Mass measurements during lymphocytic leukemia cell polyploidization decouple cell cycle- and cell size-dependent growth

Abstract

Cell size is believed to influence cell growth and metabolism. Consistently, several studies have revealed that large cells have lower mass accumulation rates per unit mass (i.e., growth efficiency) than intermediate-sized cells in the same population. Size-dependent growth is commonly attributed to transport limitations, such as increased diffusion timescales and decreased surface-to-volume ratio. However, separating cell size- and cell cycle-dependent growth is challenging. To address this, we monitored growth efficiency of pseudodiploid mouse lymphocytic leukemia cells during normal proliferation and polyploidization. This was enabled by the development of large-channel suspended microchannel resonators that allow us to monitor buoyant mass of single cells ranging from 40 pg (small pseudodiploid cell) to over 4,000 pg, with a resolution ranging from ∼1% to ∼0.05%. We find that cell growth efficiency increases, plateaus, and then decreases as cell cycle proceeds. This growth behavior repeats with every endomitotic cycle as cells grow into polyploidy. Overall, growth efficiency changes 33% throughout the cell cycle. In contrast, increasing cell mass by over 100-fold during polyploidization did not change growth efficiency, indicating exponential growth. Consistently, growth efficiency remained constant when cell cycle was arrested in G₂ Thus, cell cycle is a primary determinant of growth efficiency. As growth remains exponential over large size scales, our work finds no evidence for transport limitations that would decrease growth efficiency.

Keywords: cell cycle; cell growth; cell size; mass measurement; transport limitation.

Fig. 1.

Large-channel SMR enables buoyant mass monitoring across large size ranges with high resolution. (A) In-scale schematic of the small- and large-channel SMR cantilevers. The measurement principle of SMRs is to flow a cell through a vibrating cantilever while monitoring a change in the resonant frequency, which is directly proportional to the buoyant mass of the cell. (B) Quantification of large-channel SMR resolution based on repeated mass measurements of single polystyrene beads of different sizes (diameters provided by the manufacturer). (Insets) Zoom-in views of the 10.12- and 49.30-µm bead data along with measurement mean, SD, and coefficient of variation (CV). (C) Large-channel SMR mass measurement resolution as a function of averaging time (moving average filter length reflecting temporal resolution) over multiple measurements for different-sized polystyrene beads. Color coding is the same as in B. The measurement interval is ∼30 s, and the first data point under a gray background reflects individual measurements without any averaging. (Insets) Measurement resolution as CV. (D and E) Example mass traces of control L1210 FUCCI cells growing through multiple divisions in small-channel SMR (D) and in large-channel SMR (E). Data represent individual mass measurements without averaging. At each division, one daughter cell is randomly discarded. The mAG-Geminin signal (green) was only measured in small-channel devices, and its increase indicates G₁/S transition.

Fig. 2.

Growth efficiency of unperturbed cells correlates poorly with cell size within specific cell cycle stages. (A and B) The growth rate (A) and growth efficiency (B) of L1210 FUCCI cells as a function of mass as obtained using small-channel SMR (red traces; number of cells for each size analyzed is indicated with a color gradient at the bottom; n = 9 independent experiments, n = 64 cells) and large-channel SMR (blue traces; n = 2 independent experiments, n = 9 cells). The line and shaded area indicate mean ± SD. Average newborn size (Birth), G₁/S transition size, mitotic entry size (G₂/M), and division size are indicated with dashed vertical lines. (C) Correlations between L1210 FUCCI cell mass and growth efficiency at the beginning of G₁ (n = 9 independent experiments; n = 72 cells), at G₁/S transition (n = 5 independent experiments; n = 41 cells), and at the end of G₂ (n = 9 independent experiments; n = 72 cells). The color indicates each independent experiment. Each cell (dot) is plotted with error bars (measurement error as SD). Linear fits, Pearson correlations (R²), and P values for the correlations (two-tailed test of significance) are shown in orange.

Fig. 3.

Monitoring growth of polyploid cells over a 100-fold size range reveals that growth is not size limited. (A) Hypothesis of cell growth regulation as cells increase size during endomitotic cycles. In the size range observed during a normal cell cycle (orange), cells display nonlinear size scaling of growth efficiency. When cells grow into polyploidy, cell size-dependent (dashed blue line) and cell cycle-dependent (solid blue line) growth should result in different growth behaviors. (B) Representative DNA histograms of control (orange) and 80-h 50 nM Barasertib-treated (blue) L1210 FUCCI cells (n = 3 independent cultures). (C) Representative morphologies of control (Top) and 50 nM Barasertib-treated (Bottom) L1210 FUCCI cells based on nuclear envelope (cyan), plasma membrane (yellow), and DNA (magenta) staining (n = 2 independent experiments each with >10 fields of view). Three-dimensional projections (Left) and single z slices with orthogonal views (Right) are displayed. (Scale bars: 5 µm.) (D) Three example buoyant mass traces from 50 nM Barasertib-treated L1210 FUCCI cells obtained using the large-channel SMR. Exponential fit to the three examples and Pearson correlation (R²) are displayed in orange. (E) The combined growth efficiency of control, 50 nM Barasertib, and 10 µM H-1152–treated L1210 FUCCI cells across a large mass range as measured with both small- and large-channel SMRs (n = 76 independent experiments across all conditions, number of cells is indicated with color gradient at the bottom). Estimated ploidy level is displayed on bottom in blue. (F) Growth rate as a function of mass on a log₁₀–log₁₀ scale when analyzing only 50 nM Barasertib-treated L1210 FUCCI cells measured with the large-channel SMR (n = 31 independent experiments). Linear fit and scaling exponent b (mean ± SEM) are displayed in orange. Perfect isometric scaling (b = 1) is illustrated with dashed black line. (G) Correlations between cell mass at the beginning of each cell cycle and percent mass increase during each cell cycle (Top) and cell cycle duration (Bottom). The data represent 50 nM Barasertib-treated L1210 FUCCI cells measured with the large-channel SMR (n = 11 independent experiments, n = 16 endomitotic cycles). The dashed black line at Top represents a perfect mass doubling in each endomitotic cycle. Approximate ploidy level at the start of each cell cycle (blue); linear fits (orange) and Pearson correlations (R²) are displayed.

Fig. 4.

G₂ cell cycle arrest results in steady growth efficiency. (A) Hypothesis of cell growth regulation as cells increase size during a G₂ cell cycle arrest. In the size range observed during a normal cell cycle (orange), cells display nonlinear size scaling of growth efficiencies. When cells are arrested in G₂, cell size-dependent (dashed blue line) and cell cycle-dependent (solid blue line) growth should result in different growth behaviors. (B) Representative DNA histograms of control (orange) and 24-h 2 µM RO-3306–treated (blue) L1210 cells (n = 3 independent cultures). RO-3306 results in a G₂ arrest, and most cells do not undergo endoreplication cycles. (C) Example buoyant mass trace of a control (orange) and 2 µM RO-3306–treated (blue) L1210 cell obtained using small-channel SMR. (D) The growth efficiency of control (orange; n = 9 independent experiments, n = 64 cells) and 2 µM RO-3306–treated (blue; n = 12 independent experiments, n = 12 cells) L1210 cells. All experiments with RO-3306 lasted under 24 h to avoid cell death. The solid lines and shaded areas indicate mean ± SD. The dashed vertical line indicates the typical division size of control cells.