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. 2020 Mar 19;9:e53807. doi: 10.7554/eLife.53807

Figure 4. The microtubule crosslinker PRC1 mediates the specialized and short-lived kinetochore-fiber reinforcement near chromosomes.

See also Figure 4—figure supplements 1–2 and Figure 4—video 1. (A) Immunofluorescence images of a representative PtK2 mock RNAi (control) metaphase spindle showing where PRC1 is localized in the spindle (tubulin, yellow; PRC1, magenta). White box (bottom panel) shows the region in which intensity (B) was quantified. Scale bar = 5 μm. (B) Fluorescence intensity ratio of PRC1 to tubulin along the pole-pole axis (n = 6 cells), showing PRC1 localization in the spindle center. Plot shows mean ± SEM. (C) Immunofluorescence images of a representative PtK2 PRC1 RNAi metaphase spindle (tubulin, yellow; PRC1, magenta), showing PRC1 depletion. Scale bar = 5 μm. (D) Top: Timelapse images of a representative PtK2 metaphase PRC1 RNAi spindle (GFP-tubulin, grey) during a 60 s manipulation, showing microneedle position (white circle) and traced manipulated k-fiber (white). Scale bar = 5 μm. Time in min:sec. Bottom: Curvature mapped along traced k-fiber for each point in the top panel (blue, negative curvature; red, positive curvature), showing the absence of negative curvature near chromosomes without PRC1. (E) Local curvature of deformed k-fibers for normalized positions along the k-fiber (n = 12 k-fibers in 12 cells). Most k-fibers exhibit no negative curvature (grey) and one shows negative curvature similar to WT k-fibers (orange). Scatter plot of microneedle positions shown above (inset). (F) Left: Distribution of microneedle positions along the k-fiber in WT (n = 17 cells) and PRC1 RNAi (n = 12 cells) spindles, after datasets are minimally down-sampled to maximize microneedle position overlap between them. There is no significant difference in microneedle position in the two conditions (p=0.38, Mann-Whitney U test). Plot shows mean ± SEM. Right: Percentage of deformed k-fiber profiles showing negative curvature near chromosomes in WT and PRC1 RNAi manipulated spindles, showing loss of negative curvature without PRC1. (G) Schematic of the three measurements made in (H,I,J): Inter-kinetochore distance between the manipulated k-fiber and its sister, angle between the sister k-fiber plus-end (opposite the manipulated k-fiber) and the pole-pole axis, and the angle between sister k-fiber plus-end regions. (H) Change in inter-kinetochore distance in WT unmanipulated (control, n = 13 kinetochore pairs from 6 cells), WT manipulated (n = 8 kinetochore pairs from 8 cells) and PRC1 RNAi manipulated (n = 8 kinetochore pairs from 8 cells) spindles, measured over 60 s. Inter-kinetochore distance after manipulation is significantly higher in spindles with PRC1 RNAi than WT (p=0.003, Mann-Whitney U test). Plot shows mean ± SEM. (I) Change in angle of sister k-fiber plus-end with respect to the pole-pole axis in WT unmanipulated (control, n = 12 k-fibers from 6 cells) and WT manipulated (n = 11 k-fibers from 11 cells) and PRC1 RNAi manipulated (n = 9 k-fibers from 9 cells) spindles, measured over 60 s. The sister k-fiber moves less (smaller angle) towards the pole-pole axis after manipulation in PRC1 RNAi spindles compared to WT (p=0.004, Mann-Whitney U test). Plot shows mean ± SEM. (J) Distribution of the angle between sister k-fiber plus-end regions at the end of manipulation in WT (n = 20 cells) and PRC1 RNAi (n = 10 cells) spindles, measured between the chromosome-proximal regions of the k-fibers.

Figure 4—source data 1. This spreadsheet contains the fluorescence intensity ratio of PRC1 to tubulin along the pole-pole axis of spindles acquired by immunofluorescence (Figure 4B), the local curvature along k-fibers manipulated over 60 s in PRC1 RNAi spindles (Figure 4E), microneedle positions from 60 s manipulations in WT and PRC1 RNAi such that their positions along the k-fiber maximally overlap (Figure 4F), the change in inter-kinetochore distance (Figure 4H) and angle of sister k-fiber plus-end from the pole-pole axis (Figure 4I) in unmanipulated and manipulated spindles, and PRC1 RNAi manipulated spindles, and the angle between sister k-fiber plus-end regions in WT and PRC1 RNAi PtK2 spindles (Figure 4J).

