Cell Size Control Research Articles

What programs the size of animal cells?

The human body is programmed with definite quantities, magnitudes, and proportions. At the microscopic level, such definite sizes manifest in individual cells - different cell types are characterized by distinct cell sizes whereas cells of the same type are highly uniform in size. How do cells in a population maintain uniformity in cell size, and how are changes in target size programmed? A convergence of recent and historical studies suggest - just as a thermostat maintains room temperature - the size of proliferating animal cells is similarly maintained by homeostatic mechanisms. In this review, we first summarize old and new literature on the existence of cell size checkpoints, then discuss additional advances in the study of size homeostasis that involve feedback regulation of cellular growth rate. We further discuss recent progress on the molecules that underlie cell size checkpoints and mechanisms that specify target size setpoints. Lastly, we discuss a less-well explored teleological question: why does cell size matter and what is the functional importance of cell size control?

Analytical cell size distribution: lineage-population bias and parameter inference.

We derive analytical steady-state cell size distributions for size-controlled cells in single-lineage experiments, such as the mother machine, which are fundamentally different from batch cultures where populations of cells grow freely. For exponential single-cell growth, characterizing most bacteria, the lineage-population bias is obtained explicitly. In addition, if volume is evenly split between the daughter cells at division, we show that cells are on average smaller in populations than in lineages. For more general power-law growth rates and deterministic volume partitioning, both symmetric and asymmetric, we derive the exact lineage distribution. This solution is in good agreement with Escherichia coli mother machine data and can be used to infer cell cycle parameters such as the strength of the size control and the asymmetry of the division. When introducing stochastic volume partitioning, we derive the large-size and small-size tails of the lineage distribution and show that the lineage-population bias only depends on the single-cell growth rate. These asymptotic behaviours are extended to the adder model of cell size control. When considering noisy single-cell growth rate, we derive the large-size lineage and population distributions. Finally, we show that introducing noise, either on the volume partitioning or on the single-cell growth rate, can cancel the lineage-population bias.

The uniformity and stability of cellular mass density in mammalian cell culture.

Cell dry mass is principally determined by the sum of biosynthesis and degradation. Measurable change in dry mass occurs on a time scale of hours. By contrast, cell volume can change in minutes by altering the osmotic conditions. How changes in dry mass and volume are coupled is a fundamental question in cell size control. If cell volume were proportional to cell dry mass during growth, the cell would always maintain the same cellular mass density, defined as cell dry mass dividing by cell volume. The accuracy and stability against perturbation of this proportionality has never been stringently tested. Normalized Raman Imaging (NoRI), can measure both protein and lipid dry mass density directly. Using this new technique, we have been able to investigate the stability of mass density in response to pharmaceutical and physiological perturbations in three cultured mammalian cell lines. We find a remarkably narrow mass density distribution within cells, that is, significantly tighter than the variability of mass or volume distribution. The measured mass density is independent of the cell cycle. We find that mass density can be modulated directly by extracellular osmolytes or by disruptions of the cytoskeleton. Yet, mass density is surprisingly resistant to pharmacological perturbations of protein synthesis or protein degradation, suggesting there must be some form of feedback control to maintain the homeostasis of mass density when mass is altered. By contrast, physiological perturbations such as starvation or senescence induce significant shifts in mass density. We have begun to shed light on how and why cell mass density remains fixed against some perturbations and yet is sensitive during transitions in physiological state.

Microfluidics for long-term single-cell time-lapse microscopy: Advances and applications.

Cells are inherently dynamic, whether they are responding to environmental conditions or simply at equilibrium, with biomolecules constantly being made and destroyed. Due to their small volumes, the chemical reactions inside cells are stochastic, such that genetically identical cells display heterogeneous behaviors and gene expression profiles. Studying these dynamic processes is challenging, but the development of microfluidic methods enabling the tracking of individual prokaryotic cells with microscopy over long time periods under controlled growth conditions has led to many discoveries. This review focuses on the recent developments of one such microfluidic device nicknamed the mother machine. We overview the original device design, experimental setup, and challenges associated with this platform. We then describe recent methods for analyzing experiments using automated image segmentation and tracking. We further discuss modifications to the experimental setup that allow for time-varying environmental control, replicating batch culture conditions, cell screening based on their dynamic behaviors, and to accommodate a variety of microbial species. Finally, this review highlights the discoveries enabled by this technology in diverse fields, such as cell-size control, genetic mutations, cellular aging, and synthetic biology.

