Cell Size Control Research Articles

Growth Rate as a Direct Regulator of the Start Network to Set Cell Size

Cells are able to adjust their growth and size to external inputs to comply with specific fates and developmental programs. Molecular pathways controlling growth also have an enormous impact in cell size, and bacteria, yeast, or epithelial cells modify their size as a function of growth rate. This universal feature suggests that growth (mass) and proliferation (cell number) rates are subject to general coordinating mechanisms. However, the underlying molecular connections are still a matter of debate. Here we review the current ideas on growth and cell size control, and focus on the possible mechanisms that could link the biosynthetic machinery to the Start network in budding yeast. In particular, we discuss the role of molecular chaperones in a competition framework to explain cell size control by growth at the individual cell level.

Abstract IA02: Regulation of growth by the mTOR pathway

Abstract mTOR is the target of the immunosuppressive and anti-cancer drug rapamycin and the central component of a nutrient- and hormone-sensitive signaling pathway that regulates cell growth and proliferation. We now appreciate that this pathway becomes deregulated in many human cancers and has key roles in the control of metabolism, cell and organ size, and aging. We have identified two distinct mTOR-containing proteins complexes, one of which regulates growth through S6K and another that regulates cell survival and metabolism through Akt. These complexes, called mTORC1 and mTORC2, define both rapamycin-sensitive and insensitive branches of the mTOR pathway. Over the last few years we have focused on how nutrients, particularly amino acids, regulate mTORC1. We have identified a complex signaling pathway that relays amino acid availability to mTORC1 through the heterodimeric Rag GTPases. We recently discovered several of the amino acid sensors for the pathway. I will discuss new results from our lab on the regulation and functions of mTORC1. Citation Format: David M. Sabatini. Regulation of growth by the mTOR pathway. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr IA02.

Optimization of low frequency sound absorption by cell size control and multiscale poroacoustics modeling

Sound absorption of a polyurethane (PU) foam was predicted for various geometries to fabricate the optimum microstructure of a sound absorbing foam. Multiscale numerical analysis for sound absorption was carried out by solving flow problems in representative unit cell (RUC) and the pressure acoustics equation using Johnson-Champoux-Allard (JCA) model. From the numerical analysis, theoretical optimum cell diameter for low frequency sound absorption was evaluated in the vicinity of 400μm under the condition of 2cm-80K (thickness of 2cm and density of 80kg/m3) foam. An ultrasonic foaming method was employed to modulate microcellular structure of PU foam. Mechanical activation was only employed to manipulate the internal structure of PU foam without any other treatment. A mean cell diameter of PU foam was gradually decreased with increase in the amplitude of ultrasonic waves. It was empirically found that the reduction of mean cell diameter induced by the ultrasonic wave enhances acoustic damping efficiency in low frequency ranges. Moreover, further analyses were performed with several acoustic evaluation factors; root mean square (RMS) values, noise reduction coefficients (NRC), and 1/3 octave band spectrograms.

Ste12/Fab1 phosphatidylinositol-3-phosphate 5-kinase is required for nitrogen-regulated mitotic commitment and cell size control.

Tight coupling of cell growth and cell cycle progression enable cells to adjust their rate of division, and therefore size, to the demands of proliferation in varying nutritional environments. Nutrient stress promotes inhibition of Target Of Rapamycin Complex 1 (TORC1) activity. In fission yeast, reduced TORC1 activity advances mitotic onset and switches growth to a sustained proliferation at reduced cell size. A screen for mutants, that failed to advance mitosis upon nitrogen stress, identified a mutant in the PIKFYVE 1-phosphatidylinositol-3-phosphate 5-kinase fission yeast homolog Ste12. Ste12PIKFYVE deficient mutants were unable to advance the cell cycle to reduce cell size after a nitrogen downshift to poor nitrogen (proline) growth conditions. While it is well established that PI(3,5)P2 signalling is required for autophagy and that Ste12PIKFYVE mutants have enlarged vacuoles (yeast lysosomes), neither a block to autophagy or mutants that independently have enlarged vacuoles had any impact upon nitrogen control of mitotic commitment. The addition of rapamycin to Ste12PIKFYVE deficient mutants reduced cell size at division to suggest that Ste12PIKFYVE possibly functions upstream of TORC1. ste12 mutants display increased Torin1 (TOR inhibitor) sensitivity. However, no major impact on TORC1 or TORC2 activity was observed in the ste12 deficient mutants. In summary, Ste12PIKFYVE is required for nitrogen-stress mediated advancement of mitosis to reduce cell size at division.

The REVEILLE Clock Genes Inhibit Growth of Juvenile and Adult Plants by Control of Cell Size.

The circadian clock is a complex regulatory network that enhances plant growth and fitness in a constantly changing environment. In Arabidopsis (Arabidopsis thaliana), the clock is composed of numerous regulatory feedback loops in which REVEILLE8 (RVE8) and its homologs RVE4 and RVE6 act in a partially redundant manner to promote clock pace. Here, we report that the remaining members of the RVE8 clade, RVE3 and RVE5, play only minor roles in the regulation of clock function. However, we find that RVE8 clade proteins have unexpected functions in the modulation of light input to the clock and the control of plant growth at multiple stages of development. In seedlings, these proteins repress hypocotyl elongation in a daylength- and sucrose-dependent manner. Strikingly, adult rve4 6 8 and rve3 4 5 6 8 mutants are much larger than wild-type plants, with both increased leaf area and biomass. This size phenotype is associated with a faster growth rate and larger cell size and is not simply due to a delay in the transition to flowering. Gene expression and epistasis analysis reveal that the growth phenotypes of rve mutants are due to the misregulation of PHYTOCHROME INTERACTING FACTOR4 (PIF4) and PIF5 expression. Our results show that even small changes in PIF gene expression caused by the perturbation of clock gene function can have large effects on the growth of adult plants.

Long-term microfluidic tracking of coccoid cyanobacterial cells reveals robust control of division timing

BackgroundCyanobacteria are important agents in global carbon and nitrogen cycling and hold great promise for biotechnological applications. Model organisms such as Synechocystis sp. and Synechococcus sp. have advanced our understanding of photosynthetic capacity and circadian behavior, mostly using population-level measurements in which the behavior of individuals cannot be monitored. Synechocystis sp. cells are small and divide slowly, requiring long-term experiments to track single cells. Thus, the cumulative effects of drift over long periods can cause difficulties in monitoring and quantifying cell growth and division dynamics.ResultsTo overcome this challenge, we enhanced a microfluidic cell-culture device and developed an image analysis pipeline for robust lineage reconstruction. This allowed simultaneous tracking of many cells over multiple generations, and revealed that cells expand exponentially throughout their cell cycle. Generation times were highly correlated for sister cells, but not between mother and daughter cells. Relationships between birth size, division size, and generation time indicated that cell-size control was inconsistent with the “sizer” rule, where division timing is based on cell size, or the “timer” rule, where division occurs after a fixed time interval. Instead, single cell growth statistics were most consistent with the “adder” rule, in which division occurs after a constant increment in cell volume. Cells exposed to light-dark cycles exhibited growth and division only during the light period; dark phases pause but do not disrupt cell-cycle control.ConclusionsOur analyses revealed that the “adder” model can explain both the growth-related statistics of single Synechocystis cells and the correlation between sister cell generation times. We also observed rapid phenotypic response to light-dark transitions at the single cell level, highlighting the critical role of light in cyanobacterial cell-cycle control. Our findings suggest that by monitoring the growth kinetics of individual cells we can build testable models of circadian control of the cell cycle in cyanobacteria.

Mechanistic aspects of mammalian cell size control.

Size distribution in a group of differentiated cells often falls into a constant range. However, invitro and invivo studies have shown that cells can temporarily change their size in response to their surrounding environment and the stimuli they receive. Thus, there must be a mechanism that normally keeps cell size constant while allowing a shift to an alternative size when necessary. To investigate the molecular basis of mammalian cell size control, we conducted a genetic screen in a human T cell line to identify genes involved in cell size regulation. A prime candidate emerging from this screen increases cell size when it is overexpressed but reduces cell size when subjected to siRNA knockdown. Several lines of evidence indicate that the product of this gene, which we called "Largen", regulates mRNA translation in a manner associated with the upregulation of a specific subset of mRNAs, many of which affect mitochondrial function. In fact, cells overexpressing Largen increase both mitochondrial mass and activity, enhancing ATP production. These invitro observations have been replicated invivo using transgenic mouse models. With a focus on these findings, we discuss the possible contribution of mitochondria to the control of mammalian cell size.

Evolution of Cell Size Homeostasis and Growth Rate Diversity during Initial Surface Colonization of Shewanella oneidensis.

Cell size control and homeostasis are fundamental features of bacterial metabolism. Recent work suggests that cells add a constant size between birth and division ("adder" model). However, it is not known how cell size homeostasis is influenced by the existence of heterogeneous microenvironments, such as those during biofilm formation. Shewanella oneidensis MR-1 can use diverse energy sources on a range of surfaces via extracellular electron transport (EET), which can impact growth, metabolism, and size diversity. Here, we track bacterial surface communities at single-cell resolution to show that not only do bacterial motility appendages influence the transition from two- to three-dimensional biofilm growth and control postdivisional cell fates, they strongly impact cell size homeostasis. For every generation, we find that the average growth rate for cells that stay on the surface and continue to divide (nondetaching population) and that for cells that detach before their next division (detaching population) are roughly constant. However, the growth rate distribution is narrow for the nondetaching population, but broad for the detaching population in each generation. Interestingly, the appendage deletion mutants (ΔpilA, ΔmshA-D, Δflg) have significantly broader growth rate distributions than that of the wild type for both detaching and nondetaching populations, which suggests that Shewanella appendages are important for sensing and integrating environmental inputs that contribute to size homeostasis. Moreover, our results suggest multiplexing of appendages for sensing and motility functions contributes to cell size dysregulation. These results can potentially provide a framework for generating metabolic diversity in S. oneidensis populations to optimize EET in heterogeneous environments.

Porosity and cell size control in alumina foam preparation by thermo-foaming of powder dispersions in molten sucrose

The foaming characteristics of alumina powder dispersions in molten sucrose have been studied as a function of alumina powder to sucrose weight ratio (WA/S) and foaming temperature. The increase in foaming temperature significantly decreases the foaming and foam setting time and increases the foam volume due to an increase in the rate of –OH condensation as well as a decrease in the viscosity of the dispersion. Nevertheless, the foam collapses beyond a critical foaming temperature, which depends on the WA/S. The sintering shrinkage depends mainly on the WA/S and marginally on the foaming temperature. The porosity (83.4–94.6 vol.%) and cell size (0.55–1.6 mm) increase with an increase in foaming temperature (120–170 °C) and a decrease in WA/S (0.8–1.6). The drastic decrease in compressive strength and modulus beyond a WA/S of 1.2 is due to the pores generated on the cell walls and struts as a result of particle agglomeration. Gibson and Ashby plots show large deviation with respect to the model constants ‘C’ and ‘n’, especially at higher alumina powder to sucrose weight ratios.

The Interplay between Myc and CTP Synthase in Drosophila.

CTP synthase (CTPsyn) is essential for the biosynthesis of pyrimidine nucleotides. It has been shown that CTPsyn is incorporated into a novel cytoplasmic structure which has been termed the cytoophidium. Here, we report that Myc regulates cytoophidium formation during Drosophila oogenesis. We have found that Myc protein levels correlate with cytoophidium abundance in follicle epithelia. Reducing Myc levels results in cytoophidium loss and small nuclear size in follicle cells, while overexpression of Myc increases the length of cytoophidia and the nuclear size of follicle cells. Ectopic expression of Myc induces cytoophidium formation in late stage follicle cells. Furthermore, knock-down of CTPsyn is sufficient to suppress the overgrowth phenotype induced by Myc overexpression, suggesting CTPsyn acts downstream of Myc and is required for Myc-mediated cell size control. Taken together, our data suggest a functional link between Myc, a renowned oncogene, and the essential nucleotide biosynthetic enzyme CTPsyn.

Quantification of the Nutrient Dependence of Factors Controlling Start in Budding Yeast by 2-Photon Scanning Number and Brightness

Cell size homeostasis is a poorly understood universal property of all organisms which results from the coordination between cell growth and division. In budding yeast, cell size is modulated by nutrients. On poor nutrients cells commit to division (pass Start) at a smaller size. While many of the protein factors underlying the growth and division networks have been identified, there exists little quantitative data defining how much, when and where these proteins are present and interact at a single cell level. Even the key Start trigger, the G1 cyclin Cln3, which activates the Cdc28 kinase to phosphorylate and inactivate the Start transcription repressor Whi5, is notoriously difficult to detect and has never been observed on the single cell level. We used two-photon laser scanning Number and Brightness experiments to localize and quantify Cln3-GFPmut3 and Whi5-GFPmut3 fusions expressed from their natural loci at the single cell level in live budding yeast cells grown on rich (glucose) and poor (ethanol) nutrients. We find homogeneous Cln3 concentrations (∼3-4 nM) with no clear localization pattern or dependence on cell size or nutrients. This finding is in contrast to models suggesting changes in Cln3 concentration as a key factor in cell size control. In G1 phase cells we find Whi5 at ∼150 nM in the nucleus and ∼10-30-fold lower in the cytoplasm. In both G1 and post-Start cells we find 3-fold higher concentrations of Whi5 in the cytoplasm on ethanol, 15 +/- 3 nM, compared to glucose, 5-6 +/- 1 nM. Higher cytosolic Whi5 on ethanol is consistent with increased inactivation and export of this repressor to trigger earlier initiation of Start.

Single-Cell Analysis of Growth in Budding Yeast and Bacteria Reveals a Common Size Regulation Strategy

To maintain a constant cell size, dividing cells have to coordinate cell-cycle events with cell growth. This coordination has long been supposed to rely on the existence of size thresholds determining cell-cycle progression [1]. In budding yeast, size is controlled at the G1/S transition [2]. In agreement with this hypothesis, the size at birth influences the time spent in G1: smaller cells have a longer G1 period [3]. Nevertheless, even though cells born smaller have a longer G1, the compensation is imperfect and they still bud at smaller cell sizes. In bacteria, several recent studies have shown that the incremental model of size control, in which size is controlled by addition of a constant volume (in contrast to a size threshold), is able to quantitatively explain the experimental data on four different bacterial species [4-7]. Here, we report on experimental results for the budding yeast Saccharomyces cerevisiae, finding, surprisingly, that cell size control in this organism is very well described by the incremental model, suggesting a common strategy for cell size control with bacteria. Additionally, we argue that for S. cerevisiae the "volume increment" is not added from birth to division, but rather between two budding events.

