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Inertial picobalance reveals fast mass fluctuations in mammalian cells

Abstract

The regulation of size, volume and mass in living cells is physiologically important, and dysregulation of these parameters gives rise to many diseases1. Cell mass is largely determined by the amount of water, proteins, lipids, carbohydrates and nucleic acids present in a cell, and is tightly linked to metabolism, proliferation2 and gene expression3. Technologies have emerged in recent years that make it possible to track the masses of single suspended cells4,5 and adherent cells6,7,8. However, it has not been possible to track individual adherent cells in physiological conditions at the mass and time resolutions required to observe fast cellular dynamics. Here we introduce a cell balance (a ‘picobalance’), based on an optically excited microresonator, that measures the total mass of single or multiple adherent cells in culture conditions over days with millisecond time resolution and picogram mass sensitivity. Using our technique, we observe that the mass of living mammalian cells fluctuates intrinsically by around one to four per cent over timescales of seconds throughout the cell cycle. Perturbation experiments link these mass fluctuations to the basic cellular processes of ATP synthesis and water transport. Furthermore, we show that growth and cell cycle progression are arrested in cells infected with vaccinia virus, but mass fluctuations continue until cell death. Our measurements suggest that all living cells show fast and subtle mass fluctuations throughout the cell cycle. As our cell balance is easy to handle and compatible with fluorescence microscopy, we anticipate that our approach will contribute to the understanding of cell mass regulation in various cell states and across timescales, which is important in areas including physiology, cancer research, stem-cell differentiation and drug discovery.

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Figure 1: Working principle of the picoscopic cell balance in culture conditions.
Figure 2: Mass fluctuations of single adherent animal cells in interphase.
Figure 3: Cell mass fluctuations measured using a position-insensitive L-shaped microcantilever.
Figure 4: Infection with vaccinia virus (VACV) stops growth of animal cells.

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Acknowledgements

We thank H.-P. Lang, F. Huber, W. Junge, E. Meyer, T. Glatzel and J. Adams for discussion; E. Meyer, T. Glatzel and A. Tonin for help with the beam deflection diagram; the mechanical and electronic workshops of the Physics Department of University Basel, P. Buchmann and P. Argast for help with building our device; A. Ponti for technical support with Imaris 8.1 to determine cell position; D. Mathys for assisting with electron microscopy and focused ion beam lithography; T. Lopez and V. Jäggin for assistance with FACS operation; and Newport Corporation, Attocube Systems AG, Nanonis (SPECS Zurich GmbH), Carl Zeiss AG and Nanosurf AG for technical support. This work was funded by the Swiss Commission for Technology and Innovation (CTI; grant 17970.1 PFNM-NM to D.J.M.), the European Molecular Biology Organization (EMBO; ALTF 506-2012 to D.M.-M. and ALTF 424-2016 to B.G.), the Swiss Nanoscience Institute Basel and the NCCR Molecular Systems Engineering. C.B. and J.M. are supported by core funding to the MRC Laboratory for Molecular Cell Biology, University College London.

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Authors and Affiliations

Authors

Contributions

D.M.-M., G.F., C.G. and D.J.M. designed the experiments. D.M.-M., C.G., S.M. and D.J.M. designed and built the cell balance. R.N. generated the fibroblast cell line stably expressing H2B–eGFP and mCherry–actin. D.M.-M. and G.F. set up the controlled environmental system and conducted all experiments. B.G. performed parts of the VACV experiments. D.M.-M., G.F., C.G. and D.J.M. analysed the data. C.B. and J.M. constructed, produced and purified the VACV and provided the HeLa (ATCC CCL-2) and BSC40 (ATCC CRL-2761) cell lines. D.M.-M., G.F., B.G., R.N., J.M., C.G. and D.J.M. wrote the manuscript. All authors discussed the experiments, read and approved the manuscript.

Corresponding authors

Correspondence to David Martínez-Martín or Daniel J. Müller.

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Competing interests

D.M.-M., S.M., C.G. and D.J.M. applied for two patents related to the cell balance device compatible with optical microscopy (WO/2015/120,991 and WO/2015/120,992). D.M.-M., G.F., S.M. and D.J.M. applied for a patent for the controlled environmental system enabling cell mass measurements and optical microscopy under cell culture conditions (WO/2017/012708). D.M.-M., G.F. and D.J.M. applied for a patent for microcantilevers to measure the mass of a cell independently of its positional changes (EP17001238).

