Cell Size Control Research Articles

Cell cycle-linked vacuolar pH dynamics regulate amino acid homeostasis and cell growth

Amino acid homeostasis is critical for many cellular processes. It is well established that amino acids are compartmentalized using pH gradients generated between organelles and the cytoplasm; however, the dynamics of this partitioning has not been explored. Here we develop a highly sensitive pH reporter and find that the major amino acid storage compartment in Saccharomyces cerevisiae, the lysosome-like vacuole, alkalinizes before cell division and re-acidifies as cells divide. The vacuolar pH dynamics require the uptake of extracellular amino acids and activity of TORC1, the v-ATPase and the cycling of the vacuolar specific lipid phosphatidylinositol 3,5-bisphosphate, which is regulated by the cyclin-dependent kinase Pho85 (CDK5 in mammals). Vacuolar pH regulation enables amino acid sequestration and mobilization from the organelle, which is important for mitochondrial function, ribosome homeostasis and cell size control. Collectively, our data provide a new paradigm for the use of dynamic pH-dependent amino acid compartmentalization during cell growth/division.

Hyperactive Ras disrupts cell size control and a key step in cell cycle entry in budding yeast.

Severe defects in cell size are a nearly universal feature of cancer cells. However, the underlying causes are unknown. A previous study suggested that a hyperactive mutant of yeast Ras (ras2G19V) that is analogous to the human Ras oncogene causes cell size defects, which could provide clues to how oncogenes influence cell size. However, the mechanisms by which ras2G19V influences cell size are unknown. Here, we found that ras2G19V inhibits a critical step in cell cycle entry, in which an early G1 phase cyclin induces transcription of late G1 phase cyclins. Thus, ras2G19V drives overexpression of the early G1 phase cyclin Cln3, yet Cln3 fails to induce normal transcription of late G1 phase cyclins, leading to delayed cell cycle entry and increased cell size. ras2G19V influences transcription of late G1 phase cyclins via a poorly understood step in which Cln3 inactivates the Whi5 transcriptional repressor. Previous studies found that yeast Ras relays signals via protein kinase A (PKA); however, ras2G19V appears to influence late G1 phase cyclin expression via novel PKA-independent signaling mechanisms. Together, the data define new mechanisms by which hyperactive Ras influences cell cycle entry and cell size in yeast. Hyperactive Ras also influences expression of G1 phase cyclins in mammalian cells, but the mechanisms remain unclear. Further analysis of Ras signaling in yeast could lead to discovery of new mechanisms by which Ras family members control expression of G1 phase cyclins.

Implications of differential size-scaling of cell-cycle regulators on cell size homeostasis.

Accurate timing of division and size homeostasis is crucial for cells. A potential mechanism for cells to decide the timing of division is the differential scaling of regulatory protein copy numbers with cell size. However, it remains unclear whether such a mechanism can lead to robust growth and division, and how the scaling behaviors of regulatory proteins influence the cell size distribution. Here we study a mathematical model combining gene expression and cell growth, in which the cell-cycle activators scale superlinearly with cell size while the inhibitors scale sublinearly. The cell divides once the ratio of their concentrations reaches a threshold value. We find that the cell can robustly grow and divide within a finite range of the threshold value with the cell size proportional to the ploidy. In a stochastic version of the model, the cell size at division is uncorrelated with that at birth. Also, the more differential the cell-size scaling of the cell-cycle regulators is, the narrower the cell-size distribution is. Intriguingly, our model with multiple regulators rationalizes the observation that after the deletion of a single regulator, the coefficient of variation of cell size remains roughly the same though the average cell size changes significantly. Our work reveals that the differential scaling of cell-cycle regulators provides a robust mechanism of cell size control.

The fission yeast cell size control system integrates pathways measuring cell surface area, volume, and time

Eukaryotic cells tightly control their size, but the relevant aspect of size is unknown in most cases. Fission yeast divide at a threshold cell surface area (SA) due, in part, to the protein kinase Cdr2. We find that fission yeast cells only divide by SA under a size threshold. Mutants that divide at a larger size shift to volume-based divisions. Diploid cells divide at a larger size than haploid cells do, but they maintain SA-based divisions, and this indicates that the size threshold for changing from surface-area-based to volume-based control is set by ploidy. Within this size control system, we found that the mitotic activator Cdc25 accumulates like a volume-based sizer molecule, whereas the mitotic cyclin Cdc13 accumulates in the nucleus as a timer. We propose an integrated model for cell size control based on multiple signaling pathways that report on distinct aspects of cell size and growth, including cell SA (Cdr2), cell volume (Cdc25), and time (Cdc13). Combined modeling and experiments show how this system can generate both sizer- and adder-like properties.

