Figure 3.
Disrupting CLASP function compromises formation of ncMT perinuclear baskets and nuclear orientation and dispersion. (A) Still frames comparing perinuclear MTs in control and CLASP shRNA (CLASP) embryos reveal depleted MTs and detachment from nuclei. (B) Scatter plot of perinuclear MT intensities at GBE onset after CLASP disruption; n = 60 and 42 perinuclear regions for control and CLASP, respectively, k = 3 embryos each background. (C) Orthogonal projection showing depleted MTs (green) in CLASP embryos and defective nuclear positioning as compared to control. Arrows mark apical MTs and arrowheads mark centrosomal MTs, respectively. (D) Still frames showing enhanced centrosomal MTs and depleted perinuclear MTs in CLASP embryos compared to control. (E) Quantitation reveals enhanced MT intensities at centrosomes in CLASP as compared to control at 0 and 20 min GBE; n = 200 centrosomal regions for each background, k = 3 control embryos and k = 4 CLASP embryos. (F) Maximum-intensity projection of nuclei color-coded for position from cell apices in control and CLASP embryos. (G) Orthogonal projection of cells and nuclei in control and CLASP embryos showing apically collapsed and deformed nuclei in CLASP embryos. (H) Fraction of nuclei in apical 10 μm of cell showing nuclear crowding in apical regions in CLASP embryos; n = 236 and 258 for control and CLASP, respectively, from k = 3 embryos. (I) Still image of nucleus displaying misaligned groove after CLASP disruption. (J) Fraction of nuclei invading the apical exclusion zone in control and CLASP embryos; n = 455 and 340 nuclei for control and CLASP, respectively, at 0 min and n = 483 and 378 nuclei for control and CLASP, respectively, at 20 min, k = 3 embryos each. (K) Percent of active nuclei as detected by MSD in control and CLASP embryos. (L) Peak nuclear speeds in control and CLASP embryos. (M) Mean nuclear speeds in control and CLASP embryos. (J–L), n = 546 and 427 measured nuclei in control and CLASP, respectively, k = 3 embryos for each background. (M) Scale bar = 5 μm in A, C, D, G, I, and 10 μm in F. Fig. 3, A, C, and D; Fig. 7, A and C; Fig. 2, C and D; and Fig. 5 H reproduced for comparison purposes. All scatter plots show the mean ± SD. Statistical significance was calculated using the Mann–Whitney U-test. ****P < 0.0001.

Disrupting CLASP function compromises formation of ncMT perinuclear baskets and nuclear orientation and dispersion. (A) Still frames comparing perinuclear MTs in control and CLASP shRNA (CLASP) embryos reveal depleted MTs and detachment from nuclei. (B) Scatter plot of perinuclear MT intensities at GBE onset after CLASP disruption; n = 60 and 42 perinuclear regions for control and CLASP, respectively, k = 3 embryos each background. (C) Orthogonal projection showing depleted MTs (green) in CLASP embryos and defective nuclear positioning as compared to control. Arrows mark apical MTs and arrowheads mark centrosomal MTs, respectively. (D) Still frames showing enhanced centrosomal MTs and depleted perinuclear MTs in CLASP embryos compared to control. (E) Quantitation reveals enhanced MT intensities at centrosomes in CLASP as compared to control at 0 and 20 min GBE; n = 200 centrosomal regions for each background, k = 3 control embryos and k = 4 CLASP embryos. (F) Maximum-intensity projection of nuclei color-coded for position from cell apices in control and CLASP embryos. (G) Orthogonal projection of cells and nuclei in control and CLASP embryos showing apically collapsed and deformed nuclei in CLASP embryos. (H) Fraction of nuclei in apical 10 μm of cell showing nuclear crowding in apical regions in CLASP embryos; n = 236 and 258 for control and CLASP, respectively, from k = 3 embryos. (I) Still image of nucleus displaying misaligned groove after CLASP disruption. (J) Fraction of nuclei invading the apical exclusion zone in control and CLASP embryos; n = 455 and 340 nuclei for control and CLASP, respectively, at 0 min and n = 483 and 378 nuclei for control and CLASP, respectively, at 20 min, k = 3 embryos each. (K) Percent of active nuclei as detected by MSD in control and CLASP embryos. (L) Peak nuclear speeds in control and CLASP embryos. (M) Mean nuclear speeds in control and CLASP embryos. (J–L), n = 546 and 427 measured nuclei in control and CLASP, respectively, k = 3 embryos for each background. (M) Scale bar = 5 μm in A, C, D, G, I, and 10 μm in F. Fig. 3, A, C, and D; Fig. 7, A and C; Fig. 2, C and D; and Fig. 5 H reproduced for comparison purposes. All scatter plots show the mean ± SD. Statistical significance was calculated using the Mann–Whitney U-test. ****P < 0.0001.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal