Extended Data Fig. 2: Additional characterization of PLK1 and spindle assembly dynamics.
From: A monoastral mitotic spindle determines lineage fate and position in the mouse embryo
a,b, Live embryos show localization of PLK1-Emerald to the bridges, connecting sister cells, during interphase (b), and disappearance from the bridge prior to mitosis (b). c, Consistent with its localization on kinetochores from late prophase, PLK1-Emerald puncta are visualized at chromosomes (inset). Bona fide apical and perinuclear PLK1-Emerald-positive MTOCs are also visualized (arrows). d, Live-imaging shows the dynamics of PLK1-localization at the intercellular bridge, cell nucleus and forming spindle. e, Live imaging and graph show that PLK1-Emerald is enriched in the nucleus before NEBD. f, Live-imaging shows that the onset of microtubule growth from the apical and perinuclear MTOCs of the monoastral spindle corresponds with the timing of NEBD. The apical MTOC starts to assemble the astral array (arrows), while the perinuclear MTOC starts to assemble the first spindle half of the future interpolar parts of the spindle. g, Following NEBD, the two main MTOCs that form the monoastral mitotic spindle are moving closer. h, i, Immunofluorescence in non-injected 8-cell embryos. At prophase, microtubules are projected between the apical and perinuclear MTOCs (h) to assemble the monoastral spindle (i). j, Localization pattern of endogenous PLK1 at the monoastral mitotic spindle. k, Live-imaging. In these focal planes, only the apical MTOC is initially visible. Note how the apical MTOC joins the rest of the mitotic spindle. l, Live-imaging during the assembly of an anastral mitotic spindle. In some cases of anastral spindle assembly, an apical EB3-Tomato-positive structure is initially detected, but this fails to display significant microtubule growth and to join the rest mitotic spindle apparatus. Graph shows the proportion of cells displaying this behavior. m, Quantification of PLK1-Emerald and Cdk5rap2-Emerald fluorescence intensities at the main parts of the monoastral and anastral mitotic spindles. Note the retention of high fluorescence levels in the apical parts of the monoastral spindle. n, Immunostaining and live imaging of PLK1 and microtubules and quantification of PLK1 intensity ratio between the apical and the basal poles (arrow shows apical pole). In box plot, centre line is the median, box edges show upper and lower quartiles and whiskers represent the range. **p = 0.0025; Two-tailed Mann-Whitney U-test. o, Quantification of apical MTOC stability in monoastral and anastral spindles. Scale bars, 10 µm in a,b, d-l, n; 2 µm in c. Source numerical data are provided in source data.
