Cell Size Control Research Articles

Septal wall synthesis is sufficient to change ameba-like cells into uniform oval-shaped cells in Escherichia coli L-forms

A cell wall is required to control cell shape and size to maintain growth and division. However, some bacterial species maintain their morphology and size without a cell wall, calling into question the importance of the cell wall to maintain shape and size. It has been very difficult to examine the dispensability of cell wall synthesis in rod-shaped bacteria such as Escherichia coli for maintenance of their shape and size because they lyse without cell walls under normal culture conditions. Here, we show that wall-less E. coli L-form cells, which have a heterogeneous cell morphology, can be converted to a mostly uniform oval shape solely by FtsZ-dependent division, even in the absence of cylindrical cell wall synthesis. This FtsZ-dependent control of cell shape and size in the absence of a cell wall requires at least either the Min or nucleoid occlusion systems for positioning FtsZ at mid cell division sites.

Eukaryotic cell size regulation and its implications for cellular function and dysfunction.

Depending on cell type, environmental inputs, and disease, the cells in the human body can have widely different sizes. In recent years, it has become clear that cell size is a major regulator of cell function. However, we are only beginning to understand how the optimization of cell function determines a given cell's optimal size. Here, we review currently known size control strategies of eukaryotic cells and the intricate link of cell size to intracellular biomolecular scaling, organelle homeostasis, and cell cycle progression. We detail the cell size-dependent regulation of early development and the impact of cell size on cell differentiation. Given the importance of cell size for normal cellular physiology, cell size control must account for changing environmental conditions. We describe how cells sense environmental stimuli, such as nutrient availability, and accordingly adapt their size by regulating cell growth and cell cycle progression. Moreover, we discuss the correlation of pathological states with misregulation of cell size and how for a long time this was considered a downstream consequence of cellular dysfunction. We review newer studies that reveal a reversed causality, with misregulated cell size leading to pathophysiological phenotypes such as senescence and aging. In summary, we highlight the important roles of cell size in cellular function and dysfunction, which could have major implications for both diagnostics and treatment in the clinic.

Bacterial cell size modulation along the growth curve across nutrient conditions.

Under stable growth conditions, bacteria maintain cell size homeostasis through coordinated elongation and division. However, fluctuations in nutrient availability result in dynamic regulation of the target cell size. Using microscopy imaging and mathematical modelling, we examine how bacterial cell volume changes over the growth curve in response to nutrient conditions. We find that two rod-shaped bacteria, Escherichia coli and Salmonella enterica, exhibit similar cell volume distributions in stationary phase cultures irrespective of growth media. Cell resuspension in rich media results in a transient peak with a five-fold increase in cell volume ≈ 2h after resuspension. This maximum cell volume, which depends on nutrient composition, subsequently decreases to the stationary phase cell size. Continuous nutrient supply sustains the maximum volume. In poor nutrient conditions, cell volume shows minimal changes over the growth curve, but a markedly decreased cell width compared to other conditions. The observed cell volume dynamics translate into non-monotonic dynamics in the ratio between biomass (optical density) and cell number (colony-forming units), highlighting their non-linear relationship. Our findings support a heuristic model comparing modulation of cell division relative to growth across nutrient conditions and providing novel insight into the mechanisms of cell size control under dynamic environmental conditions.

A Generalized Adder mechanism for Cell Size Homeostasis: Implications for Stochastic Dynamics of Clonal Proliferation.

Measurements of cell size dynamics have revealed phenomeno-logical principles by which individual cells control their size across diverse organisms. One of the emerging paradigms of cell size homeostasis is the adder , where the cell cycle duration is established such that the cell size increase from birth to division is independent of the newborn cell size. We provide a mechanistic formulation of the adder considering that cell size follows any arbitrary non-exponential growth law . Our results show that the main requirement to obtain an adder regardless of the growth law (the time derivative of cell size) is that cell cycle regulators are produced at a rate proportional to the growth law and cell division is triggered when these molecules reach a prescribed threshold level. Among the implications of this generalized adder, we investigate fluctuations in the proliferation of single-cell derived colonies. Considering exponential cell size growth, random fluctuations in clonal size show a transient increase and then eventually decay to zero over time (i.e., clonal populations become asymptotically more similar). In contrast, several forms of non-exponential cell size dynamics (with adder-based cell size control) yield qualitatively different results: clonal size fluctuations monotonically increase over time reaching a non-zero value. These results characterize the interplay between cell size homeostasis at the single-cell level and clonal proliferation at the population level, explaining the broad fluctuations in clonal sizes seen in barcoded human cell lines.