Epithelial cell size dysregulation in human lung adenocarcinoma.

Human cells tightly control their dimensions, but in some cancers, normal cell size control is lost. In this study we measure cell volumes of epithelial cells from human lung adenocarcinoma progression in situ. By leveraging artificial intelligence (AI), we reconstruct tumor cell shapes in three dimensions (3D) and find airway type 2 cells display up to 10-fold increases in volume. Surprisingly, cell size increase is not caused by altered ploidy, and up to 80% of near-euploid tumor cells show abnormal sizes. Size dysregulation is not explained by cell swelling or senescence because cells maintain cytoplasmic density and proper organelle size scaling, but is correlated with changes in tissue organization and loss of a novel network of processes that appear to connect alveolar type 2 cells. To validate size dysregulation in near-euploid cells, we sorted cells from tumor single-cell suspensions on the basis of size. Our study provides data of unprecedented detail for cell volume dysregulation in a human cancer. Broadly, loss of size control may be a common feature of lung adenocarcinomas in humans and mice that is relevant to disease and identification of these cells provides a useful model for investigating cell size control and consequences of cell size dysregulation.

The histone methyltransferase SETD2 negatively regulates cell size.

Cell size varies between cell types but is tightly regulated by cell intrinsic and extrinsic mechanisms. Cell size control is important for cell function, and changes in cell size are frequently observed in cancer. Here, we uncover a role for SETD2 in regulating cell size. SETD2 is a lysine methyltransferase and a tumor suppressor protein involved in transcription, RNA processing and DNA repair. At the molecular level, SETD2 is best known for associating with RNA polymerase II through its Set2-Rbp1 interacting (SRI) domain and methylating histone H3 on lysine 36 (H3K36) during transcription. Using multiple independent perturbation strategies, we identify SETD2 as a negative regulator of global protein synthesis rates and cell size. We provide evidence that overexpression of the H3K36 demethylase KDM4A or the oncohistone H3.3K36M also increase cell size. In addition, ectopic overexpression of a decoy SRI domain increased cell size, suggesting that the relevant substrate is engaged by SETD2 via its SRI domain. These data add a central role of SETD2 in regulating cellular physiology and warrant further studies on separating the different functions of SETD2 in cancer development.

Evolution of cell size control is canalized towards adders or sizers by cell cycle structure and selective pressures.

Cell size is controlled to be within a specific range to support physiological function. To control their size, cells use diverse mechanisms ranging from 'sizers', in which differences in cell size are compensated for in a single cell division cycle, to 'adders', in which a constant amount of cell growth occurs in each cell cycle. This diversity raises the question why a particular cell would implement one rather than another mechanism? To address this question, we performed a series of simulations evolving cell size control networks. The size control mechanism that evolved was influenced by both cell cycle structure and specific selection pressures. Moreover, evolved networks recapitulated known size control properties of naturally occurring networks. If the mechanism is based on a G1 size control and an S/G2/M timer, as found for budding yeast and some human cells, adders likely evolve. But, if the G1 phase is significantly longer than the S/G2/M phase, as is often the case in mammalian cells in vivo, sizers become more likely. Sizers also evolve when the cell cycle structure is inverted so that G1 is a timer, while S/G2/M performs size control, as is the case for the fission yeast <i>S. pombe</i>. For some size control networks, cell size consistently decreases in each cycle until a burst of cell cycle inhibitor drives an extended G1 phase much like the cell division cycle of the green algae <i>Chlamydomonas</i>. That these size control networks evolved such self-organized criticality shows how the evolution of complex systems can drive the emergence of critical processes.

Cell region fingerprints enable highly precise single-cell tracking and lineage reconstruction.

Experimental studies of cell growth, inheritance and their associated processes by microscopy require accurate single-cell observations of sufficient duration to reconstruct the genealogy. However, cell tracking-assigning identical cells on consecutive images to a track-is often challenging, resulting in laborious manual verification. Here, we propose fingerprints to identify problematic assignments rapidly. A fingerprint distance compares the structural information contained in the low frequencies of a Fourier transform to measure the similarity between cells in two consecutive images. We show that fingerprints are broadly applicable across cell types and image modalities, provided the image has sufficient structural information. Our tracker (TracX) uses fingerprints to reject unlikely assignments, thereby increasing tracking performance on published and newly generated long-term data sets. For Saccharomyces cerevisiae, we propose a comprehensive model for cell size control at the single-cell and population level centered on the Whi5 regulator, demonstrating how precise tracking can help uncover previously undescribed single-cell biology.

