Figure 4.

NM-II Motor Activity Is Required for Midbody Maturation and the Generation of a Site of Constriction

(A) The sites of constriction are abolished by treatment with 7.5 μM Blebbistatin. HeLa-Kyoto cells were synchronized sequentially with thymidine, nocodazole, and MG132, and then released into fresh medium for 45 min. Two aliquots of these cells were treated with DMSO or 7.5 μM Blebbistatin for 60 min (early midbody stage) or 120 min (late midbody stage), respectively, before being fixed and stained with anti-Cep55 (green) and anti-α-tubulin (red) antibodies. Arrows, sites of constriction; arrowheads, the site of abscission. Scale bar, 5 μm.

(B) Measurements of the diameter of the midbody, the diameter at the site of constriction, as well as the distance between these cellular structures in DMSO- or Blebbistatin-treated cells during the early and late midbody stages. The samples used for this quantification include the cells synchronized at the early and late midbody stages that were double-stained with an anti-α-tubulin (red) antibody, in combination with an anti-Cep55 (green) (see A), anti-NM-IIA (green) (see Figure 5A), anti-NM-IIB (green) (see Figure 5B), phalloidin (green) (see F-actin in Figure 5C), or anti-Sept9 antibody (green) (see Figure 5D). MD, midbody; SOC, site of constriction; iSOC, illusionary SOC.

(C) Addition of Blebbistatin before the start of furrowing or at the end of furrowing causes furrow regression or a delay in abscission, respectively. HeLa cells stably expressing mCherry-H2B and EGFP-α-tubulin were treated with either DMSO or 7.5 μM Blebbistatin at the indicated times and followed by time-lapse microscopy. Maximum projection of EGFP-α-tubulin (12 z-sections with the step size of 0.7 μm) for a representative cell of each category is shown here. Regression was judged based on both the bright-field and the EGFP-α-tubulin images. Arrowhead indicates the site of constriction that becomes the site of abscission.

(D) NM-II motor activity is required for the thinning of the ICB. The same images as described in (C) were used for measuring the diameter at the midpoint of the spindle as well as the diameter at the thinnest part near the midpoint of the MT array at the ICB (presumably the “site of constriction” in DMSO-treated cells). Individual traces for individual cells of indicated categories are presented here. Time point “0” is the time when furrow ingression was completed (based on bright-field images).

(E) The same data from (D) are presented as mean ± SD.