WOX11-mediated cell size control in Arabidopsis attenuates growth and fecundity of endoparasitic cyst nematodes.

Cyst nematodes establish permanent feeding structures called syncytia inside the host root vasculature, disrupting the flow of water and minerals. In response, plants form WOX11-mediated adventitious lateral roots at nematode infection sites. WOX11 adventitious lateral rooting modulates tolerance to nematode infections; however, whether this also benefits nematode parasitism remains unknown. Here, we report on bioassays using a 35S::WOX11-SRDX transcriptional repressor mutant to investigate whether WOX11 adventitious lateral rooting promotes syncytium development and thereby female growth and fecundity. Moreover, we chemically inhibited cellulose biosynthesis to verify if WOX11 directly modulates cell wall plasticity in syncytia. Finally, we performed histochemical analyses to test if WOX11 mediates syncytial cell wall plasticity via reactive oxygen species (ROS). Repression of WOX11-mediated transcription specifically enhanced the radial expansion of syncytial elements, increasing both syncytium size and female offspring. The enhanced syncytial hypertrophy observed in the 35S::WOX11-SRDX mutant could be phenocopied by chemical inhibition of cellulose biosynthesis and was associated with elevated levels of ROS at nematode infection sites. We, therefore, conclude that WOX11 restricts radial expansion of nematode-feeding structures and female growth and fecundity, likely by modulating ROS-mediated cell wall plasticity mechanisms. Remarkably, this novel role of WOX11 in plant cell size control is distinct from WOX11 adventitious lateral rooting underlying disease tolerance.

SnapShot: Cell size control

Cell size exhibits remarkable diversity across and within organisms. Size variation correlates with DNA content and growth rates and is regulated by complex models of cell size control that are yet to be mechanistically defined. To view this SnapShot, open or download the PDF.

Abstract 4319: HSF1 inhibition induces pancreatic ductal adenocarcinoma autophagy through JNK

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related mortality, with a 5-year survival rate of 12%. Chemoresistance due to autophagy, a natural process of proteotoxic stress response, is the most common chemotherapy challenge for pancreatic cancer. Autophagy, which maintains cellular homeostasis by removing damaged or unnecessary proteins and organelles, is a self-degradation process regulated by the mammalian target of rapamycin complex (mTORC1). Excessive levels of autophagy can be detrimental and lead to cell death. Nevertheless, this dual function of autophagy in regulating pancreatic cancer remains poorly understood. It has been reported that heat shock factor 1 (HSF1), a critical pro-oncogenic transcription factor in the proteotoxic stress response, inhibits c-Jun N-terminal kinase 1/2 (JNK1/2) signaling pathway to maintain mTORC1 activity and control cell size. HSF1 is highly expressed in pancreatic tumors, and inhibition of HSF1 via small molecules enhances the chemotherapy drug effect on PDAC programmed cell death. However, the PDAC autophagy response in HSF1 inhibition is currently not fully understood. Objective: The objective of this study is to investigate the molecular mechanism of HSF1 inhibition in PDAC autophagy. Methods and Results: In human PDAC cells, Western blotting analysis revealed that HSF1 inhibition via small molecular including CCT361814/NXP800, an HSF1 inhibitor in phase I clinical trials, increased autophagy marker microtubule-associated protein 1A/1B-light chain 3 (LC3) lipidation and decreased expression of phosphorylation of HSF1 at Ser326 and total HSF1 protein, conversely using HSF1 overexpression model reversed this effect. Inhibition of autophagy via 3-methyladenine reversed HSF1 inhibition-induced autophagy. Results from transmission electron microscopy and GFP-LC3 reporter revealed that HSF1 inhibition induced autophagosome formation and accumulation. Furthermore, HSF1 inhibition induced phosphorylation of JNK1/2 at Thr183/Tyr185 and decreased mTORC1 activity, while knockdown JNK1/2 diminished HSF1 inhibition-induced LC3 lipidation. The combination of chemotherapy drugs with HSF1 inhibitors decreased PDAC cell viability. Additionally, blocking autophagy increased HSF1 inhibition-induced apoptosis. Conclusions: In conclusion, JNK1/2 is involved in HSF1 inhibition-induced PDAC autophagy, targeting autophagy could unveil a novel treatment strategy for pancreatic cancer through targeted HSF1 inhibition. Citation Format: Shruti Ghai, Alex Young, Kuo-Hui Su. HSF1 inhibition induces pancreatic ductal adenocarcinoma autophagy through JNK [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4319.

