Figure 5. Excess centrosomes randomize MT polarity and reduce nucleation capacity. (A) Representative cells expressing centrin::GFP (centrosomes) and EB3::mCherry (growing MT plus ends). (top) Single image, with red circles indicating individual MT tips. (bottom) 30-s stack to show MT paths (blue lines). see also Video 9. (B) Imposition of MT growth directions onto Rose plots to visualize the percentage of growing MTs by angle in the indicated groups. (C) Comparison of MT angle areas from Rose plots in B. (NEC, n = 12 cells; TEC 1–2 centrosomes, n = 11 cells; TEC >2 centrosomes, n = 6 cells). Error bars are SEMs. (D and E) Comparison of number of MT nucleations (D) and nucleation density (E) among groups (NEC, n = 12 cells, TEC 1–2 centrosomes, n = 11 cells; TEC >2 centrosomes, n = 6 cells). (F) MT regrowth after nocodazole washout. Indicated cells stained for MTs (α-tubulin), centrosomes (γ-tubulin), and DNA (DRAQ7). (bottom) Higher magnifications of the insets. (G and H) Mean number of MTs/centrosome (G) and MT length/centrosome (H) in the indicated groups. (TEC 1–2 centrosomes, n = 10 cells; TEC >2 centrosomes, n = 10 cells). Error bars are SEMs. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, 10 µm.
Figure 5.

Excess centrosomes randomize MT polarity and reduce nucleation capacity. (A) Representative cells expressing centrin::GFP (centrosomes) and EB3::mCherry (growing MT plus ends). (top) Single image, with red circles indicating individual MT tips. (bottom) 30-s stack to show MT paths (blue lines). see also Video 9. (B) Imposition of MT growth directions onto Rose plots to visualize the percentage of growing MTs by angle in the indicated groups. (C) Comparison of MT angle areas from Rose plots in B. (NEC, n = 12 cells; TEC 1–2 centrosomes, n = 11 cells; TEC >2 centrosomes, n = 6 cells). Error bars are SEMs. (D and E) Comparison of number of MT nucleations (D) and nucleation density (E) among groups (NEC, n = 12 cells, TEC 1–2 centrosomes, n = 11 cells; TEC >2 centrosomes, n = 6 cells). (F) MT regrowth after nocodazole washout. Indicated cells stained for MTs (α-tubulin), centrosomes (γ-tubulin), and DNA (DRAQ7). (bottom) Higher magnifications of the insets. (G and H) Mean number of MTs/centrosome (G) and MT length/centrosome (H) in the indicated groups. (TEC 1–2 centrosomes, n = 10 cells; TEC >2 centrosomes, n = 10 cells). Error bars are SEMs. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, 10 µm.

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