Single-cell monitoring of dry mass and dry density reveals exocytosis of cellular dry contents in mitosis

Cell mass and composition change with cell cycle progression. Our previous work characterized buoyant mass accumulation dynamics in mitosis (Miettinen et al., 2019), but how dry mass and cell composition change in mitosis has remained unclear. To better understand mitotic cell growth and compositional changes, we develop a single-cell approach for monitoring dry mass and the density of that dry mass every ~75 seconds with 1.3% and 0.3% measurement precision, respectively. We find that suspension grown mammalian cells lose dry mass and increase dry density following mitotic entry. These changes display large, non-genetic cell-to-cell variability, and the changes are reversed at metaphase-anaphase transition, after which dry mass continues accumulating. The change in dry density causes buoyant and dry mass to differ specifically in early mitosis, thus reconciling existing literature on mitotic cell growth. Mechanistically, the dry composition changes do not require mitotic cell swelling or elongation. Instead, cells in early mitosis increase lysosomal exocytosis, and inhibition of exocytosis prevents the dry composition from changing. Overall, our work provides a new approach for monitoring single-cell dry mass and composition and reveals that mitosis is coupled to extensive exocytosis-mediated secretion of cellular contents.

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Label-Free and Quantitative Dry Mass Monitoring for Single Cells during In Situ Culture

Cells ◽

10.3390/cells10071635 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1635

Author(s):

Ya Su ◽

Rongxin Fu ◽

Wenli Du ◽

Han Yang ◽

Li Ma ◽

...

Keyword(s):

Cell Growth ◽

Single Cell ◽

Hela Cells ◽

Quantitative Measurement ◽

Single Cells ◽

Dry Mass ◽

Quantitative Detection ◽

Label Free ◽

Mass Sensitivity

Quantitative measurement of single cells can provide in-depth information about cell morphology and metabolism. However, current live-cell imaging techniques have a lack of quantitative detection ability. Herein, we proposed a label-free and quantitative multichannel wide-field interferometric imaging (MWII) technique with femtogram dry mass sensitivity to monitor single-cell metabolism long-term in situ culture. We demonstrated that MWII could reveal the intrinsic status of cells despite fluctuating culture conditions with 3.48 nm optical path difference sensitivity, 0.97 fg dry mass sensitivity and 2.4% average maximum relative change (maximum change/average) in dry mass. Utilizing the MWII system, different intrinsic cell growth characteristics of dry mass between HeLa cells and Human Cervical Epithelial Cells (HCerEpiC) were studied. The dry mass of HeLa cells consistently increased before the M phase, whereas that of HCerEpiC increased and then decreased. The maximum growth rate of HeLa cells was 11.7% higher than that of HCerEpiC. Furthermore, HeLa cells were treated with Gemcitabine to reveal the relationship between single-cell heterogeneity and chemotherapeutic efficacy. The results show that cells with higher nuclear dry mass and nuclear density standard deviations were more likely to survive the chemotherapy. In conclusion, MWII was presented as a technique for single-cell dry mass quantitative measurement, which had significant potential applications for cell growth dynamics research, cell subtype analysis, cell health characterization, medication guidance and adjuvant drug development.

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Robust surface-to-mass coupling and turgor-dependent cell width determine bacterial dry-mass density

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2021416118 ◽

2021 ◽

Vol 118 (32) ◽

pp. e2021416118

Author(s):

Enno R. Oldewurtel ◽

Yuki Kitahara ◽

Sven van Teeffelen

Keyword(s):

Single Cell ◽

Caulobacter Crescentus ◽

Mass Density ◽

Turgor Pressure ◽

Cell Mass ◽

Dry Mass ◽

Model Organisms ◽

Cell Width ◽

Dependent Cell

During growth, cells must expand their cell volumes in coordination with biomass to control the level of cytoplasmic macromolecular crowding. Dry-mass density, the average ratio of dry mass to volume, is roughly constant between different nutrient conditions in bacteria, but it remains unknown whether cells maintain dry-mass density constant at the single-cell level and during nonsteady conditions. Furthermore, the regulation of dry-mass density is fundamentally not understood in any organism. Using quantitative phase microscopy and an advanced image-analysis pipeline, we measured absolute single-cell mass and shape of the model organisms Escherichia coli and Caulobacter crescentus with improved precision and accuracy. We found that cells control dry-mass density indirectly by expanding their surface, rather than volume, in direct proportion to biomass growth—according to an empirical surface growth law. At the same time, cell width is controlled independently. Therefore, cellular dry-mass density varies systematically with cell shape, both during the cell cycle or after nutrient shifts, while the surface-to-mass ratio remains nearly constant on the generation time scale. Transient deviations from constancy during nutrient shifts can be reconciled with turgor-pressure variations and the resulting elastic changes in surface area. Finally, we find that plastic changes of cell width after nutrient shifts are likely driven by turgor variations, demonstrating an important regulatory role of mechanical forces for width regulation. In conclusion, turgor-dependent cell width and a slowly varying surface-to-mass coupling constant are the independent variables that determine dry-mass density.

