Spindle biochemistry responds to compressive force from the nuclear envelope to tune spindle dynamics during closed mitosis

Figures

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  Figure 1:. Cerulenin-induced nuclear envelope tension alters spindle regulation in S. pombe. Treatment conditions: 300μM cerulenin. All images are representative spinning disk confocal microscopy images; scale bars, 2 μm scale bars; time stamps, min:sec. Lighter color in all beehive plot shows all data (n), darker points indicate averages of each individual cell (N), shaded region indicates mean +/− s.e.m of cellular averages σN/N. (A) Cartoon illustrates the impact of cerulenin treatment on the nuclear envelope and mitotic spindle. (B) Anaphase spindle elongation in cells expressing GFP-Atb2. Left, untreated cells; right, treated with cerulenin. (C) GFP-Atb2 fluorescence intensity in untreated, cerulenin-treated, and DMSO control cells at both the spindle midzone (Spin.) and the nucleoplasm (Nuc). (Control-Spin.: n = 60, N = 12; Control-Nuc.:n= 50, N = 10; DMSO-Spin.: n= 45, N=9; DMSO-Nuc.: n = 45, N = 9; Cer-Spin.: n=55, N = 11; Cer-Nuc.: n = 55, N = 11). (D) Spindle breakdown length in untreated (N=10), DMSO control (N=9) cerulenin-treated (N=13), and cells. (E) Representative images of untreated (left) and cerulenin-treated (right) S. pombe cells expressing GFP-Klp5. (F) Klp5-GFP…

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  Figure 2:. Optical trap assay applies increased force on the spindle through the nuclear envelope. (A) Assay schematic. (B) Color maps tracking lipid vesicle displacement across frames (left) with correlated time lapse of nuclear envelope deformation across (right). (C) Spindle elongation dynamics with optical trap turned off (blue, N=5) versus turned on without force exertion (purple, N=12). Traces show average spindle length, shaded region +/− s.e.m. (D) Change in integrated fluorescence intensity of GFP-Atb2 in cells imaged with optical trap off (N=6), cells imaged with optical trap on (N=5), and nuclei pushed on with the trap (N=10). (E) Representative example paired brightfield images (left) identifying the trapped vesicle (black circle) and scanning confocal images (right) identifying the pushing location (blue arrow). Scale bars, 2 μm; time stamps, min:sec. (F) Violin plots (normalized to same area) showing spindle curvature of spindles of length 5–7 μm without (N = 14, n = 163) and with (N = 10, n = 635) optical trapping. Shaded region refers to mean +/− s.e.m. Outliers (<0.44% of all data), with curvature values greater than 0.3 μm˘1 are not shown. (G) Spindle elongation showing…

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  Figure 3:. Loss of Klp5 minimally affects spindle response to compressive force (A) Representative time-lapse panel of spindle elongation in klp5Δ S. pombe expressing GFP-Atb2 and GFP-Cut11 (MWE75) with no treatment (top), cerulenin treatment (middle) and optical trapping (bottom, circle indicating trapped veiscle). Scale bars, 2 μm; timestamps, min:sec. (B) Left: Fluorescence intensity (of GFP-Atb2) with klp5Δ in untreated (N = 11, n = 55), DMSO control (N = 11, n= 55), and cerulenin-treated (N = 10, n = 50) cells. Lighter color in beehive plot shows all data (n), darker points indicate averages of each individual cell (N), shaded region indicates mean +/− s.e.m.. Right: Average change in fluorescence intensity in klp5Δ spindles subjected to the optical trap (as in Figure 2C) (N = 10). (C) Violin plots showing spindle curvature of klp5Δ cells at 5–7 μm of length under no treatment (magenta, N = 13, n = 130), DMSO treatment (purple, N = 9, n = 124), cerulenin treatment (gold, N = 16, n = 235), and optical trapping (light green, N = 10, n = 828). The area of each violin is normalized to the same area. Shaded region highlights the mean +/− s.e.m. Outliers (< 0.62% of all data), with curvature…

