Primary human fibroblasts progress through the S and G₂ phases and most of mitosis without cell anchorage. (A–D) NHDF were synchronized at the G₁–S phase boundary by aphidicolin and released to progress through S and G₂ phases either anchored (A) or in suspension (S). Cells were harvested at the indicated time points. Progression through S phase was analyzed by BrdU incorporation by flow cytometry (FACS), with the cell cycle profile determined as in the top exemplary plot (36) and the profiles for the below-indicated time points, with indicated percentage numbers for the distribution in G₀/G₁, S, and G₂/M phases, at 26 h after release (A) or by immunocytochemistry on cytospun samples (B). (C) Progression through G₂ and M phase was analyzed by quantifying propidium iodide FACS profiles. Shown is one representative of three experiments. (D) The fractions of cells in G₂ phase (G₂) and early mitosis (M) were analyzed by immunofluorescence detecting cyclin B1 (red) and BrdU (green). The lower images show representative examples of the immunostainings; G₂ phase cells (G₂) were determined as cells with cytoplasmic cyclin B1 without BrdU indicated by yellow arrows, cells in early mitosis (M) with nuclear cyclin B1 indicated by white arrows. The graph illustrates quantification over time after release of at least 400 cells for each condition. (E–G) NHDF were synchronized by mitotic shake-off and released either with anchorage or in suspension. (E) At 20–50 min after release, cells in suspension were harvested, spun down on glass slides, and stained to detect tubulin (green), F-actin (red), and DNA (blue). (F) The percentage of mitotic cells with central spindle or cleavage furrow was quantified; at least 100 mitotic cells were evaluated for each condition. (G) The localization of RhoA (green) and Aurora B (red) in the central spindle indicated by arrows was analyzed 1 h after release either with anchorage (A) or in suspension (S). The nucleus is in blue. (Scale bars, 10 μm.)

Cells in suspension initiate but cannot complete ingression of the cleavage furrow. NHDF were synchronized by mitotic shake-off and released with anchorage or in suspension. (A) Cells were stained to detect tubulin (green), F-actin (red), and DNA (blue). (Upper) Two representative images of binuclear cells with decondensed chromatin detected after 50 min in suspension. (Lower) Graph shows the percentage of binuclear cells. Shown is one representative of three experiments. One hundred cells were evaluated at each time point. (B–D) Cells were stained with Giemsa. B shows how the measurements of the cell waist width (M) and diameter (D) of the connected cells were performed. C shows the mean cell waist width (M) of anchored and suspended cells 30 min after release displayed as means ± SD; P < 0.0001. D shows the extent of ingression of the cleavage furrow measured 20 and 30 min after shake-off in cells incubated anchored or suspended; results displayed as the ratio between the cell waist width (M) and the mean of the two cell diameters of the connected cells (D) and is displayed as the percentage of cells in different ratio ranges. Forty to 50 cells were evaluated for each condition.

Cell attachment supports cleavage furrow ingression independent of cell spreading. (A and B) BJT fibroblasts were synchronized by shake-off and released onto the different substrata collagen type I (CI), vitronectin (VN), or Poly-l-lysine (PLL), or were kept in suspension (Susp). (A) Progression through mitosis and cytokinesis is displayed as percentage of cells in G₂/M phases analyzed 100 min after shake-off by flow cytometry of propidium iodide stainings. To enable comparison between experiments, the fraction of cells in G₂/M phases after release is expressed in relation to this fraction at the start, which was defined as 100%. Average of three experiments ± SD is shown, and statistically significant differences compared with suspension were evaluated by Student t test and are indicated; *, P < 0.05. (B Top and Middle) Representative images of BJT cells on PLL and CI at the time of maximal ingression as indicated (min) (Left) and 60 min after replating (Right). Arrows show the cleavage furrows. (Bottom) Example as above representing the fraction of the cells (≈30%) where only one of the forming cells was attached during the ingression of the furrow. Movies of these and cells on other substrates are found in SI Movies 1–4. (C) NHDF were synchronized by mitotic shake-off and released onto immobilized anti-integrin β1 mAb P4C10, anti-integrin α5β1 mAb JBS5, mouse IgG (IgG), or FCS as a control. Cells on IgG did not attach. Progression through mitosis and cytokinesis is displayed as the fraction of cells in G₂/M phases as determined 90–100 min after shake-off as in A.

Oncogenic H-Ras can override the requirement for cell anchorage during cytokinesis. (A) Exponentially growing primary fibroblasts (NHDF), M21 melanoma cells, PC3 prostate cancer cells, and B25 H-Ras-overexpressing rat embryonic fibroblasts were replated either with anchorage (A) or in suspension (S). After 24 h, the cell cycle phase distributions were analyzed by flow cytometry of propidium iodide stainings, displayed as a percentage of cells in each phase. Shown is one representative of three experiments. (B) BJT cells and derivatives thereof were synchronized with nocodazol, followed by shake-off and replating for 2 h either with anchorage (A) or in suspension (S). Cell cycle distribution was analyzed as in A. Shown is the average of three experiments ± SD of the fraction of cells in the G₂/M phases. To enable comparison between experiments, the fraction of cells in G₂/M phases after release is expressed in relation to this fraction at the start, which was defined as 100%. (C) BJT and derivatives thereof were synchronized by nocodazol, followed by shake-off and released with anchorage to progress through mitosis in the presence of inhibitors; cytochalasin B (Cytoch) inhibiting actin polymerization, Blebbistatin (Blebb) inhibiting myosin II, Y27632 inhibiting ROCK kinases, or DMSO control. Shown is the average of three experiments ± SD of the fraction of cells in the G₂/M phases displayed as in B; P values are from two-tailed Student t tests indicating statistical significance of differences between DMSO control and Y27632 treatment.