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Computationally Enhanced Quantitative Phase Microscopy Reveals Autonomous Oscillations in Mammalian Cell Growth

10.1101/631119 ◽

2019 ◽

Cited By ~ 2

Author(s):

Xili Liu ◽

Seungeun Oh ◽

Leonid Peshkin ◽

Marc W. Kirschner

Keyword(s):

Cell Cycle ◽

Growth Rate ◽

Cell Growth ◽

Single Cell ◽

Cell Lines ◽

Mammalian Cells ◽

Cell Tracking ◽

Quantitative Phase ◽

Phase Microscopy ◽

Quantitative Phase Microscopy

AbstractThe fine balance of growth and division is a fundamental property of the physiology of cells and one of the least understood. Its study has been thwarted by difficulties in the accurate measurement of cell size and the even greater challenges of measuring growth of a single-cell over time. We address these limitations by demonstrating a new computationally enhanced methodology for Quantitative Phase Microscopy (ceQPM) for adherent cells, using improved image processing algorithms and automated cell tracking software. Accuracy has been improved more than two-fold and this improvement is sufficient to establish the dynamics of cell growth and adherence to simple growth laws. It is also sufficient to reveal unknown features of cell growth previously unmeasurable. With these methodological and analytical improvements, we document a remarkable oscillation in growth rate in several different cell lines, occurring throughout the cell cycle, coupled to cell division or birth, and yet independent of cell cycle progression. We expect that further exploration with this improved tool will provide a better understanding of growth rate regulation in mammalian cells.Significance StatementIt has been a long-standing question in cell growth studies that whether the mass of individual cell grows linearly or exponentially. The two models imply fundamentally distinct mechanisms, and the discrimination of the two requires great measurement accuracy. Here, we develop a new method of computationally enhanced Quantitative Phase Microscopy (ceQPM), which greatly improves the accuracy and throughput of single-cell growth measurement in adherent mammalian cells. The measurements of several cell lines indicate that the growth dynamics of individual cells cannot be explained by either of the simple models but rather present an unanticipated and remarkable oscillatory behavior, suggesting more complex regulation and feedbacks.

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Size control in mammalian cells involves modulation of both growth rate and cell cycle duration

10.1101/152728 ◽

2017 ◽

Cited By ~ 4

Author(s):

Clotilde Cadart ◽

Sylvain Monnier ◽

Jacopo Grilli ◽

Rafaele Attia ◽

Emmanuel Terriac ◽

...

Keyword(s):

Cell Cycle ◽

Growth Rate ◽

Cell Division ◽

Cell Growth ◽

Single Cell ◽

Mammalian Cells ◽

Cell Cycle Progression ◽

Size Control ◽

Cycle Duration ◽

Mathematical Framework

SummaryDespite decades of research, it remains unclear how mammalian cell growth varies with cell size and across the cell division cycle to maintain size control. Answers have been limited by the difficulty of directly measuring growth at the single cell level. Here we report direct measurement of single cell volumes over complete cell division cycles. The volume added across the cell cycle was independent of cell birth size, a size homeostasis behavior called “adder”. Single-cell growth curves revealed that the homeostatic behavior relied on adaptation of G1 duration as well as growth rate modulations. We developed a general mathematical framework that characterizes size homeostasis behaviors. Applying it on datasets ranging from bacteria to mammalian cells revealed that a near-adder is the most common type of size control, but only mammalian cells achieve it using modulation of both cell growth rate and cell-cycle progression.

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Optical imaging of cell mass and growth dynamics

AJP Cell Physiology ◽

10.1152/ajpcell.00121.2008 ◽

2008 ◽

Vol 295 (2) ◽

pp. C538-C544 ◽

Cited By ~ 288

Author(s):

Gabriel Popescu ◽

YoungKeun Park ◽

Niyom Lue ◽

Catherine Best-Popescu ◽

Lauren Deflores ◽

...

Keyword(s):

Cell Growth ◽

Cell Model ◽

Cell Mass ◽

Dry Mass ◽

Living Cells ◽

Specific Information ◽

Surface Integral ◽

Quantitative Phase ◽

Phase Microscopy ◽

Quantitative Phase Microscopy

Using novel interferometric quantitative phase microscopy methods, we demonstrate that the surface integral of the optical phase associated with live cells is invariant to cell water content. Thus, we provide an entirely noninvasive method to measure the nonaqueous content or “dry mass” of living cells. Given the extremely high stability of the interferometric microscope and the femtogram sensitivity of the method to changes in cellular dry mass, this new technique is not only ideal for quantifying cell growth but also reveals spatially resolved cellular and subcellular dynamics of living cells over many decades in a temporal scale. Specifically, we present quantitative histograms of individual cell mass characterizing the hypertrophic effect of high glucose in a mesangial cell model. In addition, we show that in an epithelial cell model observed for long periods of time, the mean squared displacement data reveal specific information about cellular and subcellular dynamics at various characteristic length and time scales. Overall, this study shows that interferometeric quantitative phase microscopy represents a noninvasive optical assay for monitoring cell growth, characterizing cellular motility, and investigating the subcellular motions of living cells.