PI3P-dependent regulation of cell size and autophagy by phosphatidylinositol 5-phosphate 4-kinase.

Phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol 5-phosphate (PI5P) are low-abundance phosphoinositides crucial for key cellular events such as endosomal trafficking and autophagy. Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) is an enzyme that regulates PI5P in vivo but can act on both PI5P and PI3P in vitro. In this study, we report a role for PIP4K in regulating PI3P levels in Drosophila Loss-of-function mutants of the only Drosophila PIP4K gene show reduced cell size in salivary glands. PI3P levels are elevated in dPIP4K 29 and reverting PI3P levels back towards WT, without changes in PI5P levels, can rescue the reduced cell size. dPIP4K 29 mutants also show up-regulation in autophagy and the reduced cell size can be reverted by depleting Atg8a that is required for autophagy. Lastly, increasing PI3P levels in WT can phenocopy the reduction in cell size and associated autophagy up-regulation seen in dPIP4K 29 Thus, our study reports a role for a PIP4K-regulated PI3P pool in the control of autophagy and cell size.

Endoreplication controls cell size via mechanochemical signaling.

During hypocotyl development, an asymmetric auxin gradient causes differential cell elongation, leading to tissue bending and apical hook formation. Recently, Ma et al. identified a molecular pathway that links auxin with endoreplication and cell size through cell wall integrity sensing, cell wall remodeling, and regulation of cell wall stiffness.

A moonlighting role for LysM peptidoglycan binding domains underpins Enterococcus faecalis daughter cell separation

Control of cell size and morphology is of paramount importance for bacterial fitness. In the opportunistic pathogen Enterococcus faecalis, the formation of diplococci and short cell chains facilitates innate immune evasion and dissemination in the host. Minimisation of cell chain size relies on the activity of a peptidoglycan hydrolase called AtlA, dedicated to septum cleavage. To prevent autolysis, AtlA activity is tightly controlled, both temporally and spatially. Here, we show that the restricted localization of AtlA at the septum occurs via an unexpected mechanism. We demonstrate that the C-terminal LysM domain that allows the enzyme to bind peptidoglycan is essential to target this enzyme to the septum inside the cell before its translocation across the membrane. We identify a membrane-bound cytoplasmic protein partner (called AdmA) involved in the recruitment of AtlA via its LysM domains. This work reveals a moonlighting role for LysM domains, and a mechanism evolved to restrict the subcellular localization of a potentially lethal autolysin to its site of action.

Microtubule Regulation in Plants: From Morphological Development to Stress Adaptation.

Microtubules (MTs) are essential elements of the eukaryotic cytoskeleton and are critical for various cell functions. During cell division, plant MTs form highly ordered structures, and cortical MTs guide the cell wall cellulose patterns and thus control cell size and shape. Both are important for morphological development and for adjusting plant growth and plasticity under environmental challenges for stress adaptation. Various MT regulators control the dynamics and organization of MTs in diverse cellular processes and response to developmental and environmental cues. This article summarizes the recent progress in plant MT studies from morphological development to stress responses, discusses the latest techniques applied, and encourages more research into plant MT regulation.