Figure 4.

Figure 4—figure supplement 1. Validation of PRC1 depletion by RNAi.

Figure 4—figure supplement 1.

(A) Western blot depicting of the intensity of PRC1 after mock RNAi and PRC1 RNAi, with tubulin as a loading control, using PtK2 GFP-tubulin cells. There is an 88% decrease in PRC1 levels in the PRC1 RNAi lane compared to the mock RNAi lane (normalized to tubulin intensity levels). (B) Additional examples of immunofluorescence images of PtK2 spindles in mock RNAi (Luciferase) and PRC1 RNAi backgrounds (tubulin, yellow; PRC1, magenta). Scale bar = 5 μm. (C) Average fluorescence intensity of PRC1 above cytoplasmic background levels in PtK2 mock RNAi (n = 12 cells) and PRC1 RNAi (n = 24 cells) spindles. Plot shows mean ± SEM. PRC1 intensity in the spindle decreases upon PRC1 RNAi (p=2×10−6, Mann-Whitney U test). (D) Percentage of binucleated PtK2 cells in populations of mock RNAi (n = 200 cells) and PRC1 RNAi (n = 213 cells) cells.
Figure 4—figure supplement 1—source data 1. This spreadsheet contains the fluorescence intensity of PRC1 (normalized to background levels) in PtK2 mock RNAi and PRC1 RNAi spindles from immunofluorescence images (Figure 4—figure supplement 1C).
Figure 4—figure supplement 2. Immunofluorescence quantifications of inter-kinetochore distance and tubulin intensity between PRC1 RNAi and mock RNAi spindles.

Figure 4—figure supplement 2.

(A) Inter-kinetochore distance of mock (Luciferase) RNAi (n = 22 kinetochore pairs from 12 cells) and PRC1 RNAi spindles in PtK2 cells (n = 44 kinetochore pairs from 24 cells) measured from immunofluorescence images. Plot shows mean ± SEM. The inter-kinetochore distance in PRC1 RNAi spindles is smaller than that of mock RNAi spindles (p=6×10−4, Mann-Whitney U test). (B) Average fluorescence intensity of tubulin above cytoplasmic background levels in mock RNAi (n = 12 cells) and PRC1 RNAi (n = 24 cells) spindles in PtK2 cells. Regions of interest (dashed-line box) include the whole spindle excluding poles (similar to Figure 4A) and the equator region near chromosomes. Plot shows mean ± SEM. Tubulin intensity remains unchanged upon PRC1 RNAi in the whole spindle (p=0.43, Mann-Whitney U test), but slightly lower in the equator region though not significant (p=0.08, Mann-Whitney U test).
Figure 4—figure supplement 2—source data 1. This spreadsheet contains the inter-kinetochore distance of mock RNAi and PRC1 RNAi spindles (Figure 4—figure supplement 2A), and the fluorescence intensity of tubulin (normalized to background levels) in mock RNAi and PRC1 RNAi spindles (Figure 4—figure supplement 2B) in PtK2 cells.
Figure 4—video 1. The microtubule crosslinker PRC1 mediates the specialized and short-lived kinetochore-fiber reinforcement near chromosomes.
Download video file (24.6KB, mp4)
Microneedle manipulation of a metaphase spindle in a PtK2 cell depleted of PRC1 by RNAi. The microneedle (Alexa-647, white circle) pulls (time 00:00) on the spindle’s outer k-fiber (GFP-tubulin, grey) over 60 s and deforms the spindle. The k-fiber bends around the needle, similar to WT, however it shows no negative curvature near chromosomes. This suggests that PRC1 is needed for the specialized, short-lived k-fiber reinforcement near chromosomes. Scale bar = 5 μm. Time in min:sec. Video was collected using a spinning disk confocal microscope, at a rate of 1 frame every 7 s during manipulation. Video has been set to play back at constant rate of 5 frames per second. Movie corresponds to still images from Figure 4D.