Coordination of gene expression with cell size enables Escherichia coli to efficiently maintain motility across conditions

To swim and navigate, motile bacteria synthesize a complex motility machinery involving flagella, motors, and a sensory system. A myriad of studies has elucidated the molecular processes involved, but less is known about the coordination of motility expression with cellular physiology: In Escherichia coli, motility genes are strongly up-regulated in nutrient-poor conditions compared to nutrient-replete conditions; yet a quantitative link to cellular motility has not been developed. Here, we systematically investigated gene expression, swimming behavior, cell growth, and available proteomics data across a broad spectrum of exponential growth conditions. Our results suggest that cells up-regulate the expression of motility genes at slow growth to compensate for reduction in cell size, such that the number of flagella per cell is maintained across conditions. The observed four or five flagella per cell is the minimum number needed to keep the majority of cells motile. This simple regulatory objective allows E. coli cells to remain motile across a broad range of growth conditions, while keeping the biosynthetic and energetic demands to establish and drive the motility machinery at the minimum needed. Given the strong reduction in flagella synthesis resulting from cell size increases at fast growth, our findings also provide a different physiological perspective on bacterial cell size control: A larger cell size at fast growth is an efficient strategy to increase the allocation of cellular resources to the synthesis of those proteins required for biomass synthesis and growth, while maintaining processes such as motility that are only needed on a per-cell basis.

Members of SIAMESE-RELATED Class Inhibitor Proteins of Cyclin-Dependent Kinase Retard G2 Progression and Increase Cell Size in Arabidopsis thaliana.

Cell size requires strict and flexible control as it significantly impacts plant growth and development. Unveiling the molecular mechanism underlying cell size control would provide fundamental insights into plants’ nature as sessile organisms. Recently, a GRAS family transcription factor SCARECROW-LIKE28 (SCL28) was identified as a determinant of cell size in plants; specifically, SCL28 directly induces a subset of SIAMESE-RELATED (SMR) family genes encoding plant-specific inhibitors of cyclin-dependent kinases (i.e., SMR1, SMR2, SMR6, SMR8, SMR9, SMR13, and SMR14), thereby slowing down G2 phase progression to provide the time to increase cell volume. Of the SMR genes regulated by SCL28, genetic analysis has demonstrated that SMR1, SMR2, and SMR13 cooperatively regulate the cell size downstream of SCL28 in roots and leaves, whereas other SMR members’ contribution remains unexplored. This study shows that in root meristematic cells, SMR9 redundantly participates in cell size control with SMR1, SMR2, and SMR13. Moreover, our cell cycle analysis provides the first experimental evidence that SMR proteins inhibit the G2 progression of proliferating cells. Overall, these findings illuminate the diverse yet overlapping roles of SMR proteins in cell cycle regulation while reinforcing that SMRs are essential downstream effectors of SCL28 to modulate G2 progression and cell size.

The cell cycle inhibitor RB is diluted in G1 and contributes to controlling cell size in the mouse liver.

Every type of cell in an animal maintains a specific size, which likely contributes to its ability to perform its physiological functions. While some cell size control mechanisms are beginning to be elucidated through studies of cultured cells, it is unclear if and how such mechanisms control cell size in an animal. For example, it was recently shown that RB, the retinoblastoma protein, was diluted by cell growth in G1 to promote size-dependence of the G1/S transition. However, it remains unclear to what extent the RB-dilution mechanism controls cell size in an animal. We therefore examined the contribution of RB-dilution to cell size control in the mouse liver. Consistent with the RB-dilution model, genetic perturbations decreasing RB protein concentrations through inducible shRNA expression or through liver-specific Rb1 knockout reduced hepatocyte size, while perturbations increasing RB protein concentrations in an Fah −/− mouse model increased hepatocyte size. Moreover, RB concentration reflects cell size in G1 as it is lower in larger G1 hepatocytes. In contrast, concentrations of the cell cycle activators Cyclin D1 and E2f1 were relatively constant. Lastly, loss of Rb1 weakened cell size control, i.e., reduced the inverse correlation between how much cells grew in G1 and how large they were at birth. Taken together, our results show that an RB-dilution mechanism contributes to cell size control in the mouse liver by linking cell growth to the G1/S transition.