Effects of Molecular Noise on Cell Size Control.

Cells employ control strategies to maintain a stable size. Dividing at a target size (the "sizer" strategy) is thought to produce the tightest size distribution. However, this result follows from phenomenological models that ignore the molecular mechanisms required to implement the strategy. Here we investigate a simple mechanistic model for exponentially growing cells whose division is triggered at a molecular abundance threshold. We find that size noise inherits the molecular noise and is consequently minimized not by the sizer but by the "adder" strategy, where a cell divides after adding a target amount to its birth size. We derive a lower bound on size noise that agrees with publicly available data from six microfluidic studies on Escherichia coli bacteria.

Stem cell mTOR signaling directs region-specific cell fate decisions during intestinal nutrient adaptation.

The adult intestine is a regionalized organ, whose size and cellular composition are adjusted in response to nutrient status. This involves dynamic regulation of intestinal stem cell (ISC) proliferation and differentiation. How nutrient signaling controls cell fate decisions to drive regional changes in cell-type composition remains unclear. Here, we show that intestinal nutrient adaptation involves region-specific control of cell size, cell number, and differentiation. We uncovered that activation of mTOR complex 1 (mTORC1) increases ISC size in a region-specific manner. mTORC1 activity promotes Delta expression to direct cell fate toward the absorptive enteroblast lineage while inhibiting secretory enteroendocrine cell differentiation. In aged flies, the ISC mTORC1 signaling is deregulated, being constitutively high and unresponsive to diet, which can be mitigated through lifelong intermittent fasting. In conclusion, mTORC1 signaling contributes to the ISC fate decision, enabling regional control of intestinal cell differentiation in response to nutrition.

Evidence for novel mechanisms that control cell-cycle entry and cell size.

Entry into the cell cycle in late G1 phase occurs only when sufficient growth has occurred. In budding yeast, a cyclin called Cln3 is thought to link cell-cycle entry to cell growth. Cln3 accumulates during growth in early G1 phase and eventually helps trigger expression of late G1 phase cyclins that drive cell-cycle entry. All current models for cell-cycle entry assume that expression of late G1 phase cyclins is initiated at the transcriptional level. Current models also assume that the sole function of Cln3 in cell-cycle entry is to promote transcription of late G1 phase cyclins, and that Cln3 works solely in G1 phase. Here, we show that cell cycle-dependent expression of the late G1 phase cyclin Cln2 does not require any functions of the CLN2 promoter. Moreover, Cln3 can influence accumulation of Cln2 protein via posttranscriptional mechanisms. Finally, we show that Cln3 has functions in mitosis that strongly influence cell size. Together, these discoveries reveal the existence of surprising new mechanisms that challenge current models for control of cell-cycle entry and cell size.

Should I shrink or should I grow: cell size changes in tissue morphogenesis.

Cells change shape, move, divide, and die to sculpt tissues. Common to all these cell behaviours are cell size changes, which have recently emerged as key contributors to tissue morphogenesis. Cells can change their mass-the number of macromolecules they contain-or their volume-the space they encompass. Changes in cell mass and volume occur through different molecular mechanisms and at different timescales, slow for changes in mass and rapid for changes in volume. Therefore, changes in cell mass and cell volume, which are often linked, contribute to the development and shaping of tissues in different ways. Here, we review the molecular mechanisms by which cells can control and alter their size, and we discuss how changes in cell mass and volume contribute to tissue morphogenesis. The role that cell size control plays in developing embryos is only starting to be elucidated. Research on the signals that control cell size will illuminate our understanding of the cellular and molecular mechanisms that drive tissue morphogenesis.

