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, 28 (25), 3634-3646

Two Mechanisms Coordinate the Recruitment of the Chromosomal Passenger Complex to the Plane of Cell Division

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Two Mechanisms Coordinate the Recruitment of the Chromosomal Passenger Complex to the Plane of Cell Division

Jennifer Landino et al. Mol Biol Cell.

Abstract

During cytokinesis, the chromosomal passenger complex (CPC) promotes midzone organization, specifies the cleavage plane, and regulates furrow contractility. The localizations of the CPC are coupled to its cytokinetic functions. At the metaphase-to-anaphase transition, the CPC dissociates from centromeres and localizes to midzone microtubules and the equatorial cortex. CPC relocalization to the cell middle is thought to depend on MKlp2-driven, plus end-directed transport. In support of this idea, MKlp2 depletion impairs cytokinesis; however, cytokinesis failure stems from furrow regression rather than failed initiation of furrowing. This suggests that an alternative mechanism(s) may concentrate the CPC at the division plane. We show here that direct actin binding, via the inner centromere protein (INCENP), enhances CPC enrichment at the equatorial cortex, thus acting in tandem with MKlp2. INCENP overexpression rescues furrowing in MKlp2-depleted cells in an INCENP-actin binding-dependent manner. Using live-cell imaging, we also find that MKlp2-dependent targeting of the CPC is biphasic. MKlp2 targets the CPC to the anti-parallel microtubule overlap of the midzone, after which the MKlp2-CPC complex moves in a nondirected manner. Collectively, our work suggests that both actin binding and MKlp2-dependent midzone targeting cooperate to precisely position the CPC during mitotic exit, and that these pathways converge to ensure successful cleavage furrow ingression.