Increasing cell size remodels the proteome and promotes senescence

Cell size is tightly controlled in healthy tissues, but it is unclear how deviations in cell size affect cell physiology. To address this, we measured how the cell's proteome changes with increasing cell size. Size-dependent protein concentration changes are widespread and predicted by subcellular localization, size-dependent mRNA concentrations, and protein turnover. As proliferating cells grow larger, concentration changes typically associated with cellular senescence are increasingly pronounced, suggesting that large size may be a cause rather than just a consequence of cell senescence. Consistent with this hypothesis, larger cells are prone to replicative, DNA-damage-induced, and CDK4/6i-induced senescence. Size-dependent changes to the proteome, including those associated with senescence, are not observed when an increase in cell size is accompanied by an increase in ploidy. Together, our findings show how cell size could impact many aspects of cell physiology by remodeling the proteome and provide a rationale for cell size control and polyploidization.

S6 kinase 1 at the central node of cell size and ageing.

Genetic evidence in living organisms from yeast to plants and animals, including humans, unquestionably identifies the Target Of Rapamycin kinase (TOR or mTOR for mammalian/mechanistic) signal transduction pathway as a master regulator of growth through the control of cell size and cell number. Among the mTOR targets, the activation of p70 S6 kinase 1 (S6K1) is exquisitely sensitive to nutrient availability and rapamycin inhibition. Of note, in vivo analysis of mutant flies and mice reveals that S6K1 predominantly regulates cell size versus cell proliferation. Here we review the putative mechanisms of S6K1 action on cell size by considering the main functional categories of S6K1 targets: substrates involved in nucleic acid and protein synthesis, fat mass accumulation, retrograde control of insulin action, senescence program and cytoskeleton organization. We discuss how S6K1 may be involved in the observed interconnection between cell size, regenerative and ageing responses.

The role of cellular quiescence in cancer - beyond a quiet passenger.

Quiescence, the ability to temporarily halt proliferation, is a conserved process that initially allowed survival of unicellular organisms during inhospitable times and later contributed to the rise of multicellular organisms, becoming key for cell differentiation, size control and tissue homeostasis. In this Review, we explore the concept of cancer as a disease that involves abnormal regulation of cellular quiescence at every step, from malignant transformation to metastatic outgrowth. Indeed, disrupted quiescence regulation can be linked to each of the so-called 'hallmarks of cancer'. As we argue here, quiescence induction contributes to immune evasion and resistance against cell death. In contrast, loss of quiescence underlies sustained proliferative signalling, evasion of growth suppressors, pro-tumorigenic inflammation, angiogenesis and genomic instability. Finally, both acquisition and loss of quiescence are involved in replicative immortality, metastasis and deregulated cellular energetics. We believe that a viewpoint that considers quiescence abnormalities that occur during oncogenesis might change the way we ask fundamental questions and the experimental approaches we take, potentially contributing to novel discoveries that might help to alter the course of cancer therapy.

Fluorescence exclusion – a rapid, accurate and powerful method for measuring yeast cell volume

Cells exist in an astonishing range of volumes across and within species. However, our understanding of cell size control remains limited, owing in large part to the challenges associated with accurate determination of cell volume. Much of our comprehension of size regulation derives from yeast models, but even for these morphologically stereotypical cells, assessment of cell volume has mostly relied on proxies and extrapolations from two-dimensional measurements. Recently, the fluorescence exclusion method (FXm) was developed to evaluate the size of mammalian cells, but whether it could be applied to smaller cells remained unknown. Using specifically designed microfluidic chips and an improved data analysis pipeline, we show here that FXm reliably detects subtle differences in the volume of fission yeast cells, even for those with altered shapes. Moreover, it allows for the monitoring of dynamic volume changes at the single-cell level with high time resolution. Collectively, our work highlights how the coupling of FXm with yeast genetics will bring new insights into the complex biology of cell growth.