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Linking single-cell measurements of mass, growth rate, and gene expression

10.1101/331686 ◽

2018 ◽

Cited By ~ 1

Author(s):

Robert J. Kimmerling ◽

Sanjay M. Prakadan ◽

Alejandro J. Gupta ◽

Nicholas L. Calistri ◽

Mark M. Stevens ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Growth Rate ◽

T Cells ◽

Cell Growth ◽

Single Cell ◽

Cd8 T Cells ◽

Cell Mass ◽

Growth Efficiency ◽

Cellular Systems

AbstractWe introduce a microfluidic platform that enables single-cell mass and growth rate measurements upstream of single-cell RNA-sequencing (scRNA-seq) to generate paired single-cell biophysical and transcriptional data sets. Biophysical measurements are collected with a serial suspended microchannel resonator platform (sSMR) that utilizes automated fluidic state switching to load individual cells at fixed intervals, achieving a throughput of 120 cells per hour. Each single-cell is subsequently captured downstream for linked molecular analysis using an automated collection system. From linked measurements of a murine leukemia (L1210) and pro-B cell line (FL5.12), we identify gene expression signatures that correlate significantly with cell mass and growth rate. In particular, we find that both cell lines display a cell-cycle signature that correlates with cell mass, with early and late cell-cycle signatures significantly enriched amongst genes with negative and positive correlations with mass, respectively. FL5.12 cells also show a significant correlation between single-cell growth efficiency and a G1-S transition signature, providing additional transcriptional evidence for a phenomenon previously observed through biophysical measurements alone. Importantly, the throughput and speed of our platform allows for the characterization of phenotypes in dynamic cellular systems. As a proof-of-principle, we apply our system to characterize activated murine CD8+ T cells and uncover two unique features of CD8+ T cells as they become proliferative in response to activation: i) the level of coordination between cell cycle gene expression and cell mass increases, and ii) translation-related gene expression increases and shows a correlation with single-cell growth efficiency. Overall, our approach provides a new means of characterizing the transcriptional mechanisms of normal and dysfunctional cellular mass and growth rate regulation across a range of biological contexts.

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Rheology of rounded mammalian cells over continuous high-frequencies

Nature Communications ◽

10.1038/s41467-021-23158-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Gotthold Fläschner ◽

Cosmin I. Roman ◽

Nico Strohmeyer ◽

David Martinez-Martin ◽

Daniel J. Müller

Keyword(s):

High Frequency ◽

Mammalian Cells ◽

Mechanical Response ◽

Cell Mass ◽

Low Frequency ◽

Mass Measurements ◽

High Frequencies ◽

Polymer Relaxation ◽

Continuous Frequency ◽

Rheological Experiments

AbstractUnderstanding the viscoelastic properties of living cells and their relation to cell state and morphology remains challenging. Low-frequency mechanical perturbations have contributed considerably to the understanding, yet higher frequencies promise to elucidate the link between cellular and molecular properties, such as polymer relaxation and monomer reaction kinetics. Here, we introduce an assay, that uses an actuated microcantilever to confine a single, rounded cell on a second microcantilever, which measures the cell mechanical response across a continuous frequency range ≈ 1–40 kHz. Cell mass measurements and optical microscopy are co-implemented. The fast, high-frequency measurements are applied to rheologically monitor cellular stiffening. We find that the rheology of rounded HeLa cells obeys a cytoskeleton-dependent power-law, similar to spread cells. Cell size and viscoelasticity are uncorrelated, which contrasts an assumption based on the Laplace law. Together with the presented theory of mechanical de-embedding, our assay is generally applicable to other rheological experiments.

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Electrospun Nanofibrous Membranes for Tissue Engineering and Cell Growth

Applied Sciences ◽

10.3390/app11156929 ◽

2021 ◽

Vol 11 (15) ◽

pp. 6929

Author(s):

Ewin Tanzli ◽

Andrea Ehrmann

Keyword(s):

Tissue Engineering ◽

Cell Growth ◽

Polymer Blends ◽

Mammalian Cells ◽

Biological Properties ◽

Specific Substrate ◽

Electrospun Nanofiber ◽

Materials Used ◽

Nanofiber Mats ◽

Surface Morphologies

In biotechnology, the field of cell cultivation is highly relevant. Cultivated cells can be used, for example, for the development of biopharmaceuticals and in tissue engineering. Commonly, mammalian cells are grown in bioreactors, T-flasks, well plates, etc., without a specific substrate. Nanofibrous mats, however, have been reported to promote cell growth, adhesion, and proliferation. Here, we give an overview of the different attempts at cultivating mammalian cells on electrospun nanofiber mats for biotechnological and biomedical purposes. Starting with a brief overview of the different electrospinning methods, resulting in random or defined fiber orientations in the nanofiber mats, we describe the typical materials used in cell growth applications in biotechnology and tissue engineering. The influence of using different surface morphologies and polymers or polymer blends on the possible application of such nanofiber mats for tissue engineering and other biotechnological applications is discussed. Polymer blends, in particular, can often be used to reach the required combination of mechanical and biological properties, making such nanofiber mats highly suitable for tissue engineering and other biotechnological or biomedical cell growth applications.

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