Measuring single-cell density with high throughput enables dynamic profiling of immune cell and drug response from patient samples.

Cell density, the ratio of cell mass to volume, is an indicator of molecular crowding and therefore a fundamental determinant of cell state and function. However, existing density measurements lack the precision or throughput to quantify subtle differences in cell states, particularly in primary samples. Here we present an approach for measuring the density of 30,000 single cells per hour with a precision of 0.03% (0.0003 g/mL) by integrating fluorescence exclusion microscopy with a suspended microchannel resonator. Applying this approach to human lymphocytes, we discovered that cell density and its variation decrease as cells transition from quiescence to a proliferative state, suggesting that the level of molecular crowding decreases and becomes more regulated upon entry into the cell cycle. Using a pancreatic cancer patient-derived xenograft model, we found that the ex vivo density response of primary tumor cells to drug treatment can predict in vivo tumor growth response. Our method reveals unexpected behavior in molecular crowding during cell state transitions and suggests density as a new biomarker for functional precision medicine.

Cell intrinsic mechanical regulation of plasma membrane accumulation at the cytokinetic furrow.

Cytokinesis is the process where the mother cell's cytoplasm separates into daughter cells. This is driven by an actomyosin contractile ring that produces cortical contractility and drives cleavage furrow ingression, resulting in the formation of a thin intercellular bridge. While cytoskeletal reorganization during cytokinesis has been extensively studied, little is known about the spatiotemporal dynamics of the plasma membrane. Here, we image and model plasma membrane lipid and protein dynamics on the cell surface during leukemia cell cytokinesis. We reveal an extensive accumulation and folding of plasma membrane at the cleavage furrow and the intercellular bridge, accompanied by a depletion and unfolding of plasma membrane at the cell poles. These membrane dynamics are caused by two actomyosin-driven biophysical mechanisms: the radial constriction of the cleavage furrow causes local compression of the apparent cell surface area and accumulation of the plasma membrane at the furrow, while actomyosin cortical flows drag the plasma membrane towards the cell division plane as the furrow ingresses. The magnitude of these effects depends on the plasma membrane fluidity, cortex adhesion and cortical contractility. Overall, our work reveals cell intrinsic mechanical regulation of plasma membrane accumulation at the cleavage furrow that is likely to generate localized differences in membrane tension across the cytokinetic cell. This may locally alter endocytosis, exocytosis and mechanotransduction, while also serving as a self-protecting mechanism against cytokinesis failures that arise from high membrane tension at the intercellular bridge.

The human cell count and size distribution.

Cell size and cell count are adaptively regulated and intimately linked to growth and function. Yet, despite their widespread relevance, the relation between cell size and count has never been formally examined over the whole human body. Here, we compile a comprehensive dataset of cell size and count over all major cell types, with data drawn from >1,500 published sources. We consider the body of a representative male (70 kg), which allows further estimates of a female (60 kg) and 10-y-old child (32 kg). We build a hierarchical interface for the cellular organization of the body, giving easy access to data, methods, and sources (https://humancelltreemap.mis.mpg.de/). In total, we estimate total body counts of ≈36 trillion cells in the male, ≈28 trillion in the female, and ≈17 trillion in the child. These data reveal a surprising inverse relation between cell size and count, implying a trade-off between these variables, such that all cells within a given logarithmic size class contribute an equal fraction to the body's total cellular biomass. We also find that the coefficient of variation is approximately independent of mean cell size, implying the existence of cell-size regulation across cell types. Our data serve to establish a holistic quantitative framework for the cells of the human body, and highlight large-scale patterns in cell biology.

Single-cell mass distributions reveal simple rules for achieving steady-state growth.

IMPORTANCE: Microbiologists have watched clear liquid turn cloudy for over 100 years. While the cloudiness of a culture is proportional to its total biomass, growth rates from optical density measurements are challenging to interpret when cells change size. Many bacteria adjust their size at different steady-state growth rates, but also when shifting between starvation and growth. Optical density cannot disentangle how mass is distributed among cells. Here, we use single-cell mass measurements to demonstrate that a population of cells in batch culture achieves a stable mass distribution for only a short period of time. Achieving steady-state growth in rich medium requires low initial biomass concentrations and enough time for individual cell mass accumulation and cell number increase via cell division to balance out. Steady-state growth is important for reliable cell mass distributions and experimental reproducibility. We discuss how mass variation outside of steady-state can impact physiology, ecology, and evolution experiments.

Metabolic regulation of species-specific developmental rates.

