Functional interplay between the cell cycle and cell phenotypes

Abstract

Cell cycle distribution of adherent cells is typically assessed using flow cytometry, which precludes the measurements of many cell properties and their cycle phase in the same environment. Here we develop and validate a microscopy system to quantitatively analyze the cell-cycle phase of thousands of adherent cells and their associated cell properties simultaneously. This assay demonstrates that population-averaged cell phenotypes can be written as a linear combination of cell-cycle fractions and phase-dependent phenotypes. By perturbing the cell cycle through inhibition of cell-cycle regulators or changing nuclear morphology by depletion of structural proteins, our results reveal that cell cycle regulators and structural proteins can significantly interfere with each other's prima facie functions. This study introduces a high-throughput method to simultaneously measure the cell cycle and phenotypes at single-cell resolution, which reveals a complex functional interplay between the cell cycle and cell phenotypes.

Figure 1. Measurement of cell cycle phase distribution in situ – comparison with flow cytometry (FC)

A. Schematic showing that a common procedure to extract cell information is to run parallel experiments with different instruments. However, whether cell cycle and cell properties are linked, it still needs direct measurement to address. B. Our Microscopy-based high-throughput assay used in these studies to understand the question in panel A. Eighty one fields of four-channel fluorescence/phase contrast images were automatically collected (only DNA channel in blue and actin channel in green are shown here) to analyze the intensity of ~1,200 nuclei and simultaneously measure cell and nuclear properties (cell size, nuclear size, nuclear shape, etc.) in the same individual cells through edge detection of cell boundaries (green contours) and nuclear boundaries (blue contours). Inset. This analysis produced a DNA stain intensity distribution (blue profile). C. Normalized DNA stain intensity distribution of c2c12 cells obtained from FC analysis (magenta profile) and our microscopy-based assay (blue profile). D. Proportion of cells in the G₀/G₁, S, and G₂/M cell-cycle phases, as measured by conventional FC analysis (magenta bars) and by microscopy-based analysis (blue bars). NS: non-significant differences; P > 0.05 (t-test for phase-to-phase comparison). For panel E, three biological repeats on different cells were conducted for both FC analysis and microscopy-based analysis.

Figure 2. Simultaneous measurements of cell cycle phase and cell properties in adherent cells

A. c2c12 mouse myoblasts in culture were stained with DNA and F-actin stains and DNA content was quantified following a calibration step (see Methods section); nuclei were color-coded according to their cell cycle phase. This illustrates how the position of cells in the culture dish is not lost, unlike for FC for which cells are detached before cell-cycle and phenotypic assessments. B. For sake of clarity, c2c12 cells were computationally placed on a grid, depending on their cell-cycle phase. This illustrates how DNA content distribution is measured using direct measurement of DNA stain intensity in adherent cells. Cells contours are in green, nuclear contours are in blue. C–H. Distributions and averaged values of nuclear size (C and D), cell size (E and F), nuclear size (G and H) and nuclear shape (I and J) measured by our assay. Population-based distributions of cell and nuclear properties, for which no distinction among phases is made, are shown in black. Distributions of cell and nuclear properties for the G₀/G₁ phase, the S phase, and the G₂/M phase are shown in red (panels C, E, G). In panel D, F, H, J, all apparent differences are statistically significant, P < 0.0001 (one-way ANOVA) as compared to population-averaged values of the considered phenotype. Three biological repeats conducted on different cells were analyzed for a total of >3,000 cells for each tested condition.

Figure 3. Conventional cell cycle synchronization methods cannot be used to measure cell-cycle-dependent nuclear/cellular properties

A. Ubiquitously used methods were applied to synchronize the phase of c2c12 cells. These include serum-starvation which enriches cells in the G₁ phase (green), nocodazole treatment which enriches cells in the M phase (purple), and thymidine treatment which enriches cells in the early S phase (orange). B. Cell-cycle phase distributions obtained by microscopy-based analysis of untreated control asynchronized cells (blue bars), and serum-starved cells (green bars), thymidine-treated cells (orange), and nocodazole-treated cells (purple). The contour of the cell-cycle distribution for control cells (blue) is shown in each case to help visual comparison. C. Proportion of cells in the G₀/G₁, S, and G₂/M phases for control (blue bars), serum-starved (green), thymidine-treated (orange bars), and nocodazole–treated cells (purple bars). D–F. Population-averaged values and cell-cycle-phase-dependent mean values of nuclear size (D), cell size (E), and nuclear shape (F), compared phase-to-phase, induced in serum-starved (green), thymidine-treated (orange), and nocodazole–treated cells (purple) in each phase compared to control cells (blue). All apparent differences are statistically significant, P < 0.0001 (one-way ANOVA) as compared to phenotypic values for control cells in each corresponding phase. For panels B–F, three biological repeats conducted on different cells were analyzed for a total of >3,000 cells for each tested condition.