A c-di-GMP binding effector controls cell size in a cyanobacterium

Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule. It is also a critical player in the regulation of cell size and cell behaviors such as cell aggregation and phototaxis in cyanobacteria, which constitute an important group of prokaryotes for their roles in the ecology and evolution of the Earth. However, c-di-GMP receptors have never been revealed in cyanobacteria. Here, we report the identification of a c-di-GMP receptor, CdgR, from the filamentous cyanobacterium Anabaena PCC 7120. Crystal structural analysis and genetic studies demonstrate that CdgR binds c-di-GMP at the dimer interface and this binding is required for the control of cell size in a c-di-GMP-dependent manner. Different functions of CdgR, in ligand binding and signal transmission, could be separated genetically, allowing us to dissect its molecular signaling functions. The presence of the apo-form of CdgR triggers cell size reduction, consistent with the similar effects observed with a decrease of c-di-GMP levels in cells. Furthermore, we found that CdgR exerts its function by interacting with a global transcription factor DevH, and this interaction was inhibited by c-di-GMP. The lethal effect triggered by conditional depletion of DevH or by the production of several point-mutant proteins of CdgR in cells indicates that this signaling pathway plays critical functions in Anabaena. Our studies revealed a mechanism of c-di-GMP signaling in the control of cell size, an important and complex trait for bacteria. CdgR is highly conserved in cyanobacteria, which will greatly expand our understanding of the roles of c-di-GMP signaling in these organisms.

Stochastic threshold in cell size control

Classic models of cell size control consider that cells divide when reaching a threshold, e.g., size, age, or size extension. The molecular basis of the threshold involves multiple layers of regulation as well as gene noise. In this paper, we study the cell cycle as a first-passage problem with a stochastic threshold and discover that such stochasticity affects the intergeneration statistics, which bewilders the criteria to distinguish the types of size control models. The analytic results show that the autocorrelation in the threshold can drive a sizer model to the adderlike and even timerlike intergeneration statistics, which is supported by simulations. Following the picture that the autocorrelation in the threshold can propagate to the intergeneration statistics, we further show that the adder model can be driven to the timerlike one by a positively autocorrelated threshold, and even to the sizerlike one when the threshold is negatively autocorrelated. This work highlights the importance of examining gene noise in size control.

A Swi4 positive feedback loop that regulates timing of start revealed by two photon scanning number and brightness microscopy.

The irreversible commitment to division in Saccharomyces cerevisiae takes place at the G1/S cell cycle transition (Start). While many Start regulators are known, the mechanisms controlling its timing, and hence cell size, remain to be determined. Start is activated by the SBF and MBF transcriptional factor complexes which bind to target sites in G1/S promoters and activate the transcription of ∼200 genes in the G1/S regulon. SBF and MBF contain the same activator subunit, Swi6 but a distinct DNA binding subunit, Swi4 or Mbp1, respectively. SBF is repressed during G1 phase by Whi5. Cyclin dependent kinases produced as part of the G1/S regulon in a positive feedback loop phosphorylate Whi5, leading to its dissociation from SBF and export from the nucleus. We previously reported that G1/S transcription factors, particularly Swi4, are sub-saturating with respect to their promoters in newborn daughter cells. Their increasing copy numbers throughout G1 phase gradually titrate their target promoters, representing a critical determinant of the timing of Start. The positive phosphorylation feedback loop ensures a sharp Start transition once sufficient SBF is present at G1/S promoters. A possible positive feedback mechanism of Swi4 transcription has been suggested and could contribute to the strong Swi4 copy number increase in G1 phase. However, the existence of this feedback loop had never been established. In this study, we demonstrate the existence of this positive feedback loop using two photon scanning number and brightness fluorescence microscopy on yeast strains expressing Swi4-GFP from its natural locus and deleted for key features of the putative feedback loop or ectopically expressing another copy of Swi4. These results are interpreted within the framework of a model for cell size control.

Control of Cell Size by c-di-GMP Requires a Two-Component Signaling System in the Cyanobacterium Anabaena sp. Strain PCC 7120.

Each bacterial species possesses a specific cell size and morphology, which constitute important and recognizable physical traits. How bacteria maintain their particular cell size and morphology remains an essential question in microbiology. Cyanobacteria are oxygen-evolving photosynthetic prokaryotes. Although monophyletic, these organisms are highly diverse in their cell morphology and cell size. How these physical traits of cyanobacteria are controlled is poorly understood. Here, we report the identification of a two-component signaling system, composed of a histidine kinase CdgK and a response regulator CdgS, involved in cell size regulation in the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Inactivation of cdgK or cdgS led to reduction of cell length and width with little effect on cell growth capacity. CdgS has a GGDEF domain responsible for the synthesis of the second messenger c-di-GMP. Based on genetic and biochemical studies, we proposed a signaling pathway initiated by CdgK, leading to the phosphorylation of CdgS, and thereby an enhanced enzymatic activity for c-di-GMP synthesis of the latter. The GGDEF domain of CdgS was essential in cell size control, and the reduction of cell size observed in various mutants could be rescued by the expression of a c-di-GMP synthetase from E. coli. These results provided evidence that a minimal threshold of c-di-GMP level was required for maintaining cell size in Anabaena. IMPORTANCE Cyanobacteria are considered the first organisms to produce oxygen on Earth, and their activities shaped the evolution of our ecosystems. Cell size is an important trait fixed early in evolution, with the diversification of micro- and macrocyanobacterial species during the Great Oxidation Event. However, the genetic basis underlying cell size control in cyanobacteria was not understood. Our studies demonstrated that the CdgK-CdgS signaling pathway participates in the control of cell size, and their absence did not affect cell growth. CdgK has multiple domains susceptible to signal input, which are necessary for cell size regulation. This observation suggests that cell size in Anabaena could respond to environmental signals. These studies paved the way for genetic dissection of cell size regulation in cyanobacteria.