Animals display substantial inter-species variation in the rate of embryonic development despite a broad conservation of the overall sequence of developmental events. Differences in biochemical reaction rates, including the rates of protein production and degradation, are thought to be responsible for species-specific rates of development1-3. However, the cause of differential biochemical reaction rates between species remains unknown. Here, using pluripotent stem cells, we have established an in vitro system that recapitulates the twofold difference in developmental rate between mouse and human embryos. This system provides a quantitative measure of developmental speed as revealed by the period of the segmentation clock, a molecular oscillator associated with the rhythmic production of vertebral precursors. Using this system, we show that mass-specific metabolic rates scale with the developmental rate and are therefore higher in mouse cells than in human cells. Reducing these metabolic rates by inhibiting the electron transport chain slowed down the segmentation clock by impairing the cellular NAD+/NADH redox balance and, further downstream, lowering the global rate of protein synthesis. Conversely, increasing the NAD+/NADH ratio in human cells by overexpression of the Lactobacillus brevis NADH oxidase LbNOX increased the translation rate and accelerated the segmentation clock. These findings represent a starting point for the manipulation of developmental rate, with multiple translational applications including accelerating the differentiation of human pluripotent stem cells for disease modelling and cell-based therapies.

Nucleotide imbalance decouples cell growth from cell proliferation.

Nucleotide metabolism supports RNA synthesis and DNA replication to enable cell growth and division. Nucleotide depletion can inhibit cell growth and proliferation, but how cells sense and respond to changes in the relative levels of individual nucleotides is unclear. Moreover, the nucleotide requirement for biomass production changes over the course of the cell cycle, and how cells coordinate differential nucleotide demands with cell cycle progression is not well understood. Here we find that excess levels of individual nucleotides can inhibit proliferation by disrupting the relative levels of nucleotide bases needed for DNA replication and impeding DNA replication. The resulting purine and pyrimidine imbalances are not sensed by canonical growth regulatory pathways like mTORC1, Akt and AMPK signalling cascades, causing excessive cell growth despite inhibited proliferation. Instead, cells rely on replication stress signalling to survive during, and recover from, nucleotide imbalance during S phase. We find that ATR-dependent replication stress signalling is activated during unperturbed S phases and promotes nucleotide availability to support DNA replication. Together, these data reveal that imbalanced nucleotide levels are not detected until S phase, rendering cells reliant on replication stress signalling to cope with this metabolic problem and disrupting the coordination of cell growth and division.

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

Cell mass and composition change with cell cycle progression. Our previous work characterized buoyant mass 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 s with 1.3% and 0.3% measurement precision, respectively. We find that suspension grown mammalian cells lose dry mass and increase dry mass 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 mass density causes buoyant and dry mass to differ specifically in early mitosis, thus reconciling existing literature on mitotic cell growth. Mechanistically, cells in early mitosis increase lysosomal exocytosis, and inhibition of lysosomal exocytosis decreases the dry mass loss and dry mass density increase in mitosis. Overall, our work provides a new approach for monitoring single-cell dry mass and dry mass density, and reveals that mitosis is coupled to extensive exocytosis-mediated secretion of cellular contents.

Cell size is a determinant of stem cell potential during aging.

Stem cells are remarkably small. Whether small size is important for stem cell function is unknown. We find that hematopoietic stem cells (HSCs) enlarge under conditions known to decrease stem cell function. This decreased fitness of large HSCs is due to reduced proliferation and was accompanied by altered metabolism. Preventing HSC enlargement or reducing large HSCs in size averts the loss of stem cell potential under conditions causing stem cell exhaustion. Last, we show that murine and human HSCs enlarge during aging. Preventing this age-dependent enlargement improves HSC function. We conclude that small cell size is important for stem cell function in vivo and propose that stem cell enlargement contributes to their functional decline during aging.

Directed evolution of rRNA improves translation kinetics and recombinant protein yield.

In bacteria, ribosome kinetics are considered rate-limiting for protein synthesis and cell growth. Enhanced ribosome kinetics may augment bacterial growth and biomanufacturing through improvements to overall protein yield, but whether this can be achieved by ribosome-specific modifications remains unknown. Here, we evolve 16S ribosomal RNAs (rRNAs) from Escherichia coli, Pseudomonas aeruginosa, and Vibrio cholerae towards enhanced protein synthesis rates. We find that rRNA sequence origin significantly impacted evolutionary trajectory and generated rRNA mutants with augmented protein synthesis rates in both natural and engineered contexts, including the incorporation of noncanonical amino acids. Moreover, discovered consensus mutations can be ported onto phylogenetically divergent rRNAs, imparting improved translational activities. Finally, we show that increased translation rates in vivo coincide with only moderately reduced translational fidelity, but do not enhance bacterial population growth. Together, these findings provide a versatile platform for development of unnatural ribosomal functions in vivo.