Heat tolerance in Drosophila melanogaster is influenced by oxygen conditions and mutations in cell size control pathways.

Understanding metabolic performance limitations is key to explaining the past, present and future of life. We investigated whether heat tolerance in actively flying Drosophila melanogaster is modified by individual differences in cell size and the amount of oxygen in the environment. We used two mutants with loss-of-function mutations in cell size control associated with the target of rapamycin (TOR)/insulin pathways, showing reduced (mutant rictorΔ2) or increased (mutant Mnt1) cell size in different body tissues compared to controls. Flies were exposed to a steady increase in temperature under normoxia and hypoxia until they collapsed. The upper critical temperature decreased in response to each mutation type as well as under hypoxia. Females, which have larger cells than males, had lower heat tolerance than males. Altogether, mutations in cell cycle control pathways, differences in cell size and differences in oxygen availability affected heat tolerance, but existing theories on the roles of cell size and tissue oxygenation in metabolic performance can only partially explain our results. A better understanding of how the cellular composition of the body affects metabolism may depend on the development of research models that help separate various interfering physiological parameters from the exclusive influence of cell size. This article is part of the theme issue 'The evolutionary significance of variation in metabolic rates'.

Cell size homeostasis is tightly controlled throughout the cell cycle.

To achieve a stable size distribution over multiple generations, proliferating cells require a means of counteracting stochastic noise in the rate of growth, the time spent in various phases of the cell cycle, and the imprecision in the placement of the plane of cell division. In the most widely accepted model, cell size is thought to be regulated at the G1/S transition, such that cells smaller than a critical size pause at the end of G1 phase until they have accumulated mass to a predetermined size threshold, at which point the cells proceed through the rest of the cell cycle. However, a model, based solely on a specific size checkpoint at G1/S, cannot readily explain why cells with deficient G1/S control mechanisms are still able to maintain a very stable cell size distribution. Furthermore, such a model would not easily account for stochastic variation in cell size during the subsequent phases of the cell cycle, which cannot be anticipated at G1/S. To address such questions, we applied computationally enhanced quantitative phase microscopy (ceQPM) to populations of cultured human cell lines, which enables highly accurate measurement of cell dry mass of individual cells throughout the cell cycle. From these measurements, we have evaluated the factors that contribute to maintaining cell mass homeostasis at any point in the cell cycle. Our findings reveal that cell mass homeostasis is accurately maintained, despite disruptions to the normal G1/S machinery or perturbations in the rate of cell growth. Control of cell mass is generally not confined to regulation of the G1 length. Instead mass homeostasis is imposed throughout the cell cycle. In the cell lines examined, we find that the coefficient of variation (CV) in dry mass of cells in the population begins to decline well before the G1/S transition and continues to decline throughout S and G2 phases. Among the different cell types tested, the detailed response of cell growth rate to cell mass differs. However, in general, when it falls below that for exponential growth, the natural increase in the CV of cell mass is effectively constrained. We find that both mass-dependent cell cycle regulation and mass-dependent growth rate modulation contribute to reducing cell mass variation within the population. Through the interplay and coordination of these 2 processes, accurate cell mass homeostasis emerges. Such findings reveal previously unappreciated and very general principles of cell size control in proliferating cells. These same regulatory processes might also be operative in terminally differentiated cells. Further quantitative dynamical studies should lead to a better understanding of the underlying molecular mechanisms of cell size control.

Cytoophidia Influence Cell Cycle and Size in Schizosaccharomyces pombe.