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  Figure 4:. Loss of Klp6 minimally affects spindle response to compressive force. (A) Time-lapse panel of spindle elongation in klp6Δ S. pombe expressing GFP-Atb2 (MWE76) with no treatment (top), cerulenin treatment (middle) and optical trapping (bottom, circle indicates trapped vesicle). Scale bars, 2 μm; timestamps, min:sec. (B) Left: Fluorescence intensity (of GFP-Atb2) in untreated (N = 11, n = 55), DMSO control (N = 10, n = 50), and cerulenin-treated ( N = 10, n = 50) cells. Lighter color in beehive plot shows all data points (n), darker points indicate averages of each individual cell (N), shaded region indicates mean +/− s.e.m of cellular averages σN/N Right: Average change in fluorescence intensity in klp6Δ spindles subjected to the optical trap (as in Figure 3C) (N = 8). (C) Violin plots showing spindle curvature of klp6Δ cells at 5–7 μm in length under no treatment (magenta, N = 12, n = 223), DMSO control (purple, N = 11, n = 157), cerulenin treatment (gold, N = 19, n = 330), and optical trapping (light green, N = 10, n = 1006). Violins are normalized to the same area. Shaded region highlights the mean +/− s.e.m. Outliers (< 0.57% of all data), with curvature values greater than…

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  Figure 5:. While Ase1 is crucial for spindle stability, compressive force rescues spindle and microtubule dynamics in its absence. (A) Representative time-lapse panel of spindle elongation in ase1Δ S. pombe expressing GFP-Atb2 (MWE3) with no treatment (top), cerulenin treatment (middle) and optical trapping (bottom, circle indicates trapped vesicle). Scale bars, 2 μm; timestamps, min:sec. (B) Left: Fluorescence intensity (of GFP-Atb2) in untreated (N = 10, n = 50), DMSO control (N = 9, n = 45, and cerulenin-treated (N= 13, n = 65) cells. Lighter color in beehive plot shows all data (n), darker points indicate averages of each individual cell (N), shaded region indicates mean +/− s.e.m of cellular averages σN/N. Right: Average change in fluorescence intensity in ase1Δ spindles subjected to the optical trap (N = 7). (C) Violin plots showing spindle curvature of ase1Δ cells at 5–7 μm in length under no treatment (magenta, N = 8, n = 118), DMSO treatment (purple, N = 11, n = 84), cerulenin treatment (gold, N = 10, n = 190), and optical trapping (light green, N = 14, n = 1217). Violin are normalized to the same area. Shaded region highlights mean +/− s.e.m. Outliers (<0.57% of all data), with…

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  Figure 6:. Ase1 prevents depolymerization and stabilizes microtubules in response to laser ablation (A) Time-lapse panel of spindle repair response to ablation in cells expressing GFP-Atb2. Control (blue), ase1Δ (orange), klp6Δ (cyan) and klp5Δ (green). Scale bars, 2 μm and timestamps, min:sec. (B) Counts showing splay events for control, ase1Δ klp5Δ, and klp6Δ S. pombe, with Ase1 displaying significantly more splaying events. (C) The change in spindle half length in control (blue, N = 13, n = 26), klp5Δ (green, N = 11, n = 21), ase1Δ (orange, N = 13, n = 20), and klp6Δ (cyan, N = 26, n = 52). Traces represent the average change in spindle half length +/− s.e.m. (shaded region). Traces are aligned such that t=0 is the frame after ablation is complete.

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  Figure 7:. In normal cells, compressive force reduces spindle bending and causes a reduction in microtubule intensity and increased recruitment of Ase1 and Klp5 to the midzone. In klp5Δ and klp6Δ cells, the mechanical response is unchanged relative to control spindles. In contrast, with ase1Δ, compressive force induces an increase in microtubule intensity. The mechanism by which force triggers this biochemical response remains an open question for future research.