Variations of intracellular density during the cell cycle arise from tip-growth regulation in fission yeast.

Intracellular density impacts the physical nature of the cytoplasm and can globally affect cellular processes, yet density regulation remains poorly understood. Here, using a new quantitative phase imaging method, we determined that dry-mass density in fission yeast is maintained in a narrow distribution and exhibits homeostatic behavior. However, density varied during the cell cycle, decreasing during G2, increasing in mitosis and cytokinesis, and dropping rapidly at cell birth. These density variations were explained by a constant rate of biomass synthesis, coupled to slowdown of volume growth during cell division and rapid expansion post-cytokinesis. Arrest at specific cell-cycle stages exacerbated density changes. Spatially heterogeneous patterns of density suggested links between density regulation, tip growth, and intracellular osmotic pressure. Our results demonstrate that systematic density variations during the cell cycle are predominantly due to modulation of volume expansion, and reveal functional consequences of density gradients and cell-cycle arrests.

IFITM3 functions as a PIP3 scaffold to amplify PI3K signalling in B cells.

Interferon-induced transmembrane protein 3 (IFITM3) has previously been identified as an endosomal protein that blocks viral infection1-3. Here we studied clinical cohorts of patients with B cell leukaemia and lymphoma, and identified IFITM3 as a strong predictor of poor outcome. In normal resting B cells, IFITM3 was minimally expressed and mainly localized in endosomes. However, engagement of the B cell receptor (BCR) induced both expression of IFITM3 and phosphorylation of this protein at Tyr20, which resulted in the accumulation of IFITM3 at the cell surface. In B cell leukaemia, oncogenic kinases phosphorylate IFITM3 at Tyr20, which causes constitutive localization of this protein at the plasma membrane. In a mouse model, Ifitm3-/- naive B cells developed in normal numbers; however, the formation of germinal centres and the production of antigen-specific antibodies were compromised. Oncogenes that induce the development of leukaemia and lymphoma did not transform Ifitm3-/- B cells. Conversely, the phosphomimetic IFITM3(Y20E) mutant induced oncogenic PI3K signalling and initiated the transformation of premalignant B cells. Mechanistic experiments revealed that IFITM3 functions as a PIP3 scaffold and central amplifier of PI3K signalling. The amplification of PI3K signals depends on IFITM3 using two lysine residues (Lys83 and Lys104) in its conserved intracellular loop as a scaffold for the accumulation of PIP3. In Ifitm3-/- B cells, lipid rafts were depleted of PIP3, which resulted in the defective expression of over 60 lipid-raft-associated surface receptors, and impaired BCR signalling and cellular adhesion. We conclude that the phosphorylation of IFITM3 that occurs after B cells encounter antigen induces a dynamic switch from antiviral effector functions in endosomes to a PI3K amplification loop at the cell surface. IFITM3-dependent amplification of PI3K signalling, which in part acts downstream of the BCR, is critical for the rapid expansion of B cells with high affinity to antigen. In addition, multiple oncogenes depend on IFITM3 to assemble PIP3-dependent signalling complexes and amplify PI3K signalling for malignant transformation.

Monitoring and modeling of lymphocytic leukemia cell bioenergetics reveals decreased ATP synthesis during cell division.

The energetic demands of a cell are believed to increase during mitosis, but the rates of ATP synthesis and consumption during mitosis have not been quantified. Here, we monitor mitochondrial membrane potential of single lymphocytic leukemia cells and demonstrate that mitochondria hyperpolarize from the G2/M transition until the metaphase-anaphase transition. This hyperpolarization was dependent on cyclin-dependent kinase 1 (CDK1) activity. By using an electrical circuit model of mitochondria, we quantify mitochondrial ATP synthesis rates in mitosis from the single-cell time-dynamics of mitochondrial membrane potential. We find that mitochondrial ATP synthesis decreases by approximately 50% during early mitosis and increases back to G2 levels during cytokinesis. Consistently, ATP levels and ATP synthesis are lower in mitosis than in G2 in synchronized cell populations. Overall, our results provide insights into mitotic bioenergetics and suggest that cell division is not a highly energy demanding process.