Nutritional Control of Cell Size by the Greatwall-Endosulfine-PP2A·B55 Pathway

Proliferating cells adjust their cell size depending on the nutritional environment. Cells are large in rich media and small in poor media. This physiological response has been demonstrated in both unicellular and multicellular organisms. Here we show that the greatwall-endosulfine (Ppk18-Igo1 in fission yeast) pathway couples the nutritional environment to the cell-cycle machinery by regulating the activity of PP2A·B55. In the presence of nutrients, greatwall (Ppk18) protein kinase is inhibited by TORC1 and PP2A·B55 is active. High levels of PP2A·B55 prevent the activation of mitotic Cdk1·Cyclin B, and cells increase in size in G2 before they undergo mitosis. When nutrients are limiting, TORC1 activity falls off, and the activation of greatwall (Ppk18) leads to the phosphorylation of endosulfine (Igo1) and inhibition of PP2A·B55, which in turn allows full activation of Cdk1·CyclinB and entry into mitosis with a smaller cell size. Given the conservation of this pathway, it is reasonable to assume that this mechanism operates in higher eukaryotes, as well.

Targeting Insulin Receptor with a Novel Internalizing Aptamer

Nucleic acid-based aptamers are emerging as therapeutic antagonists of disease-associated proteins such as receptor tyrosine kinases. They are selected by an in vitro combinatorial chemistry approach, named Systematic Evolution of Ligands by Exponential enrichment (SELEX), and thanks to their small size and unique chemical characteristics, they possess several advantages over antibodies as diagnostics and therapeutics. In addition, aptamers that rapidly internalize into target cells hold as well great potential for their in vivo use as delivery tools of secondary therapeutic agents. Here, we describe a nuclease resistant RNA aptamer, named GL56, which specifically recognizes the insulin receptor (IR). Isolated by a cell-based SELEX method that allows enrichment for internalizing aptamers, GL56 rapidly internalizes into target cells and is able to discriminate IR from the highly homologous insulin-like growth factor receptor 1. Notably, when applied to IR expressing cancer cells, the aptamer inhibits IR dependent signaling. Given the growing interest in the insulin receptor as target for cancer treatment, GL56 reveals a novel molecule with great translational potential as inhibitor and delivery tool for IR-dependent cancers.

Control of Skeletal Muscle Cell Growth and Size Through Adhesion GPCRs.

Skeletal muscle homeostasis is regulated by a constant influx of chemicals and exposure to mechanical stimuli. A number of key signaling pathways that translate these stimuli into changes in muscle physiology have been established. The GPCR family known as adhesion GPCRs (aGPCRs) has largely elusive roles in skeletal muscle biology; however, their unique capacity to activate adhesion and G protein signaling pathways makes them an attractive point of investigation. The skeletal muscle myofiber contains a highly organized cytoarchitecture to ensure contractile function. This requires intricate interactions with components of the extracellular matrix (ECM) surrounding each fiber. aGPCRs possess extended N-termini known to interact with ECM proteins and complexes suggesting a compatible role in skeletal muscle biology. Furthermore, recent work demonstrated the involvement of certain aGPCRs in whole muscle hypertrophy and differentiation of muscle progenitor cells. Signaling pathways downstream of aGPCRs are still incompletely understood; however, initial findings show involvement of the Gα12/13 subunit signaling to the pro-anabolic Akt/mTOR pathway. Together, this chapter will review the emerging role of aGPCRs in skeletal muscle biology and putative mechanism(s) employed to regulate skeletal muscle growth.

Actin-dependent vacuolar occupancy of the cell determines auxin-induced growth repression

The cytoskeleton is an early attribute of cellular life, and its main components are composed of conserved proteins. The actin cytoskeleton has a direct impact on the control of cell size in animal cells, but its mechanistic contribution to cellular growth in plants remains largely elusive. Here, we reveal a role of actin in regulating cell size in plants. The actin cytoskeleton shows proximity to vacuoles, and the phytohormone auxin not only controls the organization of actin filaments but also impacts vacuolar morphogenesis in an actin-dependent manner. Pharmacological and genetic interference with the actin-myosin system abolishes the effect of auxin on vacuoles and thus disrupts its negative influence on cellular growth. SEM-based 3D nanometer-resolution imaging of the vacuoles revealed that auxin controls the constriction and luminal size of the vacuole. We show that this actin-dependent mechanism controls the relative vacuolar occupancy of the cell, thus suggesting an unanticipated mechanism for cytosol homeostasis during cellular growth.