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Reviewer Information Nature thanks Y. Dufrene, G. Wuite and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 Control mass measurement operating the picobalance in cell culture conditions.

ae, Side and top view SEM images of an FIB-manufactured microcantilever with and without a weight sculpted from a silicon block. Theoretically, the mass sensitivity of the cantilever is highest if the mass is added to the free end of the cantilever and lowest (that is, zero) if the mass is added to the fixed end (Supplementary Note 3). To experimentally evaluate the mass sensitivity of the cantilever as a function of the location of the added mass, the weight is shifted by 10 μm from the free end of the cantilever. From the dimensions of the silicon block (10.1 × 21.2 × 1.8 μm3) a mass of 0.90 ± 0.12 ng (value ± propagated error) is calculated using the density of silicon (2.33 g cm−3). After removal of the block by FIB milling, SEM shows a residual volume of 0.9 × 21.2 × 1.8 μm3, which corresponds to a mass mresidual ≈ 0.08 ± 0.01 ng. Therefore, a mass of 0.90 − 0.08 = 0.82 ± 0.12 ng is expected to be added by the silicon block. f, Phase curves recorded in cell culture medium for the cantilever with (b) and without (c) the silicon block. The natural resonance frequency of the cantilever (arrows) increases by 4.542 kHz after the block is removed. Taking into account the position of the block on the cantilever, this frequency shift accounts for a mass difference of 0.81 ± 0.02 ng, which is in good agreement with the mass expected from volume measurements by SEM. Thin solid lines on top of the experimental data in f correspond to fittings of a driven and damped harmonic oscillator. Equations used for the fit are introduced in Supplementary Note 2. The measurements are complementary to the control mass measurements shown in Fig. 1d, e. Scale bars, 20 μm (a), 5 μm (b, c) and 10 μm (d, e).

Extended Data Figure 2 The controlled environmental system provides culture conditions for mammalian cells adhering to the substrate-coated microcantilever.

a, MDCK II cells adhering to a collagen type I functionalized cantilever20,21 in culture medium. Culture conditions are provided by the controlled environmental system of the balance (Fig. 1a). Shown are superimposed DIC and fluorescence images recorded at the times indicated by time stamps at the bottom of each image. Time stamps correspond to hours:minutes. The time-lapse images show that the cells progress through multiple cell cycles and remain alive and viable for several days until the mass measurement (the mass measured is shown in other examples) is terminated. Cells were cultured in phenol red-free, high-glucose DMEM, supplemented with 1 mM sodium pyruvate, 4 mM GlutaMAX, 10% (v/v) FCS, 100 units ml–1 penicillin and 10 μg ml–1 streptomycin. Scale bar, 20 μm. The MDCK II cell line stably expressing H2B–eGFP was created by subcloning sequences encoding H2B–eGFP into a lentiviral vector (pRRLsincPPT-PGK.WPRE) using a standardized protocol32. Lentiviruses carrying the H2B–eGFP transgenes were obtained in our laboratory and used to transduce MDCK cells (clone II, provided by B. Roska). FACS was used to select cells stably expressing H2B–eGFP. b, Mass measurement acquired simultaneously with a. In mitosis, cells detach partially from the cantilever and reattach after anaphase, producing the dips observed in the measurements. Dashed lines (red) and time stamps mark the first three dividing cells, which can be seen in a. Owing to the lack of space to accommodate the growing number of cells, some cells migrate towards the fixed end of the cantilever after about 48 h. For this reason, the mass measurement was stopped. Long-term experiments such as these were conducted at least ten times with biologically independent cells.

Extended Data Figure 3 Mass fluctuation analysis of single fibroblasts and HeLa cells.

a, Schematic demonstrating the decomposition into two oscillations that result in mass fluctuations. The fluctuation shown on the left (black line) is composed of two oscillations differing in period (fast and slow) and amplitude (fast and slow mass extrema). The fast oscillation has a period tfast and amplitude Afast, the slow oscillation has a period tslow and amplitude Aslow. b, Analysing the fast and slow components of experimentally determined cell mass fluctuations. The top row shows fibroblasts (data from Fig. 2c) and the bottom row HeLa cells (data from Fig. 2d). Black lines represent the cell mass recorded over time. The faster mass fluctuations (red line) overlapping the slower mass fluctuations (green line) are obtained by smoothing the cell mass over time. Orange and blue dots show extremes (peaks and valleys) of the faster and slower mass fluctuations, respectively. The data analysis reveals that fibroblasts experience fast mass fluctuations (red lines) with period 2.2 ± 0.1 s and amplitude 14 ± 1 pg (mean ± s.e.; n = 132) and slow mass fluctuations (green lines) with period 17.6 ± 2.0 s and amplitude 12 ± 1 pg (n = 10). HeLa cells experience fast mass fluctuations with period 2.1 ± 0.1 s and amplitude 15 ± 1 pg (n = 177) and slow mass fluctuations with period 18.0 ± 1.4 s and amplitude 14 ± 2 pg (n = 17). The average amplitude of the mass fluctuations for each regime (fast or slow) is calculated by the mass difference between two adjacent extrema divided by two. The average time period is calculated from the time difference between adjacent mass peaks and valleys (mass extrema) multiplied by two. n is the number of fluctuations analysed from at least three biologically independent cells per cell line.

