Figure 1.
M-phase kinases are present and properly localized on mitotic spindles formed in G1 daughters with impaired APC-Cdh1 activity.
(A) Histogram of G1-phase duration in daughters of CDK1WT-expressing HeLa cells. (B) Histogram of G1-phase duration in daughters of CDK1AF-expressing HeLa cells. (C) CDK1WT M-phase cell and M-phase-like G1 cell (CDK1AF daughter) probed for Aurora A, (D) Aurora B, and (E) Plk1; mCherry-α-tubulin (red) and kinases (green). (F) Pseudo-color scatter plots of DNA content vs. phospho-histone H3 (pHH3) content in 50000 CDK1WT-transfected (left) or CDK1AF-transfected (right) HeLa cells, with DNA content histogram. (G) Histogram of Cdc20 contents in cell cycle phases of CDK1WT-expressing cells and the low DNA-content/high pHH3-content CDK1AF-expressing cells (left); same histogram shown as percent of max (right).
Figure 2.
Bipolar spindle maintenance does not require completed DNA replication or condensation.
(A) CDK1-CFP, microtubules (MT), DNA, and the overlay of MT and DNA in CDK1WT mitotic cells and daughters of CDK1AF expressers. Microtubules (green) and DNA (blue) are shown. (B) Histogram of spindle morphology observed in M-phase-like G1 cells (CDK1AF daughters), with percent bipolar (light gray) and monopolar spindles (dark gray). (C) Kinetochores (ACA; magenta), microtubules (MT), and the overlay of both of these plus DNA from cells in the same population described in (A). Scale bars = 10 µM.
Figure 3.
Premature spindle formation in G1 cells requires CDK1 and AurB activities.
(A) Representative mitotic (M) and interphase (I) CDK1WT-expressing cells and a G1 cell produced by division of a CDK1AF-expresser (CDK1AF daughter), presenting microtubules (MT; red), DNA (blue), and nuclear pore complexes (Mab414; green). (B) Different cells from the population in (A), probed for pHH3 content (green). (C) Histogram of percent CDK1WT mitotic or CDK1AF M-phase-like G1 cells with spindles and/or pHH3 content after DMSO or roscovitine treatment. (D) Histogram of percent CDK1WT mitotic or CDK1AF M-phase-like G1 cells with spindles and/or pHH3 content after DMSO or hesperadin treatment. Unsynchronized HeLa cells stably expressing histone H2B-GFP (green) and mCherry-α-tubulin (red) were transfected with CFP-CDK1AF, then 24 h later were treated with DMSO, roscovitine, or hesperadin, and imaged live for 75 min (E–G). (E) M-phase-like G1 cell treated with DMSO. (F) M-phase-like G1 cell treated with roscovitine. (G) M-phase-like G1 cell treated with hesperadin. Scale bars in (A–C) = 10 µM. White arrowheads in (E–G) indicate prematurely formed spindle; gray arrowheads in (F–G) indicate last image of apparent spindle structure.
Figure 4.
Mad2 stabilizes prematurely formed spindles in M-phase-like G1 cells.
(A) Mad2 (green) with kinetochores (ACA; magenta) and DNA (blue) (left), or their overlay of MT (green) and DNA with kinetochores (ACA; magenta) (right) in a representative CDK1WT mitotic cell (top) and daughter produced by division of a CDK1AF-expresser (bottom). Scale bar = 10 µM. (B) Mad2 (top) and tubulin (bottom) immunoblots in a cell line stably expressing histone H2B-GFP (green) and mCherry-α-tubulin (red), following co-transfection with CFP-CDK1AF and Diced siRNA pools to firefly luciferase (GL3) or human Mad2 3′ UTR. (C) Montage of live-cell images from transfections described in (B) showing a representative GL3 d-siRNA-treated cell (top) and Mad2 d-siRNA-treated cell (bottom). White arrowheads indicate prematurely formed spindle; gray arrowheads indicate last image of apparent spindle structure. (D) Scatter plot of spindle duration times for CDK1AF/GL3-siRNA (dark gray) and CDK1AF/Mad2-siRNA (light gray) co-transfected cells (left) and box plot (right). Transfections and imaging were performed in triplicate (N = 3; nGL3 and nMad2 = 167 representative cells). GL3-siRNA spindle duration: range, 60–990 min; first quartile, 225 min; median 345 min; third quartile, 450 min. Mad2-siRNA spindle duration: range, 30–240 min; first quartile, 45 min, median, 60 min; third quartile, 75 min.
Figure 5.
Mad2 knockdown in G1 cells with impaired APC-Cdh1 activity increases cyclin B1-YFP oscillation frequency.
