Figure S5.
Reducing dynein-driven cortical astral MT-based pulling forces can rescue central spindle assembly in hcp-1Δ but not hcp-1/2 codisrupted embryos. (A) Representative image (left; white dashed line outlines the embryo) showing method used to measure chromosome segregation rates in B and schematic (center) showing the balance between central spindle versus dynein-driven cortical astral MT-based pulling forces. Inset (right) depicts the molecular players in dynein-driven cortical astral MT-based pulling forces. (B) Measurement of chromosome separation as a proxy for the mechanical integrity of the central spindle in embryos expressing mCherry::H2B and GFP::PHPLC1δ1 (see Fig. 3 A). Time is relative to chromosome segregation, and n is listed to the right of the graph in the indicated color for each genotype [n = 13 control, n = 10 hcp-1(RNAi); hcp-2Δ, n = 14 hcp-1Δ; hcp-2(RNAi), n = 12 hcp-1Δ, n = 8 hcp-2Δ, n = 9 spd-1(oj5ts), n = 9 cls-2(RNAi), and n = 9 zen-4(or153ts)]; error bars represent the SEM. (C) Schematic of method used for quantification of AIR-2Aurora-B::GFP fluorescence intensity at the central spindle in D and E. (D) Representative images at 45 s after chromosome segregation of control (top; gray outline), hcp-1Δ; hcp-2(RNAi) (middle; red outline), and hcp-1Δ (bottom; orange outline) embryos expressing AIR-2Aurora-B::GFP (green) and mCherry::H2B (magenta) with and without disruption of cortical pulling forces via RNAi-mediated depletion of LIN-5NuMA or GPR-1/2. Note that central spindle assembly is rescued upon depletion of LIN-5NuMA or GPR-1/2 in hcp-1Δ but not in hcp-1Δ; hcp-2(RNAi) codisrupted embryos, which do not assemble central spindle MTs (Maton et al., 2015). Representative individual line scans of AIR-2Aurora-B::GFP (green) and mCherry::H2B (magenta) are shown to the right of each image. (E) Quantification of AIR-2Aurora-B::GFP levels at the spindle midzone 45 s after chromosome segregation in control (left), hcp-1Δ; hcp-2(RNAi) (middle), and hcp-1Δ (right) embryos with and without RNAi-mediated depletion of LIN-5NuMA or GPR-1/2. (F) Measurement of chromosome separation with and without cortical astral MT-pulling forces (see A). Time is relative to chromosome segregation, and control(RNAi) analysis is included on all graphs for comparison. Keys (right) indicate genotype; n is listed to the right of graphs in the indicated color for each genotype [n = 12 control(RNAi), n = 8 lin-5(RNAi), n = 6 gpr-1/2(RNAi), n = 13 hcp-1Δ; hcp-2(RNAi); control(RNAi), n = 7 hcp-1Δ; hcp-2(RNAi); lin-5(RNAi), n = 9 hcp-1Δ; hcp-2(RNAi); gpr-1/2(RNAi), n = 8 hcp-1Δ; control(RNAi), n = 10 hcp-1Δ; lin-5(RNAi), n = 7 hcp-1Δ; gpr-1/2(RNAi)]; error bars represent the SEM (E and F). All fluorescence intensity analysis was done on sum projections of all Z-planes. All scale bars, 10 µm.

Reducing dynein-driven cortical astral MT-based pulling forces can rescue central spindle assembly in hcp-1Δ but not hcp-1/2 codisrupted embryos. (A) Representative image (left; white dashed line outlines the embryo) showing method used to measure chromosome segregation rates in B and schematic (center) showing the balance between central spindle versus dynein-driven cortical astral MT-based pulling forces. Inset (right) depicts the molecular players in dynein-driven cortical astral MT-based pulling forces. (B) Measurement of chromosome separation as a proxy for the mechanical integrity of the central spindle in embryos expressing mCherry::H2B and GFP::PHPLC1δ1 (see Fig. 3 A). Time is relative to chromosome segregation, and n is listed to the right of the graph in the indicated color for each genotype [n = 13 control, n = 10 hcp-1(RNAi); hcp-2Δ, n = 14 hcp-1Δ; hcp-2(RNAi), n = 12 hcp-1Δ, n = 8 hcp-2Δ, n = 9 spd-1(oj5ts), n = 9 cls-2(RNAi), and n = 9 zen-4(or153ts)]; error bars represent the SEM. (C) Schematic of method used for quantification of AIR-2Aurora-B::GFP fluorescence intensity at the central spindle in D and E. (D) Representative images at 45 s after chromosome segregation of control (top; gray outline), hcp-1Δ; hcp-2(RNAi) (middle; red outline), and hcp-1Δ (bottom; orange outline) embryos expressing AIR-2Aurora-B::GFP (green) and mCherry::H2B (magenta) with and without disruption of cortical pulling forces via RNAi-mediated depletion of LIN-5NuMA or GPR-1/2. Note that central spindle assembly is rescued upon depletion of LIN-5NuMA or GPR-1/2 in hcp-1Δ but not in hcp-1Δ; hcp-2(RNAi) codisrupted embryos, which do not assemble central spindle MTs (Maton et al., 2015). Representative individual line scans of AIR-2Aurora-B::GFP (green) and mCherry::H2B (magenta) are shown to the right of each image. (E) Quantification of AIR-2Aurora-B::GFP levels at the spindle midzone 45 s after chromosome segregation in control (left), hcp-1Δ; hcp-2(RNAi) (middle), and hcp-1Δ (right) embryos with and without RNAi-mediated depletion of LIN-5NuMA or GPR-1/2. (F) Measurement of chromosome separation with and without cortical astral MT-pulling forces (see A). Time is relative to chromosome segregation, and control(RNAi) analysis is included on all graphs for comparison. Keys (right) indicate genotype; n is listed to the right of graphs in the indicated color for each genotype [n = 12 control(RNAi), n = 8 lin-5(RNAi), n = 6 gpr-1/2(RNAi), n = 13 hcp-1Δ; hcp-2(RNAi); control(RNAi), n = 7 hcp-1Δ; hcp-2(RNAi); lin-5(RNAi), n = 9 hcp-1Δ; hcp-2(RNAi); gpr-1/2(RNAi), n = 8 hcp-1Δ; control(RNAi), n = 10 hcp-1Δ; lin-5(RNAi), n = 7 hcp-1Δ; gpr-1/2(RNAi)]; error bars represent the SEM (E and F). All fluorescence intensity analysis was done on sum projections of all Z-planes. All scale bars, 10 µm.

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