Sizing up to divide: mitotic cell-size control in fission yeast.

Schizosaccharomyces pombe is a good model to study cell-size control. These cells integrate size information into cell cycle controls at both the G1/S and G2/M transitions, although the primary control operates at the entry into mitosis. At G2/M there is both a size threshold, demonstrated by the fact that cells divide when they reach 14 μm in length, and also correction around this threshold, evident from the narrow distribution of sizes within a population. This latter property is referred to as size homeostasis. It has been argued that a population of cells accumulating mass in a linear fashion will have size homeostasis in the absence of size control, if cycle time is controlled by a fixed timer. Because fission yeast cells do not grow in a simple linear fashion, they require a size-sensing mechanism. However, current models do not fully describe all aspects of this control, especially the coordination of cell size with ploidy.

The Biosynthetic Basis of Cell Size Control.

Cell size is an important physiological trait that sets the scale of all biosynthetic processes. Although physiological studies have revealed that cells actively regulate their size, the molecular mechanisms underlying this regulation have remained unclear. Here we review recent progress in identifying the molecular mechanisms of cell size control. We focus on budding yeast, where cell growth dilutes a cell cycle inhibitor to couple growth and division. We discuss a new model for size control based on the titration of activator and inhibitor molecules whose synthesis rates are differentially dependent on cell size.

Active Control of Cell Size Generates Spatial Detail during Plant Organogenesis

SummaryHow cells regulate their dimensions is a long-standing question [1, 2]. In fission and budding yeast, cell-cycle progression depends on cell size, although it is still unclear how size is assessed [3, 4, 5]. In animals, it has been suggested that cell size is modulated primarily by the balance of external signals controlling growth and the cell cycle [1], although there is evidence of cell-autonomous control in cell cultures [6, 7, 8, 9]. Regardless of whether regulation is external or cell autonomous, the role of cell-size control in the development of multicellular organisms remains unclear. Plants are a convenient system to study this question: the shoot meristem, which continuously provides new cells to form new organs, maintains a population of actively dividing and characteristically small cells for extended periods [10]. Here, we used live imaging and quantitative, 4D image analysis to measure the sources of cell-size variability in the meristem and then used these measurements in computer simulations to show that the uniform cell sizes seen in the meristem likely require coordinated control of cell growth and cell cycle in individual cells. A genetically induced transient increase in cell size was quickly corrected by more frequent cell division, showing that the cell cycle was adjusted to maintain cell-size homeostasis. Genetically altered cell sizes had little effect on tissue growth but perturbed the establishment of organ boundaries and the emergence of organ primordia. We conclude that meristem cells actively control their sizes to achieve the resolution required to pattern small-scale structures.

Bacterial growth, detachment and cell size control on polyethylene terephthalate surfaces

In medicine and food industry, bacterial colonisation on surfaces is a common cause of infections and severe illnesses. However, the detailed quantitative information about the dynamics and the mechanisms involved in bacterial proliferation on solid substrates is still lacking. In this study we investigated the adhesion and detachment, the individual growth and colonisation, and the cell size control of Escherichia coli (E. coli) MG1655 on polyethylene terephthalate (PET) surfaces. The results show that the bacterial growth curve on PET exhibits the distinct lag and log phases, but the generation time is more than twice longer than in bulk medium. Single cells in the lag phase are more likely to detach than clustered ones in the log phase; clustered bacteria in micro-colonies have stronger adhesive bonds with surfaces and their neighbours with the progressing colonisation. We show that the cell size is under the density-dependent pathway control: when the adherent cells are at low density, the culture medium is responsible for coordinating cell division and cell size; when the clustered cells are at high population density, we demonstrate that the effect of quorum sensing causes the cell size decrease as the cell density on surfaces increases.