Extended Data Figure 4 Time-lapse microscopy of single spread and unspread HeLa cells, from which mass fluctuations are characterized.

a, Time-lapse DIC images correspond to the HeLa cell from which mass was measured in Fig. 2d (right). Images at higher magnification are presented below. b, Time-lapse DIC images corresponding to the VACV-infected HeLa cell from which mass was measured in Fig. 4d (right). The first DIC image of the time-lapse experiment shows an overlaid fluorescent channel confirming that the cell was infected with VACV. The fluorescence signal of the eGFP-tagged VACV is shown in green. Images of the VACV-infected cell are presented at higher magnification below. In neither experiment did the cells observably change position on the cantilever. c, Time-lapse of a HeLa cell freshly attached to a microcantilever. The rounded cell is not yet spread, which prevents the cell from migrating. The cell is fluorescently labelled with CellTracker Green CMFDA (cytoplasmic dye) and NucBlue (nuclear dye). DIC and fluorescent images are recorded every 10 s. Upper and lower rows show DIC images overlapped with cytoplasmic (green) and nuclear (blue) fluorescent channels, respectively. No movement of the cell or nucleus is detected within the optical resolution limit. d, Mass fluctuations of two different unspread HeLa cells adhering to a microcantilever. The mass curve on the left corresponds to the cell shown in c and was recorded simultaneously with the images in c. The fast fluctuations (red lines) have an amplitude of 15 ± 1 pg (mean ± s.e.; n = 129) with a period of 1.9 ± 0.1 s while the slow fluctuations (green lines) have an amplitude of 16 ± 2 pg (n = 10) with a period of 19.0 ± 1.2 s. Values are means obtained from the identified extremes (peaks and valleys), which are marked with orange and blue dots for the fast and slow fluctuations, respectively. n is the number of fluctuations analysed from at least three biologically independent cells. e, To generate fast mass fluctuations (30 pg from amplitude peak to peak; Fig. 2), a cell adhering to the microcantilever would need to change position along the long cantilever axis by around 0.6 μm with a frequency of around 0.5 Hz (Supplementary Note 4). Such fast movements have not been reported so far, nor were they observed in our experiments by monitoring the cell morphology over time. The cantilever used in the experiment in c had a length of 135 μm. Scale bars 20 μm (a, b), 10 μm (c).

Extended Data Figure 5 Cell adhesion and spreading do not affect the cantilever spring constant.

a, Force-displacement curves recorded using a cantilever with and without an adherent mouse fibroblast. To record force-displacement curves, a cantilever used for mass measurements is pressed against a calibrated reference cantilever. The system, which behaves as two springs in series, determines whether the presence of an adherent cell changes the spring constant of the cantilever. b, Optical microscopy image showing the cantilever used for mass measurements (right) pushed against a reference cantilever (left). ce, Cantilevers with an adherent fibroblast used for mass measurements. Overlaid DIC and fluorescence images of mCherry–actin (red) and H2B–eGFP (green) show the fibroblast in different spread states. The slopes of the force-displacement curves in a reveal the effective spring constant keff of the coupled spring system. As the slopes of the force curves do not change with the presence and spreading of the fibroblast, we conclude that the cantilever spring constant remains unaffected by the fibroblast. Measurements were made using a commercial AFM (CellHesion 200, JPK Instruments) under cell culture conditions. The reference cantilever (Olympus OMCL-RC) had dimensions of 100 × 40 × 0.8 μm3 and a spring constant of 0.65 N m–1. The cantilever used for mass measurements had dimensions of 76.3 × 30.0 × 1.0 μm3 and a spring constant of 1.85 N m–1. The experiment was repeated at least three times with biologically independent cells.

