Figure 4. Acute inhibition of PLK-1 and... | Rockefeller University Press

Figure 4.

$Acute inhibition of PLK-1 and SPD-2 induces premature weakening and disassembly of the PCM scaffold. (A) PCM was subjected to high-flow FLUCS during metaphase in wild-type embryos or permeabilized embryos treated with 10 µM BI-2536 (inhibitor of Polo kinases). Permeabilized embryos behaved as wild-type embryos during the first cell division (see Materials and methods; Carvalho et al., 2011). (B) PCM deformation rates in metaphase using low, medium, and high flow in wild-type and BI-2536–treated embryos. Wild-type data are from experiments in Fig. 1. Individual data points are plotted showing mean ± 95% CI; n = 6–7 (wild-type) and n = 5–7 (BI-2536 treated). P values were calculated using a Mann–Whitney U test. (C) PCM fracture probabilities from experiments in B. (D) Permeabilized embryos were arrested in metaphase using 10 µM MG-132, then treated with 0.1% ethanol (no drug) or 10 µM BI-2536. Data are plotted as normalized lines representing mean ± 95% CI; n = 8 (no drug) and n = 10 (BI-2536 treated). (E) Embryos expressing GFP::SPD-5 and a temperature-sensitive version of SPD-2 (spd-2(or188ts)) were allowed to assemble centrosomes at the permissive temperature (16°C), upshifted to the nonpermissive temperature (25°C) for 1 min during prometaphase, and then subjected to high-flow FLUCS during metaphase. (F) For each experiment in spd-2(or188ts) embryos, one centrosome was subjected to FLUCS and the other left alone (control). Integrated fluorescent intensities of the SPD-5 signal were tracked over time and then normalized to the starting value. Each curve represents a single experiment. (G) PCM fracture probabilities using low, medium, and high flow. Wild-type data are reproduced from Fig. 1. n = 7, 7, and 8 (wild-type) and 5, 6, and 8 (spd-2(or188ts)). (H) Embryos were upshifted from 16°C to 23°C during metaphase and then imaged during anaphase. Data show integrated fluorescence densities of PCM-localized signal, plotted as normalized lines representing mean ± 95% CI; n = 24 centrosomes in both wild-type and spd-2(or188ts) embryos.$

Acute inhibition of PLK-1 and SPD-2 induces premature weakening and disassembly of the PCM scaffold. (A) PCM was subjected to high-flow FLUCS during metaphase in wild-type embryos or permeabilized embryos treated with 10 µM BI-2536 (inhibitor of Polo kinases). Permeabilized embryos behaved as wild-type embryos during the first cell division (see Materials and methods; Carvalho et al., 2011). (B) PCM deformation rates in metaphase using low, medium, and high flow in wild-type and BI-2536–treated embryos. Wild-type data are from experiments in Fig. 1. Individual data points are plotted showing mean ± 95% CI; n = 6–7 (wild-type) and n = 5–7 (BI-2536 treated). P values were calculated using a Mann–Whitney U test. (C) PCM fracture probabilities from experiments in B. (D) Permeabilized embryos were arrested in metaphase using 10 µM MG-132, then treated with 0.1% ethanol (no drug) or 10 µM BI-2536. Data are plotted as normalized lines representing mean ± 95% CI; n = 8 (no drug) and n = 10 (BI-2536 treated). (E) Embryos expressing GFP::SPD-5 and a temperature-sensitive version of SPD-2 (spd-2(or188ts)) were allowed to assemble centrosomes at the permissive temperature (16°C), upshifted to the nonpermissive temperature (25°C) for 1 min during prometaphase, and then subjected to high-flow FLUCS during metaphase. (F) For each experiment in spd-2(or188ts) embryos, one centrosome was subjected to FLUCS and the other left alone (control). Integrated fluorescent intensities of the SPD-5 signal were tracked over time and then normalized to the starting value. Each curve represents a single experiment. (G) PCM fracture probabilities using low, medium, and high flow. Wild-type data are reproduced from Fig. 1. n = 7, 7, and 8 (wild-type) and 5, 6, and 8 (spd-2(or188ts)). (H) Embryos were upshifted from 16°C to 23°C during metaphase and then imaged during anaphase. Data show integrated fluorescence densities of PCM-localized signal, plotted as normalized lines representing mean ± 95% CI; n = 24 centrosomes in both wild-type and spd-2(or188ts) embryos.