Expression of oncogenic HRAS G12V causes defects in control of cell size in NIH 3T3 cells.

Severe defects in control of cell size are closely associated with cancer. However, the mechanisms that drive cell size defects in cancer remain unknown and it is unclear whether they are a direct consequence of signals from primary oncogenic drivers or a secondary consequence of mutations that accumulate during evolution of cancer cells. Here, we report that expression of oncogenic HRAS G12V is sufficient to cause cell size defects in NIH 3T3 cells, which suggests that the cell size defects of cancer cells are a direct consequence of primary oncogenic drivers.

MYB3R-SCL28-SMR module with a role in cell size control negatively regulates G2 progression in Arabidopsis

ABSTRACT Cell size control is one of the prerequisites for plant growth and development. Recently, a GRAS family transcription factor, SCARECROW-LIKE28 (SCL28), was identified as a critical regulator for both mitotic and postmitotic cell-size control. Here, we show that SCL28 is specifically expressed in proliferating cells and exerts its function to delay G2 progression during mitotic cell cycle in Arabidopsis thaliana. Overexpression of SCL28 provokes a significant enlargement of cells in various organs and tissues, such as leaves, flowers and seeds, to different extents depending on the type of cells. The increased cell size is most likely due to a delayed G2 progression and accelerated onset of endoreplication, an atypical cell cycle repeating DNA replication without cytokinesis or mitosis. Unlike DWARF AND LOW-TILLERING, a rice ortholog of SCL28, SCL28 may not have a role in brassinosteroid (BR) signaling because sensitivity against brassinazole, a BR biosynthesis inhibitor, was not dramatically altered in scl28 mutant and SCL28-overexpressing plants. Collectively, our findings strengthen a recently proposed model of cell size control by SCL28 and suggest the presence of diversified evolutionary mechanisms for the regulation and action of SCL28.

Endoreplication mediates cell size control via mechanochemical signaling from cell wall

Endoreplication is an evolutionarily conserved mechanism for increasing nuclear DNA content (ploidy). Ploidy frequently scales with final cell and organ size, suggesting a key role for endoreplication in these processes. However, exceptions exist, and, consequently, the endoreplication-size nexus remains enigmatic. Here, we show that prolonged tissue folding at the apical hook in Arabidopsis requires endoreplication asymmetry under the control of an auxin gradient. We identify a molecular pathway linking endoreplication levels to cell size through cell wall remodeling and stiffness modulation. We find that endoreplication is not only permissive for growth: Endoreplication reduction enhances wall stiffening, actively reducing cell size. The cell wall integrity kinase THESEUS plays a key role in this feedback loop. Our data thus explain the nonlinearity between ploidy levels and size while also providing a molecular mechanism linking mechanochemical signaling with endoreplication-mediated dynamic control of cell growth.