Cytidine triphosphate synthase (CTPS) forms cytoophidia in all three domains of life. Here we focus on the function of cytoophidia in cell proliferation using Schizosaccharomyces pombe as a model system. We find that converting His359 of CTPS into Ala359 leads to cytoophidium disassembly. By reducing the level of CTPS protein or specific mutation, the loss of cytoophidia prolongs the G2 phase and expands cell size. In addition, the loss-filament mutant of CTPS leads to a decrease in the expression of genes related to G2/M transition and cell growth, including histone chaperone slm9. The overexpression of slm9 alleviates the G2 phase elongation and cell size enlargement induced by CTPS loss-filament mutants. Overall, our results connect cytoophidia with cell cycle and cell size control in Schizosaccharomyces pombe.

Cell size: Single cells illuminate the rules of cell size control

A new study uses Chlamydomonas reinhardtii to understand how cell size homeostasis emerges from stochastic individual cell behaviors within a population. The authors find that a simple power law model was a poor predictor of cell size regulation; rather, it is better explained by a modified threshold model.

Spatiotemporal deposition of cell wall polysaccharides in oat endosperm during grain development.

Oat (Avena sativa) is a cereal crop whose grains are rich in (1,3;1,4)-β-D-glucan (mixed-linkage glucan or MLG), a soluble dietary fiber. In our study, we analyzed oat endosperm development in 2 Canadian varieties with differing MLG content and nutritional value. We confirmed that oat undergoes a nuclear type of endosperm development but with a shorter cellularization phase than barley (Hordeum vulgare). Callose and cellulose were the first polysaccharides to be detected in the early anticlinal cell walls at 11 days postemergence (DPE) of the panicle. Other polysaccharides such as heteromannan and homogalacturonan were deposited early in cellularization around 12 DPE after the first periclinal walls are laid down. In contrast to barley, heteroxylan deposition coincided with completion of cellularization and was detected from 14 DPE but was only detectable after demasking. Notably, MLG was the last polysaccharide to be laid down at 18 DPE within the differentiation phase, rather than during cellularization. In addition, differences in the spatiotemporal patterning of MLG were also observed between the 2 varieties. The lower MLG-containing cultivar AC Morgan (3.5% w/w groats) was marked by the presence of a discontinuous pattern of MLG labeling, while labeling in the same walls in CDC Morrison (5.6% w/w groats) was mostly even and continuous. RNA-sequencing analysis revealed higher transcript levels of multiple MLG biosynthetic cellulose synthase-like F (CSLF) and CSLH genes during grain development in CDC Morrison compared with AC Morgan that likely contributes to the increased abundance of MLG at maturity in CDC Morrison. CDC Morrison was also observed to have smaller endosperm cells with thicker walls than AC Morgan from cellularization onwards, suggesting the processes controlling cell size and shape are established early in development. This study has highlighted that the molecular processes influencing MLG content and deposition are more complex than previously imagined.

Modelling how plant cell-cycle progression leads to cell size regulation.

Populations of cells typically maintain a consistent size, despite cell division rarely being precisely symmetrical. Therefore, cells must possess a mechanism of "size control", whereby the cell volume at birth affects cell-cycle progression. While size control mechanisms have been elucidated in a number of other organisms, it is not yet clear how this mechanism functions in plants. Here, we present a mathematical model of the key interactions in the plant cell cycle. Model simulations reveal that the network of interactions exhibits limit-cycle solutions, with biological switches underpinning both the G1/S and G2/M cell-cycle transitions. Embedding this network model within growing cells, we test hypotheses as to how cell-cycle progression can depend on cell size. We investigate two different mechanisms at both the G1/S and G2/M transitions: (i) differential expression of cell-cycle activator and inhibitor proteins (with synthesis of inhibitor proteins being independent of cell size), and (ii) equal inheritance of inhibitor proteins after cell division. The model demonstrates that both these mechanisms can lead to larger daughter cells progressing through the cell cycle more rapidly, and can thus contribute to cell-size control. To test how these features enable size homeostasis over multiple generations, we then simulated these mechanisms in a cell-population model with multiple rounds of cell division. These simulations suggested that integration of size-control mechanisms at both G1/S and G2/M provides long-term cell-size homeostasis. We concluded that while both size independence and equal inheritance of inhibitor proteins can reduce variations in cell size across individual cell-cycle phases, combining size-control mechanisms at both G1/S and G2/M is essential to maintain size homeostasis over multiple generations. Thus, our study reveals how features of the cell-cycle network enable cell-cycle progression to depend on cell size, and provides a mechanistic understanding of how plant cell populations maintain consistent size over generations.

