yellow arrowheads: GFP:Mud on the spindle and its poles, respectively. The same panels -22min to 4min are shown in Fig. 1a. See also Supplementary Video 1.
(g) Apical-basal (AB) sections of the cell in (f) at t=-22min (top) and t=-1min (bottom). White arrows: GFP:Mud at TCJ. n=21 cells.
(h) GFP:Mud kymograph along the cortex (x axis) from t=-22 to t=0 min of the cell in (f). TCJ numbered as in (f). The kymograph shows that during mitotic rounding GFP:Mud spread only modestly along the cortex of the dividing cell. n=21 cells.
(i) AB sections of GFP:Mud, adherens junction marker E-Cad and septate junction marker Dlg (top, n=16 cells) or septate TCJ marker Gli (bottom, n=30 cells).
(j-m) Localizations of GFP:Mud (white in j-m and green in j”-m”) and Gli (white in j’-m’ and red in j”-m”) in fixed pupal wing (j-k) and larval wing disc (l-m) tissues. GFP:Mud colocalizes with Gli at TCJ in G2 interphase and mitotic cells in both the pupal wing and larval wing disc epithelium. Asterisks mark Mud punctate structures present on the nuclear envelope of early G1 cells. Yellow arrows indicate GFP:Mud on the spindle poles. n=20 cells (j-j”); n=5 cells (k-k”); n=63 cells (l-l”) and n=12 cells (m-m”).
(n-o) Localizations of Mud (white in n, o and green in n”, o”) and Gli (white in n’, o’ and red in n”, o”) detected by antibody staining in G2 interphase and mitotic cells in the pupal dorsal thorax tissue. As observed for GFP:Mud (Fig. 1b and Extended data Fig. 1j-m), the endogenous Mud is enriched at TCJ where it co-localizes with Gli in G2 interphase and mitotic cells. Yellow arrows indicate Mud on the spindle poles. n=37 cells (n-n”) and n=21 cells (o-o”).
Scale bars: 1μm (a, c, d, e, f, g, i, j, k, l, m, n, o). 3 min (h).