The transcription factor Ahr1 links cell size control to amino acid metabolism in the opportunistic yeast Candida albicans

In most eukaryotes, size homeostasis is exerted in late G1 phase as cells commit to division, called Start in yeast and the Restriction Point in metazoans. At the cellular level, size is dictated by the balance between cellular growth and division such that each cell division is accompanied by a doubling in cell mass. Our systematic screen for size mutants revealed that hundreds of genes markedly altered cell size in the opportunistic yeast Candida albicans, but only few of these overlapped with size control genes in the model yeast Saccharomyces cerevisiae. Here, we characterized one of the potent size regulators in C. albicans, the zinc-finger transcription factor Ahr1 that is unique to Candida yeasts of the CTG-clade. We found that Ahr1 acts as both a repressor of Start and a transcriptional regulator of amino acid metabolic genes. Consistently, Ahr1 was required for amino acid and nitrogen-source modulation of cell size. Genetic interactions with deletions of different known Start regulators in C. albicans revealed functional relationship of Ahr1 with the AGC family protein kinase Sch9. Collectively, this work uncovered a novel network of the nutrient-dependent size control in C. albicans and emphasizes the impact of nitrogen and amino acid metabolisms in size homeostasis in this pathogenic fungus.

Eukaryotic Cell Size Control and Its Relation to Biosynthesis and Senescence.

The most fundamental feature of cellular form is size, which sets the scale of all cell biological processes. Growth, form, and function are all necessarily linked in cell biology, but we often do not understand the underlying molecular mechanisms nor their specific functions. Here, we review progress toward determining the molecular mechanisms that regulate cell size in yeast, animals, and plants, as well as progress toward understanding the function of cell size regulation. It has become increasingly clear that the mechanism of cell size regulation is deeply intertwined with basic mechanisms of biosynthesis, and how biosynthesis can be scaled (or not) in proportion to cell size. Finally, we highlight recent findings causally linking aberrant cell size regulation to cellular senescence and their implications for cancer therapies.

A hierarchical transcriptional network activates specific CDK inhibitors that regulate G2 to control cell size and number in Arabidopsis

How cell size and number are determined during organ development remains a fundamental question in cell biology. Here, we identified a GRAS family transcription factor, called SCARECROW-LIKE28 (SCL28), with a critical role in determining cell size in Arabidopsis. SCL28 is part of a transcriptional regulatory network downstream of the central MYB3Rs that regulate G2 to M phase cell cycle transition. We show that SCL28 forms a dimer with the AP2-type transcription factor, AtSMOS1, which defines the specificity for promoter binding and directly activates transcription of a specific set of SIAMESE-RELATED (SMR) family genes, encoding plant-specific inhibitors of cyclin-dependent kinases and thus inhibiting cell cycle progression at G2 and promoting the onset of endoreplication. Through this dose-dependent regulation of SMR transcription, SCL28 quantitatively sets the balance between cell size and number without dramatically changing final organ size. We propose that this hierarchical transcriptional network constitutes a cell cycle regulatory mechanism that allows to adjust cell size and number to attain robust organ growth.

Cellular Flocculation Using Concentrated Polymer Brush-Modified Cellulose Nanofibers with Different Fiber Lengths.

In this study, concentrated polymer brush-modified cellulose nanofibers (CNFs) with different fiber lengths were used for the flocculation of cells for systematically studying the mechanism of this unique cellular flocculation based on colloidal flocculation theory. Concentrated poly(p-styrenesulfonic acid sodium salt) brush-grafted CNF (CNF-PSSNa) with different fiber lengths were cultured with three different cell types to examine their influence on floc (cell clusters formed by cellular flocculation) characteristics. The floc size and survival rate could be controlled by modifying the CNF-PSSNa fiber lengths. The three cell types showed the same flocculation tendency after culture, indicating the applicability of the method in different cell lines. After 2 weeks of culture, CNF-PSSNa increased the specific expression of hepatocytes compared to the two-dimensional cell culture. Thus, owing to its wide applicability, high cell viability, and ability to control cell size and improve cell function, this technology could be used as a new three-dimensional cell culture method.

Branching processes with resetting as a model for cell division

We study the stochastic thermodynamics of cell growth and division using a theoretical framework based on branching processes with resetting. Cell division may be split into two sub-processes: branching, by which a given cell gives birth to an identical copy of itself, and resetting, by which some properties of the daughter cells (such as their size or age) are reset to new values following division. We derive the first and second laws of stochastic thermodynamics for this process, and identify separate contributions due to branching and resetting. We apply our framework to well-known models of cell size control, such as the sizer, the timer, and the adder. We show that the entropy production of resetting is negative and that of branching is positive for these models in the regime of exponential growth of the colony. This property suggests an analogy between our model for cell growth and division and heat engines, and the introduction of a thermodynamic efficiency, which quantifies the conversion of one form of entropy production to another.