Figures

FIGURE 1:
FIGURE 1:
CPC localization to the division plane requires both MTs and actin. (A) Schematic of MT populations within the division plane of a bipolar C-phase cell. Midzone MTs (green) create a barrier between segregated chromosomes, whereas furrow-associated astral MTs extend toward the equatorial cortex (pink). These MTs can associate end-on (red) or laterally (purple) with the cortex. (B) Volume projection of an anaphase HeLa cell rotated 0° (XY dimension), 30°, 60°, and 90° (YZ dimension) and fixed and stained for tubulin (green) and ABK (red). Scale bar, 10 μm. (C) Maximum z-projections of the XY dimensions (top) and volume projections of ABK in the XY and YZ dimensions (middle) of cells treated with DMSO, 5 μg/ml cytochalasin B, 5 μM nocodazole, or both drugs simultaneously. Cells were stained with antibodies to tubulin (green) and ABK (red). DNA was counterstained with Hoechst 33342 (blue). Scale bars, 10 μm. Bottom, fold enrichment of ABK immunofluorescence parallel to the spindle axis for each condition. Immunofluorescence was measured using line scans 100 pixels wide (∼6 µm) and 230 pixels long (∼14 µm). Data represent the mean ± SD; n = 7 (DMSO), 6 (cytochalasin B), 7 (nocodazole), and 7 (cytochalasin + nocodazole) cells.
FIGURE 2:
FIGURE 2:
The CPC localizes to the division site in the cells depleted of MKlp2. (A) Left, maximum z-projections of HeLa cells transfected with control (top) or MKlp2 (bottom) siRNA. Cells were stained with antibodies to tubulin (green), MKlp2 (teal), and ABK (red). DNA was counterstained with Hoechst 33342. Dashed boxes indicate sample regions used for quantitation of ABK fluorescence intensity. Dashed lines represent 10-pixel-wide (∼0.5 µm) line scans. Scale bar, 10 μm. Right, line scans of ABK fluorescence intensity along the spindle axis in cells treated with control or MKlp2 siRNA. AU, arbitrary units. (B) Quantification of ABK fluorescence intensity at the division plane in cells treated as described in A. Data represent the mean ± SE, n > 50 cells from three independent experiments, *p < 0.05. (C) Top, volume projections (YZ dimensions) of the division plane of cells shown in A. Dashed lines represent 10-pixel-wide (∼0.5 µm) line scans. Scale bars, 10 μm. Bottom, line scans of MKlp2 and ABK fluorescence intensity across the YZ projection of the division plane. (D) Maximum z-projections of HeLa cells transfected with control or MKlp2 siRNA and treated with DMSO or 5 μg/ml cytochalasin B (Cyto B). Cells were stained with antibodies to tubulin (green), MKlp2 (teal), and ABK (red). DNA (blue) was counterstained with Hoechst 33342. Scale bar, 10 μm.
FIGURE 3:
FIGURE 3:
Cortical enrichment of GFP-INCENP at the division plane requires MKlp2 and INCENP-actin binding. (A) Top, single z-plane micrographs in the XY and YZ dimensions of the division plane in a HeLa cell expressing GFP-INCENP treated with control siRNA. Pink arrowhead denotes cortical GFP-INCENP fluorescence; yellow arrowhead marks GFP-INCENP fluorescence on a midzone bundle. Scale bars, 10 μm. Middle, line scans across XY or YZ projections of the division plane in the cell shown above. Bottom, fold enrichment of GFP-INCENP fluorescence intensity, relative to cytoplasmic fluorescence, from line scans across the YZ projection of cells treated with control siRNA. Asterisks mark peaks of GFP-INCENP localized to midzone bundles. n = 10 cells. (B) Top, single z-plane micrographs in the XY and YZ dimensions of the division plane in a cell expressing GFP-INCENP treated with MKlp2 siRNA. Pink arrowhead denotes cortical GFP-INCENP fluorescence. Scale bars, 10 μm. Middle, line scans across XY or YZ projections of the division plane in the cell shown above. Bottom, fold enrichment of GFP-INCENP fluorescence intensity, relative to cytoplasmic fluorescence, from line scans across the YZ projection of cells treated with MKlp2 siRNA. (C) Top, single z-plane micrographs in the XY and YZ dimensions of the division plane in a HeLa cell expressing GFP-INCENP CR treated with control siRNA. Pink arrowhead denotes cortical GFP-INCENP CR fluorescence; yellow arrowhead marks GFP-INCENP CR fluorescence on a midzone bundle. Scale bars, 10 μm. Middle, line scans across XY or YZ projections of the division plane in the cell shown above. Bottom, fold enrichment of GFP-INCENP CR fluorescence intensity, relative to cytoplasmic fluorescence, from line scans across the YZ projection of cells treated with control siRNA. Asterisks mark peaks of GFP-INCENP CR localized to midzone bundles. n = 10 cells. (D) Top, single z-plane micrographs in the XY and YZ dimensions of the division plane in a HeLa cell expressing GFP-INCENP CR treated with MKlp2 siRNA. Scale bars, 10 μm. Middle, line scans across XY or YZ projections of the division plane in the cell shown above. Bottom, fold enrichment of GFP-INCENP CR fluorescence intensity, relative to cytoplasmic fluorescence, from line scans across the YZ projection of cells treated with MKlp2 siRNA. n = 9 cells. (E) Quantification of cortical peak intensities of GFP-INCENP or GFP-INCENP CR from line scans across the YZ projection as described in A–D. Three adjacent peak intensity values were averaged for each individual cell. n ≥ 9 cells,*p < 0.05.
FIGURE 4:
FIGURE 4:
The CPC localizes to the early midzone at anaphase onset. (A) Single z-plane micrographs taken from a time-lapse movie of a HeLa cell transiently expressing GFP-INCENP (green) and MKlp2-mCherry (red). Time is indicated in seconds relative to anaphase onset (0 s). Dashed lines indicate regions used to generate line scans. Scale bar, 10 μm. (B) Line scans of GFP-INCENP fluorescence intensity on the midzone and the cortex as indicated in A. (C) Single z-plane micrographs taken from a time-lapse movie of HeLa cells transiently expressing GFP-INCENP and treated with control or MKlp2-targeting siRNA. Time is indicated in seconds relative to anaphase onset (0 s). Scale bar, 10 μm. (D) Single z-plane micrographs taken from a time-lapse movie of a HeLa cell transiently expressing GFP-PRC1 (green) and MKlp2-mCherry (red). Time is indicated in seconds relative to anaphase onset (0 s). Scale bar, 10 μm. (E) Single z-plane micrographs taken from a time-lapse movie of a HeLa cell transiently expressing GFP-KIF4A (green) and MKlp2-mCherry (red). Time is indicated in seconds relative to anaphase onset (0 s). Scale bar, 10 μm. (F) Top, maximum intensity projections of three 1-µm optical sections taken from a time-lapse movie of a HeLa cell stably expressing INCENP-PA-GFP (green) after 48 h in 125 ng/ml doxycycline. Chromosomes were visualized using DIC. Time is indicated in seconds relative to photo activation (0 s). The final frame shows total photoactivation of INCENP. Dashed line represents region used to generate line scans. Bottom, line scans represent INCENP-PA-GFP fluorescence over time along the spindle midzone after anaphase onset. (G) Fold enrichment of INCENP-PA-GFP fluorescence parallel to the spindle axis at 30 s (red) or 120 s (blue) after photoactivation and total cellular INCENP (green). Schematic indicates the orientation of the line scan. Data represent the mean ± SD, n = 6 cells. (H) Fold enrichment of INCENP-PA-GFP fluorescence parallel to the division plane at 30 s (red) or 120 s (blue) after photoactivation and total cellular INCENP (green). Schematic indicates the orientation of the line scan. Data represent the mean ± SD, n = 6 cells.
FIGURE 5:
FIGURE 5:
GFP-INCENP and MKlp2-mCherry are colocalized on the midzone throughout mitotic exit. (A) Single z-plane micrographs of the midzone taken from a time-lapse movie of a HeLa cell transiently expressing GFP-INCENP and MKlp2-mCherry plated on 100 µg/ml fibronectin. Time is indicated in minutes:seconds relative to anaphase onset (0:00). Scale bar, 10 μm. (B) Top, single z-plane micrograph taken from the time-lapse movie shown in A representing a time point 6 min after anaphase onset. The “tracks” panel highlights examples of spots and tracks used to quantify MSD. Numbers indicate tracks that correspond to kymographs below. Scale bar, 10 μm. Bottom, kymographs of GFP-INCENP and MKlp2-mCherry fluorescence generated from the spots indicated above. Scale bars, 100 s (y-axis) and 1 μm (x-axis). (C) Pearson’s r of GFP-INCENP and MKlp2-mCherry on the midzone (colored circles). Colocalization was quantified from immediately after anaphase onset (t = 0) to immediately before cleavage furrow ingression (t = 1). Control cells expressing GFP-INCENP and mCherry are represented by grayscale circles. n = 3 cells (control) and 5 cells (experimental) from three independent experiments. (D) MSD of GFP-INCENP (green circles) and MKlp2-mCherry (red circles) spots in the cortical plane and in the midzone during anaphase. Data represent mean ± SE. Linear regression represents best fit used to calculate the diffusion coefficient. Diffusion coefficient = 1.9 × 10−3 µm2/s (GFP-INCENP) or 2.1 × 10−3 µm2/s (MKlp2-mCherry). n = 626 tracks (GFP-INCENP) or 424 tracks (MKlp2-mCherry) from six cells from three independent experiments.
FIGURE 6:
FIGURE 6:
GFP-INCENP and MKlp2-mCherry are colocalized in the plane of the cell cortex. (A) Single z-plane micrographs in the plane of the cell cortex taken from a time-lapse movie of a HeLa cell transiently expressing GFP-INCENP and MKlp2-mCherry plated on 100 µg/ml fibronectin. Time is indicated in minutes:seconds relative to anaphase onset (0:00). Scale bar, 10 μm. (B) Top, single z-plane micrographs in the plane of cell cortex taken from a time-lapse movie of a HeLa cell transiently expressing GFP-INCENP and MKlp2-mCherry plated on 100 μg/ml fibronectin. Micrographs represent a time point 6 min after anaphase onset. The “tracks” panel highlights examples of spots and tracks used to quantify MSD. Numbers indicate tracks that correspond to kymographs below. Scale bar, 10 μm. Bottom, kymographs of GFP-INCENP and MKlp2-mCherry generated from the spots indicated above. Scale bars, 100 s (y-axis) and 1 μm (x-axis). (C) Pearson’s r of GFP-INCENP and MKlp2-mCherry in the plane of the cell cortex (colored circles). Colocalization was quantified from immediately after anaphase onset (t = 0) to immediately before cleavage furrow ingression (t = 1). Control cells expressing GFP-INCENP and mCherry are represented by grayscale circles. n = 3 (control) and 5 cells (experimental) from three independent experiments. (D) MSD of GFP-INCENP (green circles) and MKlp2-mCherry (red circles) spots in the cortical plane during C phase. Data represent mean ± SE. Linear regression represents best fit used to calculate the diffusion coefficient. n = 625 tracks (GFP-INCENP) or 537 tracks (MKlp2-mCherry) from six cells from three independent experiments.
FIGURE 7:
FIGURE 7:
Overexpression of GFP-INCENP rescues cleavage furrow ingression in MKlp2-depleted cells. (A) Top, single z-plane micrographs taken from time-lapse movies of cells transiently expressing mCherry-Utrophin after treatment with a control or MKlp2-targeting siRNA. Time is indicated in minutes:seconds. Dashed lines were used to generate kymographs. Scale bar, 10 μm. Bottom, kymographs of mCherry-Utrophin fluorescence across the division plane. Yellow arrows represent cleavage furrow regression. Scale bars, 2.5 min (y-axis) and 10 μm (x-axis). (B) Top, single z-plane micrographs taken from time-lapse movies of cells transiently expressing GFP-INCENP after treatment with a control or MKlp2-targeting siRNA. Time is indicated in minutes:seconds. Dashed lines were used to generate kymographs. Scale bar, 10 μm. Bottom, kymographs of GFP-INCENP fluorescence across the division plane. Scale bars, 2.5 min (y-axis) and 10 μm (x-axis). (C) Top, single z-plane micrographs taken from time-lapse movies of cells transiently expressing GFP-INCENP CR after treatment with a control or MKlp2-targeting siRNA. Time is indicated in minutes:seconds. Dashed lines were used to generate kymographs. Scale bar, 10 μm. Bottom, kymographs of GFP-INCENP CR fluorescence across the division plane. Scale bars, 2.5 min (y-axis) and 10 μm (x-axis). (D) Quantification of the percentage of cells from A that successfully completed furrow ingression. n = 12 (control) and 15 (MKlp2 siRNA) cells, *p < 0.05. (E) Quantification of total percentage of cells from B that successfully completed furrow ingression. n = 14 cells for each condition, n = 8 (control) and 12 (MKlp2 siRNA) cells. (F) Quantification of total percentage of cells from C that successfully completed furrow ingression. n = 14 cells for each condition, *p < 0.05.

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