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. 2020 Mar 7;23(3):100964. doi: 10.1016/j.isci.2020.100964

Figure 5.

Figure 5

Enrichment of Dynein in the Anterior Surface of the Cells before the Spindle Rotation

(A) Images of anti-dynein antibody staining of the cells in the row PP are shown. Anterior is to the left. (Left) Dynein signal is observed at the cellular surface of the cells and around the nucleus. (Middle) Cell boundary (white line) is identified by separating cell-cell contact after squishing the specimen. (Right) A merged image of the left and middle images indicating the enriched cell cortex signal is from the anterior of the cells (arrowheads). Scale bar, 10 μm.

(B) Quantification of the dynein protein enrichment. (Top) Schematic diagram showing how to measure the signal intensity. (Bottom) Relative signal intensity of the anterior to the posterior surfaces of the cells. All the cells with cell boundaries identified by squishing were measured. See Transparent Methods for the calculation methods. Data are represented as individual values and boxplot. The mean is indicated as a red horizontal bar. The median is indicated with dashed line. Box indicates the second and third quartiles, and whiskers indicate the maximum and minimum values.

(C) Images of DCIC-EGFP and PH-dTomato fluorescent signals in the row PP cells. (C1) Dynein signal is observed in the surface of PP cells (arrowheads). Scale bar, 10 μm. (C2) PH-dTomato signal (magenta) is detected at the cell membrane of PP cells. (C3) A merged image of C1 and C2. (C4, left) A close-up view of the adjacent cell surfaces between PP cells (a dot-lined square in C3). Brightness and contrast were enhanced for visualization. (C4, right) Average signal intensities of DCIC-EGFP and PH-dTomato from 26 different cell surfaces. See Transparent Methods for the calculation method. Note that DCIC-EGFP signal was peaked posterior to the PH-dTomato peak, indicating that DCIC-EGFP signal is enriched in the anterior surface of the cell.

(D) Selected snapshot images from Video S7 showing the expression of DCIC-EGFP (for dynein, green) and MAP7-mCherry (for spindle orientation, red) in posterior epidermal cells. Anterior is to the left. P and PP represent cells in the rows P and PP, respectively. Cell boundaries between different rows are not straight perpendicular to the A–P axis due to different timing of cell divisions (also see Figure 3B). Enrichment of the dynein is observed in the anterior surface of the cells (arrowheads). Time after the start of observation is shown in the bottom left of each image. The yellow P and PP indicate the cells in which dynein is observed in the anterior surface before spindle rotation. Scale bar, 10 μm.