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

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.

Mammalian cell growth dynamics in mitosis.

The extent and dynamics of animal cell biomass accumulation during mitosis are unknown, primarily because growth has not been quantified with sufficient precision and temporal resolution. Using the suspended microchannel resonator and protein synthesis assays, we quantify mass accumulation and translation rates between mitotic stages on a single-cell level. For various animal cell types, growth rates in prophase are commensurate with or higher than interphase growth rates. Growth is only stopped as cells approach metaphase-to-anaphase transition and growth resumes in late cytokinesis. Mitotic arrests stop growth independently of arresting mechanism. For mouse lymphoblast cells, growth in prophase is promoted by CDK1 through increased phosphorylation of 4E-BP1 and cap-dependent protein synthesis. Inhibition of CDK1-driven mitotic translation reduces daughter cell growth. Overall, our measurements counter the traditional dogma that growth during mitosis is negligible and provide insight into antimitotic cancer chemotherapies.

Publisher Correction: Noninvasive monitoring of single-cell mechanics by acoustic scattering.

The version of this paper originally published online contained an error in the x-axis of Fig. 2c: the LatB concentrations should be 0.4 and 1 µM, but during typesetting, the 1 µM label was incorrectly changed to 0.1 µM. The label is now correct in the print, PDF, and HTML versions of the paper. In addition, in the article's online Supplementary Information, Supplementary Video 2 was a duplicate of Supplementary Video 1. The correct versions of both videos are now available online.

Excessive Cell Growth Causes Cytoplasm Dilution And Contributes to Senescence.

Cell size varies greatly between cell types, yet within a specific cell type and growth condition, cell size is narrowly distributed. Why maintenance of a cell-type specific cell size is important remains poorly understood. Here we show that growing budding yeast and primary mammalian cells beyond a certain size impairs gene induction, cell-cycle progression, and cell signaling. These defects are due to the inability of large cells to scale nucleic acid and protein biosynthesis in accordance with cell volume increase, which effectively leads to cytoplasm dilution. We further show that loss of scaling beyond a certain critical size is due to DNA becoming limiting. Based on the observation that senescent cells are large and exhibit many of the phenotypes of large cells, we propose that the range of DNA:cytoplasm ratio that supports optimal cell function is limited and that ratios outside these bounds contribute to aging.

Noninvasive monitoring of single-cell mechanics by acoustic scattering.

The monitoring of mechanics in a single cell throughout the cell cycle has been hampered by the invasiveness of mechanical measurements. Here we quantify mechanical properties via acoustic scattering of waves from a cell inside a fluid-filled vibrating cantilever with a temporal resolution of < 1 min. Through simulations, experiments with hydrogels and the use of chemically perturbed cells, we show that our readout, the size-normalized acoustic scattering (SNACS), measures stiffness. To demonstrate the noninvasiveness of SNACS over successive cell cycles, we used measurements that resulted in deformations of < 15 nm. The cells maintained constant SNACS throughout interphase but showed dynamic changes during mitosis. Our work provides a basis for understanding how growing cells maintain mechanical integrity, and demonstrates that acoustic scattering can be used to noninvasively probe subtle and transient dynamics.

Thermal proteome profiling of breast cancer cells reveals proteasomal activation by CDK4/6 inhibitor palbociclib.

Palbociclib is a CDK4/6 inhibitor approved for metastatic estrogen receptor-positive breast cancer. In addition to G1 cell cycle arrest, palbociclib treatment results in cell senescence, a phenotype that is not readily explained by CDK4/6 inhibition. In order to identify a molecular mechanism responsible for palbociclib-induced senescence, we performed thermal proteome profiling of MCF7 breast cancer cells. In addition to affecting known CDK4/6 targets, palbociclib induces a thermal stabilization of the 20S proteasome, despite not directly binding to it. We further show that palbociclib treatment increases proteasome activity independently of the ubiquitin pathway. This leads to cellular senescence, which can be counteracted by proteasome inhibitors. Palbociclib-induced proteasome activation and senescence is mediated by reduced proteasomal association of ECM29. Loss of ECM29 activates the proteasome, blocks cell proliferation, and induces a senescence-like phenotype. Finally, we find that ECM29 mRNA levels are predictive of relapse-free survival in breast cancer patients treated with endocrine therapy. In conclusion, thermal proteome profiling identifies the proteasome and ECM29 protein as mediators of palbociclib activity in breast cancer cells.