Cell size induced bias of current density in hypertrophic cardiomyocytes.

Alterations in ion channel expression and function known as "electrical remodeling" contribute to the development of hypertrophy and to the emergence of arrhythmias and sudden cardiac death. However, comparing current density values - an electrophysiological parameter commonly utilized to assess ion channel function - between normal and hypertrophied cells may be flawed when current amplitude does not scale with cell size. Even more, common routines to study equally sized cells or to discard measurements when large currents do not allow proper voltage-clamp control may introduce a selection bias and thereby confound direct comparison. To test a possible dependence of current density on cell size and shape, we employed whole-cell patch-clamp recording of voltage-gated sodium and calcium currents in Langendorff-isolated ventricular cardiomyocytes and Purkinje myocytes, as well as in cardiomyocytes derived from trans-aortic constriction operated mice. Here, we describe a distinct inverse relationship between voltage-gated sodium and calcium current densities and cell capacitance both in normal and hypertrophied cells. This inverse relationship was well fit by an exponential function and may be due to physiological adaptations that do not scale proportionally with cell size or may be explained by a selection bias. Our study emphasizes the need to consider cell size bias when comparing current densities in cardiomyocytes of different sizes, particularly in hypertrophic cells. Conventional comparisons based solely on mean current density may be inadequate for groups with unequal cell size or non-proportional current amplitude and cell size scaling.

NMY-2, TOE-2 and PIG-1 regulate Caenorhabditis elegans asymmetric cell divisions.

Asymmetric cell division is an important mechanism that generates cellular diversity during development. Not only do asymmetric cell divisions produce daughter cells of different fates, but many can also produce daughters of different sizes, which we refer to as Daughter Cell Size Asymmetry (DCSA). In Caenorhabditis elegans, apoptotic cells are frequently produced by asymmetric divisions that exhibit DCSA, where the smaller daughter dies. We focus here on the divisions of the Q.a and Q.p neuroblasts, which produce larger surviving cells and smaller apoptotic cells and divide with opposite polarity using both distinct and overlapping mechanisms. Several proteins regulate DCSA in these divisions. Previous studies showed that the PIG-1/MELK and TOE-2 proteins regulate DCSA in both the Q.a and Q.p divisions, and the non-muscle myosin NMY-2 regulates DCSA in the Q.a division but not the Q.p division. In this study, we examined endogenously tagged NMY-2, TOE-2, and PIG-1 reporters and characterized their distribution at the cortex during the Q.a and Q.p divisions. In both divisions, TOE-2 localized toward the side of the dividing cell that produced the smaller daughter, whereas PIG-1 localized toward the side that produced the larger daughter. As previously reported, NMY-2 localized to the side of Q.a that produced the smaller daughter and did not localize asymmetrically in Q.p. We used temperature-sensitive nmy-2 mutants to determine the role of nmy-2 in these divisions and were surprised to find that these mutants only displayed DCSA defects in the Q.p division. We generated double mutant combinations between the nmy-2 mutations and mutations in toe-2 and pig-1. Because previous studies indicate that DCSA defects result in the transformation of cells fated to die into their sister cells, the finding that the nmy-2 mutations did not significantly alter the Q.a and Q.p DCSA defects of toe-2 and pig-1 mutants but did alter the number of daughter cells produced by Q.a and Q.p suggests that nmy-2 plays a role in specifying the fates of the Q.a and Q.p that is independent of its role in DCSA.

Sepal shape variability is robust to cell size heterogeneity in Arabidopsis.

How organisms produce organs with robust shapes and sizes is still an open question. In recent years, the Arabidopsis sepal has been used as a model system to study this question because of its highly reproducible shape and size. One interesting aspect of the sepal is that its epidermis contains cells of very different sizes. Previous reports have qualitatively shown that sepals with more or less giant cells exhibit comparable final size and shape. Here, we investigate this question using quantitative approaches. We find that a mixed population of cell size modestly contribute to the normal width of the sepal but is not essential for its shape robustness. Furthermore, in a mutant with increased cell and organ growth variability, the change in final sepal shape caused by giant cells is exaggerated but the shape robustness is not affected. This formally demonstrates that sepal shape variability is robust to cell size heterogeneity.

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.

Mechanisms of cell size regulation in slow-growing Escherichia coli cells: discriminating models beyond the adder.

Under ideal conditions, Escherichia coli cells divide after adding a fixed cell size, a strategy known as the adder. This concept applies to various microbes and is often explained as the division that occurs after a certain number of stages, associated with the accumulation of precursor proteins at a rate proportional to cell size. However, under poor media conditions, E. coli cells exhibit a different size regulation. They are smaller and follow a sizer-like division strategy where the added size is inversely proportional to the size at birth. We explore three potential causes for this deviation: degradation of the precursor protein and two models where the propensity for accumulation depends on the cell size: a nonlinear accumulation rate, and accumulation starting at a threshold size termed the commitment size. These models fit the mean trends but predict different distributions given the birth size. To quantify the precision of the models to explain the data, we used the Akaike information criterion and compared them to open datasets of slow-growing E. coli cells in different media. We found that none of the models alone can consistently explain the data. However, the degradation model better explains the division strategy when cells are larger, whereas size-related models (power-law and commitment size) account for smaller cells. Our methodology proposes a data-based method in which different mechanisms can be tested systematically.