(A) Box plots of cyclin B1-YFP oscillation peak frequencies in DMSO- and nocodazole-treated cells, respectively. DMSO (oscillations per experiment (OPE)): range, 0.92–8.35; first quartile, 3.71; median, 5.56, third quartile, 6.49. Nocodazole (OPE): range, 0.92–3.71; first quartile, 1.85; median, 1.85; third quartile, 2.78. (B) Box plots of cyclin B1-YFP oscillation amplitudes in DMSO- and nocodazole-treated cells, respectively. DMSO (relative fluorescence units (RFU)): range, 1.9–7.3; first quartile, 2.7; median, 3.6, third quartile, 4.5. Nocodazole (RFU): range, 2.4–10.9; first quartile, 4.4; median, 6.0; third quartile, 8.1. (C, D) Representative cells oscillating at the median frequency in the DMSO and nocodazole treatments, respectively. (E) Box plots of cyclin B1-YFP oscillation peak frequencies in DMSO- and hesperadin-treated cells. DMSO (OPE): range, 0.96–6.74; first quartile, 1.92; median, 2.88, third quartile, 3.85. Hesperadin (OPE): range, 0.96–7.7; first quartile, 2.4; median, 3.85; third quartile, 5.77. (F) Box plots of cyclin B1-YFP oscillation amplitudes in DMSO- and hesperadin-treated cells, respectively. DMSO (RFU): range, 2.0–8.5; first quartile, 3.9; median, 4.4, third quartile, 5.8. Hesperadin (RFU): range, 1.0–11.2; first quartile, 3.5; median, 4.8; third quartile, 6.6. (G, H) Representative cells oscillating at median frequency in the DMSO and hesperadin treatments, respectively. (I) Box plots of cyclin B1-YFP oscillation peak frequencies in GL3- and Mad2 d-siRNA-treated cells. GL3 (OPE): range, 0.99–6.94; first quartile, 1.98; median, 2.97, third quartile, 4.96. Mad2 (OPE): range, 0.99–8.92; first quartile, 3.96; median, 4.96; third quartile, 5.95. (J) Box plots of cyclin B1-YFP oscillation amplitudes in GL3- and Mad2 d-siRNA-treated cells, respectively. GL3 (RFU): range, 3.5–32.4; first quartile, 9.9; median, 18.0, third quartile, 23.2. Mad2 (RFU): range, 0.9–14.9; first quartile, 2.9; median, 6.9; third quartile, 8.3. (K, L) Representative cells oscillating at median frequency in the GL3- and Mad2-d-siRNA treatments, respectively. In (A), (F), and (J), outliers that are at least 1.5 times the interquartile distance away from their respective quartiles are presented as circles.
Figure 6.
Premature M-phase initiation can be reduced in G1 cells with impaired APC-Cdh1 activation by enforced non-degradable Wee1 expression.
(A) Fifty-thousand CDK1WT-transfected cells and (B) 50000 CDK1AF-transfected cells on a pseudo-color scatter plot of PI (abscissa) and pHH3 content (ordinate). High DNA and high pHH3 content (M phase) gates (blue “M”) and low DNA and high pHH3 (abnormal G1/S cells) gates (green “G1/S”) are shown with their respective frequencies. (C) Cells gated in (A) are overlaid on a pseudo-color scatter plot of PI (abscissa) and Wee1 content (ordinate) of CDK1WT-transfected cells. (D) Cells gated in (B) are overlaid on a pseudo-color scatter plot of PI (abscissa) and Wee1 content (ordinate) of CDK1AF-transfected cells. The M-phase Wee1 level (gray dashed line) is defined in both (C) and (D) by the horizontal axis centered on the Wee1-stained M-phase population that was gated in (A). (E) Histogram of relative Wee1 contents in CDK1AF-transfected cells in normal S phase (pHH3-negative; red), in normal M phase (pHH3-positive; blue), and in precocious M phase (G1-S DNA content, pHH3-positive; green) (F) Histogram of percent oscillating daughters generated in HeLa cells co-transfected with cyclin B1-YFP and CDK1AF alone, or together along with ΔN214Wee1.
Figure 7.
Cyclin B1 overexpression and proteasome inhibition fails to induce premature mitosis in Cdh1-ablated cells.
(A) Western blots for Cdh1, cyclin B1 (CycB), and tubulin, of lysates from cells transfected with GL2 or Cdh1 siRNAs, followed by transfection with cyclin B1-YFP (CycB1-YFP) and/or treatment with proteasome inhibitor MG132. One asterisk indicates endogenous CycB, two asterisks indicates ectoptic CycB1-YFP. (B) Cell cycle distribution of GL2 and Cdh1 siRNA-transfected cells from (A) as determined by PI staining followed by flow cytometry. (C) Fifty thousand GL2-siRNA-transfected cells (top) and Cdh1-siRNA-transfected cells (bottom), either untreated or treated with MG132 and/or transfected with CycB1-YFP, on pseudo-color scatter plots of PI (abscissa) and pHH3 content (ordinate). High DNA and high pHH3 (M phase) gates (blue “M”) and low DNA and high pHH3 (abnormal G1/S cells) gates (green “G1/S”) are shown with their respective cell frequencies.
Figure 8.
Overview of stabilized APC-Cdh1 targets and the absence of Wee1 in G1 daughters following division of cells with or without CDK1AF expression.
Dark gray inhibitory symbols represent complete activation of APC-Cdh1; light gray inhibitory symbols (with CDK1AF expression) and light gray APC-Cdh1 represent a lack of sustained activation. Asterisks represent active CDK.