Extended Data Figure 6 Cantilever deflection induced by a contractingadherent mammalian cell shows no influence on cell mass measurement.

a, Deflection of the cantilever with an adherent fibroblast. During the measurement, the fibroblast contracted, causing mechanical stress, and deflected the cantilever. b, Mass of the fibroblast recorded simultaneously with a. The lack of correlation between a and b demonstrates that the deflection of the cantilever does not affect the mass measurement. The FIB-manufactured cantilever had dimensions of 75.5 × 27.9 × 1.0 μm3 and a spring constant of 1.86 N m–1. Measurements were made under cell culture conditions. The medium was high-glucose DMEM with phenol red, supplemented with 1 mM sodium pyruvate, 4 mM GlutaMAX and 10% (v/v) FCS. The medium also contained 100 units per ml penicillin and 10 μg ml–1 streptomycin (Gibco Life technologies). The experiment was repeated at least three times with biologically independent cells.

Extended Data Figure 7 Mass fluctuations of HeLa cells depend on water transport and ATP synthesis.

Shown are perturbation experiments correlating the fast and slow mass fluctuations with biological processes. Each curve corresponds to a single HeLa cell. Every experimental condition was repeated at least three times using different cells. The numerical and statistical analysis of the fluctuations for each condition is shown in Table 1. a, Mass fluctuations of a single HeLa cell under cell culture conditions (data from Fig. 2) as a control. b, HeLa cell under the effect of 2,4-dinitrophenol, which lowers ATP production by dissipating the proton gradient in mitochondria38. No significant changes were detected with respect to the control. 2,4-dinitrophenol was added 1 h before the measurements. c, HeLa cell perturbed with oligomycin A, which inhibits ATP synthase39. The mass fluctuations observed were similar to the control. Oligomycin A was added 30 min before the measurements40. d, HeLa cell cultured in glucose-free culture medium (Methods). Prior to the mass measurements the cells were cultured in glucose-free medium for 12 h to prevent glycolysis. However, other sources of energy such as glutamine were accessible for the cells40. No significant changes in the mass fluctuations were observed, see Table 1. e, HeLa cell perturbed with oligomycin A as in c but cultured in glucose-free medium as in d to more efficiently deplete the energy of the cell. Significant changes were observed in the amplitudes of the slow and fast mass fluctuations, which decreased by around 79% and 33%, respectively (Table 1). f, HeLa cell under the effect of mercury(II) chloride, which blocks aquaporins from transporting water41. Compared to the control cells, the amplitudes of the slow and fast mass fluctuations decreased by around 71% and 27%, respectively.

Extended Data Figure 8 Mass fluctuations of VACV-infected HeLa cells.

Each graph corresponds to a different VACV-infected HeLa cell. Black lines depict the cell mass recorded with a time resolution of 10 ms. VACV-infected HeLa cells show fast mass fluctuations (red lines) with a period of 1.9 ± 0.1 s (mean ± s.e.; n = 402) and an amplitude of 15 ± 1 pg and slow mass fluctuations (green lines) with a period of 17.0 ± 1.5 s (n = 31) and an amplitude of 15 ± 2 pg. n is the number of fluctuations analysed from at least ten biologically independent cells. Single cell mass measurements include those shown in the main manuscript.

Extended Data Figure 9 Long-term mass measurements of single VACV-infected and uninfected HeLa cells.

The black curve shows the growth and proliferation of an uninfected HeLa cell adhering to a microcantilever. After about 9 h, the cell progresses through mitosis and the two daughter cells keep growing. Red curves show the mass of single HeLa cells that have been infected with VACV. Superimposed DIC and fluorescence images show the HeLa cells at the beginning of the experiment and after several hours. The green fluorescence signal indicates the presence of eGFP-tagged VACV as a result of virus production. The mass of VACV-infected HeLa cells remains largely unchanged during virus production. The experiment with virus-infected cells was repeated ten times with biologically independent cells. Additional data are shown in Fig. 4.

Extended Data Figure 10 Setup and block diagram of the picobalance.

The intensity-modulated blue laser excites an oscillatory movement of the microcantilever, which is detected by an infrared laser (red) reflected from the free cantilever end onto a four-quadrant photodiode. To measure the amplitude and phase of the cantilever movement the signal from the photodiode is analysed by a lock-in amplifier. For high time resolution measurements, a phase-locked loop instantaneously tracks the natural resonance frequency of the cantilever.

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Martínez-Martín, D., Fläschner, G., Gaub, B. et al. Inertial picobalance reveals fast mass fluctuations in mammalian cells. Nature 550, 500–505 (2017). https://doi.org/10.1038/nature24288

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