Extended Data Figure 5: A large proportion of mitotic spindles remain planar in the absence of Mud, Dlg and Dynein function.
From: Epithelial tricellular junctions act as interphase cell shape sensors to orient mitosis
Loss of Mud activity is known to induce defects in mitotic spindle orientation relative to the apical–basal axis (AB) of the cell31,34,56. Nonetheless, in mud, dlg, dynein (glDN) mutant cells around 50% of the epithelial cell divide with an AB angle (αAB) in the range of the wild-type tissue (a–e). Since a large proportion of the spindles remain within the plane of the tissue, all analyses reported in the manuscript were performed on cell divisions that occurred within the plane of the tissue. Furthermore, in a central region of the tissue (box in f and f′), 88% of the divisions in mud tissue occur with an αAB in the range of the wild-type tissue (g). This region was analysed to compare TCJ bipolarity and cell-shape-based predictions of division orientation in wild-type and mud tissue (Fig. 3h). a, AB views of a dividing epithelial cell in wild-type (left panel, out of 257 cells quantified in b) or mud (right panels, out of 176 cells quantified in b) tissue. The spindle is labelled using Jupiter–GFP (green) and the centrosomes using Sas-4–RFP (red). αAB varies from 0° (spindle parallel to the plane of the tissue) to 90° (spindle perpendicular to the plane of the tissue). b, Quantification of αAB in wild-type, mud and in mud tissue expressing GFP–Mud (mud, GFP–Mud). In wild-type tissue, αAB varies between 0 and 22° (blue dashed line). In mud tissue, 56% of cells divide with a αAB angle lower than 22° (dashed red lines). The expression of GFP–Mud in mud tissue rescues the spindle AB orientation phenotype caused by Mud loss of function. Numbers of cells (n) for each genotype are indicated. The distribution of angles in mud tissue is significantly different from wild type (P < 1 × 10−4), and is restored in mud, GFP–mud (P < 1 × 10−4). P values, Kolmogorov–Smirnov test. c, Quantification of αAB in wild-type, Gαi and pins. The loss of either Gαi or Pins function does not affect the orientation of the spindle relative to the plane of tissue (P > 0.3) in agreement with our findings that Mud localization at TCJs is independent of Pins and Gαi. The analysis in pins tissue confirmed previously published findings46. Number of cells (n) are indicated. P values, Kolmogorov–Smirnov test. d, e, Quantification of αAB in wild-type, Gli and dlg tissues at 25 °C (d) and in wild-type and glDN tissues at 29 °C (e). Gli loss of function does not affect αAB orientation, whereas 46% of the dlg cells (P < 1 × 10−4) and 59% of the glDN cells (P < 1 × 10−4) divide with αAB lower than 22° and 24°, respectively. Numbers of cells (n) are indicated. P values, Kolmogorov–Smirnov test. f, f′, Identification of a region of the notum where AB orientation of the spindle is not affected in mud mutant tissue. Defects in AB orientation of the mitotic spindle result in size asymmetry of the two daughter cells57. Therefore daughter cell size was initially used as a proxy for the magnitude of spindle misorientation along the AB axis in mud tissue. The maps of daughter cell size asymmetry in wild-type (f) and mud (f′) tissues (green, no size asymmetry; purple, strong size asymmetry) revealed that a region (highlighted by the black box, f, f′) in the mud notum tissue exhibits almost no defects in daughter cell size asymmetry. Accordingly the quantification of spindle AB orientation within the region in wild-type and mud tissue revealed that 88% of the cells of the region divide within the range of the wild-type cells (see g). Anterior is to the right and the dashed back line indicates the midline. Colour coding: purple, daughter cells with strong size asymmetry; green, daughter cells with normal size symmetry; cyan, cells for which no division was detected; grey, cells which left the field of view and were not analysed; yellow, macrocheatae; white, sensory organ precursors (SOPs). g, Quantification of αAB in wild-type and mud tissue in the boxed regions in f and f′ was performed as in b–e. Numbers of cells (n) for each genotype are indicated. Scale bars, 1 μm (a), 100 μm (f, f′).