Coupling gene expression dynamics to cell size dynamics and cell cycle events: Exact and approximate solutions of the extended telegraph model

The standard model describing the fluctuations of mRNA numbers in single cells is the telegraph model which includes synthesis and degradation of mRNA, and switching of the gene between active and inactive states. While commonly used, this model does not describe how fluctuations are influenced by the cell cycle phase, cellular growth and division, and other crucial aspects of cellular biology. Here, we derive the analytical time-dependent solution of an extended telegraph model that explicitly considers the doubling of gene copy numbers upon DNA replication, dependence of the mRNA synthesis rate on cellular volume, gene dosage compensation, partitioning of molecules during cell division, cell-cycle duration variability, and cell-size control strategies. Based on the time-dependent solution, we obtain the analytical distributions of transcript numbers for lineage and population measurements in steady-state growth and also find a linear relation between the Fano factor of mRNA fluctuations and cell volume fluctuations. We show that generally the lineage and population distributions in steady-state growth cannot be accurately approximated by the steady-state solution of extrinsic noise models, i.e. a telegraph model with parameters drawn from probability distributions. This is because the mRNA lifetime is often not small enough compared to the cell cycle duration to erase the memoryof division and replication. Accurate approximations are possible when this memory is weak, e.g. for genes with bursty expression and for which there is sufficient gene dosage compensation when replication occurs.

Novel determinants of cell size homeostasis in the opportunistic yeast Candida albicans.

The basis for commitment to cell division in late G1 phase, called Start in yeast, is a critical but still poorly understood aspect of eukaryotic cell proliferation.Most dividing cells accumulate mass and grow to a critical cell size before traversing the cell cycle. This size threshold couples cell growth to division and thereby establishes long-term size homeostasis.At present, mechanisms involved in cell size homeostasis in fungal pathogens are not well described. Our previous survey of the size phenome in Candida albicans focused on 279 unique mutants enriched mainly in kinases and transcription factors (Sellam et al. PLoS Genet 15:e1008052, 2019). To uncover novel size regulators in C. albicans and highlight potential innovation within cell size control in pathogenic fungi, we expanded our genetic survey of cell size to include 1301 strains from the GRACE (Gene Replacement and Conditional Expression) collection. The current investigation uncovered both known and novel biological processes required for cell size homeostasis in C. albicans. We also confirmed the plasticity of the size control network as few C. albicans size genes overlapped with those of the budding yeast Saccharomyces cerevisiae. Many new size genes of C. albicans were associated with biological processes that were not previously linked to cell size control and offer an opportunity for future investigation. Additional work is needed to understand if mitochondrial activity is a critical element of the metric that dictates cell size in C. albicans and whether modulation of the onset of actomyosin ring constriction is an additional size checkpoint.

Make Your Own Sprites

Pixel art is a unique art style with the appearance of low resolution images. In this paper, we propose a data-driven pixelization method that can produce sharp and crisp cell effects with controllable cell sizes. Our approach overcomes the limitation of existing learning-based methods in cell size control by introducing a reference pixel art to explicitly regularize the cell structure. In particular, the cell structure features of the reference pixel art are used as an auxiliary input for the pixelization process, and for measuring the style similarity between the generated result and the reference pixel art. Furthermore, we disentangle the pixelization process into specific cell-aware and aliasing-aware stages, mitigating the ambiguities in joint learning of cell size, aliasing effect, and color assignment. To train our model, we construct a dedicated pixel art dataset and augment it with different cell sizes and different degrees of anti-aliasing effects. Extensive experiments demonstrate its superior performance over state-of-the-arts in terms of cell sharpness and perceptual expressiveness. We also show promising results of video game pixelization for the first time. Code and dataset are available at https://github.com/WuZongWei6/Pixelization.

Phytoplankton cell size control can be affected by photosynthetic light energy utilization.

Phytoplankton cell size is well known as an essential functional trait, but its control factors are still unclear. Considering light provides the necessary energy for phytoplankton survival, we hypothesized that photosynthetic light energy utilization could influence phytoplankton cell size control. Several scenarios were conducted to understand the relationship between Fv /Fm and cell size for phytoplankton interspecies, and metatranscriptome in the field and transcriptome in the laboratory were used to understand relevant molecular mechanisms. The results indicated that there was a universal significant positive relationship between Fv /Fm and cell volume in general. The molecular evidence demonstrated that light utilization by phytoplankton regulates their cell size by harmonizing the generation and allocation of chemical energy and fixed carbon in the cell. Phytoplankton cell size would cease to enlarge once the increased light energy conversion and subsequent fixed carbon could no longer satisfy the increasing demand of size enlargement. This unity of energy and matter in shaping phytoplankton size results in cell size being an important functional trait. This study is the first to discover the above molecular mechanisms and is helpful to deepen the understanding on the cell size control of phytoplankton.

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?