Figure 6. Disrupting salt-bridge residues in Borealin diminishes phase separation in cells.

(A) Endogenous Aurora B is recruited to nuclear Borealin foci upon exposure to 488 nm light. Images within the nucleus are shown (see Figure 6—figure supplement 1 for images of the entire cell for each of these enlarged views). The positions of the line scans (below) are indicated in the images by a white line. Scale bar = 5 μm. (B) Fluorescent detection of Borealin_WT or Borealin_Mut, each fused to mCherry-Cry2 in an optoDroplet assay. Images were collected before and after (at the indicated timepoints) exposure to 488 nm light to induce Cry2 dimerization (note that the images were acquired with the same imaging conditions and scaled in the same manner for display). Scale bar = 10 μm. (C) Quantification of the intensity of foci. n=2 experiments, and 18 (WT) and 16 (Mut) cells. The results of an unpaired, non-parametric t-test, Mann-Whitney test is shown, wherein **** equates to a p-value <0.0001. The lines represent the median and the interquartile range.

Figure 6—figure supplement 1. Endogenous Aurora B is recruited to Borealin-mCherry-Cry2 droplets in the nucleus upon exposure to white light.

(A) Cells expressing the indicated construct with and without exposure to white light were fixed and assessed for Aurora B and mCherry localization. Note that to expose the entire coverslip, the light in this experiment is performed on a light box for 10 min, which explains why the difference in intensity between wild-type and mutant versions is not as clear as in Figure 6 where a microscope was used to excite Cry2 in a controlled manner. Scale bar = 5 μm. (B) The intensities of Borealin^WT-mCherry-Cry2 and Borealin^Mut-mCherry-Cry2-expressing cells were similar before exposure to light. Intensity measurements of mCherry channel in the nucleus of all cells measured in Figure 6B before light exposure as a control to show that the differences in intensity of the resulting foci were not a function of the amount of starting Borealin expression. Note the images were acquired with the same imaging conditions. Nuclear mCherry intensity per cell was quantified using ImageJ software, and plotted as a scatter plot. Nucleus: n=2 experiments, m=18 (WT) and m=16 (Mut) cells. The statistical significance was calculated using unpaired, non-parametric t-test, Mann-Whitney test, p-value 0.9192, ns. The lines represent the median and the interquartile range.

Figure 6—figure supplement 2. Evidence supporting the engagement of the Borealin-mCherry-Cry2 with the endogenous chromosome passenger complex (CPC).

(A) Borealin-mCherry-Cry2-expressing cells were exposed to white light analyzed as in Figure 6A and Figure 6—figure supplement 1. Scale bar = 5 μm. (B) Mitotic cells expressing the indicated constructs were fixed without exposure to white light and assessed for Aurora B and mCherry localization. Co-localization at the inner centromere was observed with Borealin-mCherry-Cry2 but not with mCherry-Cry2. Scale bar = 5 μm.