The molecular underpinnings of body size regulation: Transcriptional changes in foxo and mTor in Drosophila melanogaster selected for fast development

One of biology's most perplexing ideas is the biological control of an organism's size. Even if many questions remain unanswered, it can be logically implied that the size of an individual is clearly correlated with its cell number and cell size. Size regulation, which happens through the control of cell size and number, is crucial for the development of multicellular organisms. Long term selection of Drosophila melanogaster for fast development has led to reduction in body size. In this study, we compared three types of D. melanogaster populations- the first were selected for fast development and short effective lifespan (FEJs), second were selected for fast development and longer effective lifespan (FLJs) and the third were their ancestral control populations (JBs), and answered whether up-regulation of foxo (forkhead box, subgroup-o) and/or down regulation of mTor (mechanistic Target of rapamycin) is responsible for reduction in organismal size in fast developing D. melanogaster. Our results show that upregulation of foxo might be responsible for reduction in size in both FLJs and FEJs. Further, higher mTor in FEJs but no change in mTor in FLJs in comparison to JBs confirms that selection for fast development does not lead to starvation like phenotype or stress like conditions in D. melanogaster. Furthermore, this difference in mTor in FEJs and FLJs might be due to selection for short effective lifespan and long effective lifespan respectively, as suppression or blocking mTor expression has been reported to extend lifespan. These results suggest that potentially deleterious allele for late life is under positive selection in FEJs but not in FLJs.

Bacterial cell-size changes resulting from altering the relative expression of Min proteins

The timing of cell division, and thus cell size in bacteria, is determined in part by the accumulation dynamics of the protein FtsZ, which forms the septal ring. FtsZ localization depends on membrane-associated Min proteins, which inhibit FtsZ binding to the cell pole membrane. Changes in the relative concentrations of Min proteins can disrupt FtsZ binding to the membrane, which in turn can delay cell division until a certain cell size is reached, in which the dynamics of Min proteins frees the cell membrane long enough to allow FtsZ ring formation. Here, we study the effect of Min proteins relative expression on the dynamics of FtsZ ring formation and cell size in individual Escherichia coli bacteria. Upon inducing overexpression of minE, cell size increases gradually to a new steady-state value. Concurrently, the time required to initiate FtsZ ring formation grows as the size approaches the new steady-state, at which point the ring formation initiates as early as before induction. These results highlight the contribution of Min proteins to cell size control, which may be partially responsible for the size fluctuations observed in bacterial populations, and may clarify how the size difference acquired during asymmetric cell division is offset.

LAMMER Kinase Modulates Cell Cycle by Phosphorylating the MBF Repressor, Yox1, in Schizosaccharomyces pombe

Lkh1, a LAMMER kinase homolog in the fission yeast Schizosaccharomyces pombe, acts as a negative regulator of filamentous growth and flocculation. It is also involved in the response to oxidative stress. The lkh1-deletion mutant displays slower cell growth, shorter cell size, and abnormal DNA content compared to the wild type. These phenotypes suggest that Lkh1 controls cell size and cell cycle progression. When we performed microarray analysis using the lkh1-deletion mutant, we found that only four of the up-regulated genes in the lkh1-deletion were associated with the cell cycle. Interestingly, all of these genes are regulated by the Mlu1 cell cycle box binding factor (MBF), which is a transcription complex responsible for regulating the expression of cell cycle genes during the G1/S phase. Transcription analyses of the MBF-dependent cell-cycle genes, including negative feedback regulators, confirmed the up-regulation of these genes by the deletion of lkh1. Pull-down assay confirmed the interaction between Lkh1 and Yox1, which is a negative feedback regulator of MBF. This result supports the involvement of LAMMER kinase in cell cycle regulation by modulating MBF activity. In vitro kinase assay and NetPhosK 2.0 analysis with the Yox1T40,41A mutant allele revealed that T40 and T41 residues are the phosphorylation sites mediated by Lkh1. These sites affect the G1/S cell cycle progression of fission yeast by modulating the activity of the MBF complex.