Figure 4. Inhibition of cell cycle regulator, cdk4/6, causes changes on cell properties

A. Cell-cycle distributions of control cells and cells treated with Cdk4/6 inhibitor IV. Blue represents control. Black represents Cdk4/6 inhibitor IV treatment. B–D. Population-averaged (first bars) and cell-cycle-dependent nuclear size (B), cell size (C), and nuclear shape factor (D). Three biological repeats on different cells were analyzed for a total of >3,000 cells for each tested condition.

Figure 5. Combined measurements of cell cycle phase and cell properties reveal bona fide regulators of cell phenotypes and cycle phase

A. Cell cycle distributions obtained by microscopy-based analysis of control cells (blue) and cells depleted of nuclear envelope-associated proteins Lamin A/C (red), Nesprin3 (black), or Nesprin2giant (grey). The profile of the cell cycle distribution for control cells (blue) is shown for visual comparison. B. Proportions of cells in the G₀/G₁, S, and G₂/M phases for control (blue bars), Lamin A/C-depleted cells (red), Nesprin3-depleted cells (black), and Nesprin2giant-depleted cells (grey) (B). C–E. Cell-cycle-phase-dependent mean values of nuclear size (C), cell size (D), and nuclear shape (E), compared phase to phase, induced in each phase by depletion of Lamin A/C (red), depletion of Nesprin3 (grey), and depletion of Nesprin2giant (black) compared to control cells (blue). All apparent differences are statistically significant, P < 0.0001 (one-way ANOVA) as compared to phenotypic values for control cells in each corresponding phase. Three biological replicates were analyzed for all tested conditions (panels A–E).

Figure 6. Contribution of cell cycle redistribution to population-averaged changes in cell properties

A. Changes in the population-averaged values of cell/nuclear properties can be expressed as a sum of three major contributions: $\mathrm{\Delta }x={\langle x\rangle }_{\mathit{control}}-{\langle x\rangle }_{\mathit{KDorDrug}}={\sum }_{i=Go/G1}^{G2/M}\mathrm{\Delta }{x}_i{f}_i{\mid }_{\mathit{control}}+{\displaystyle \sum _i}\mathrm{\Delta }{f}_i{x}_i{\mid }_{\mathit{control}}+{\displaystyle \sum _i}\mathrm{\Delta }{f}_i\mathrm{\Delta }{x}_i$. Here Δx is the total change in the population-averaged value of the cell/nucleus property of interest caused by the depletion (denoted by lowercase KD) of either Nesprin2giant, Nesprin3, or Lamin A/C or induced by forced synchronization (lowercase Drug) compared to control cells; Δx_i are the same differences but evaluated for cells in each cell-cycle phase i; and Δf_i are the changes in cell-cycle fractions for each phase i. The summation Overall changes in cell properties, Δx, may stem from three distinct contributions: changes in intrinsic cell properties independent of changes in cell cycle (first term), indirect changes in cell properties due a change in cell cycle distribution (second term), and coupled changes in cell cycle and cell properties (third term), which are expected to be second-order in magnitude. B–D. Contributions to global changes in population-averaged nuclear size (B), cell size (C) and nuclear shape (D) due to intrinsic cell-cycle-independent changes in these properties (black), due to cell cycle redistribution (white), and due to coupled effects of cell cycle redistribution and intrinsic cell-cycle-independent changes in nuclear size (grey). This analysis was applied to c2c12 cells depleted of Lamin A/C, cells depleted of Nesprin2giant, and cells depleted of Nesprin3, as well as c2c12 cells subjected to serum –starvation, cells treated with thymidine, and cells treated with nocodazole. Three biological replicates were analyzed for all tested conditions (panels B–D).