In Vitro Titan Cell Generation in Cryptococcus neoformans and Automated Cell Size Measurements.

A special feature of the human fungal pathogen Cryptococcus neoformans is its morphological changes triggered by the interaction with the host. During infection, a specific increase in cell size is observed, particularly in lung tissue, from a typical cell size of 5-7 µm cells to cells larger than 10 µm, dubbed titan cells (TCs). However, the study of this specific cell subpopulation was, until now, only possible via recovery of TCs from lungs of mice during experimental infections where stable and reproducible generation of TCs occurs.The protocol described here generates TCs using in vitro conditions and measures cell size using a rapid, automated method. TC generation in vitro is robust and reproducible, generating yeast cells harboring the same characteristics of TCs generated in vivo.

Development of an Undergraduate Cell Biology Laboratory to Assess Pigmentation and Cell Size in a Zebrafish Model of Uveal Melanoma.

This study outlines a 2-week laboratory module for an authentic cell biology undergraduate research experience that uses zebrafish (Danio rerio), a popular model organism for research. Previous research has indicated that course-based undergraduate research experiences such as this one increase student confidence, active learning, and retention. During this research experience, students investigate variations in pigmentation in the caudal fins of wild type (WT) and transgenic fish [Tg(mitfa:GNAQQ209L)]. The transgenic fish express a hyperactive Gα protein, GNAQQ209L, under the melanocyte-specific mitfa promoter, offering insights into uveal melanoma, a common eye cancer. Students specifically analyze the black pigmented cells, melanophores, within the caudal fin. We determined that the transgenic zebrafish have increased pigmentation in their caudal fins, but smaller melanophores. These results suggest there are more melanophores in the Tg(mitfa:GNAQQ209L) fish compared to the WT. Future undergraduate research could investigate these cellular differences. This research experience imparts microscopy and image analysis skills and instills the ability to grapple with large datasets, statistical tests, and data interpretation in alignment with biology education principles. Post-laboratory surveys reveal students attain confidence in the above skills and in handling animals, along with a deeper appreciation for model organism research and its relevance to cancer cell biology.

Anisotropic power-law viscoelasticity of living cells is dominated by cytoskeletal network structure.

During physiological and pathological processes, cells experience significant morphological alterations with the re-arrangement of cytoskeletal filaments, resulting in anisotropic viscoelasticity. Here, a structure-based cell model is proposed to study the anisotropic viscoelastic mechanical behaviors of living cells. We investigate how cell shape affects its creep responses in longitudinal and perpendicular directions. It is shown that cells exhibit power-law rheological behavior in both longitudinal and perpendicular directions under step stress, with a more solid-like behavior along the longitudinal direction. We reveal that the cell volume and cytoskeletal filament orientation, which have been neglected in most existing models, play a critical role in regulating cellular anisotropic viscoelasticity. The stiffness of the cell in both directions increases linearly with increasing its aspect ratio, due to the decrease of cell volume. Moreover, the increase in the cell's aspect ratio produces the aggregation of cytoskeletal filaments along the longitudinal direction, resulting in higher stiffness in this direction. It is also shown that the increase in cell's aspect ratio corresponds to a process of cellular ordering, which can be quantitatively characterized by the orientational entropy of cytoskeletal filaments. In addition, we present a simple yet robust method to establish the relationship between cell's aspect ratio and cell volume, thus providing a theoretical framework to capture the anisotropic viscoelastic behavior of cells. This study suggests that the structure-based cell models may be further developed to investigate cellular rheological responses to external mechanical stimuli and may be extended to the tissue scale. STATEMENT OF SIGNIFICANCE: The viscoelastic behaviors of cells hold significant importance in comprehending the roles of mechanical forces in embryo development, invasion, and metastasis of cancer cells. Here, a structure-based cell model is proposed to study the anisotropic viscoelastic mechanical behaviors of living cells. Our study highlights the crucial role of previously neglected factors, such as cell volume and cytoskeletal filament orientation, in regulating cellular anisotropic viscoelasticity. We further propose an orientational entropy of cytoskeletal filaments to quantitatively characterize the ordering process of cells with increasing aspect ratios. Moreover, we derived the analytical interrelationships between cell aspect ratio, cell stiffness, cell volume, and cytoskeletal fiber orientation. This study provides a theoretical framework to describe the anisotropic viscoelastic mechanical behavior of cells.

Guard cell and subsidiary cell sizes are key determinants for stomatal kinetics and drought adaptation in cereal crops.

Climate change-induced drought is a major threat to agriculture. C4 crops have a higher water use efficiency (WUE) and better adaptability to drought than C3 crops due to their smaller stomatal morphology and faster response. However, our understanding of stomatal behaviours in both C3 and C4 Poaceae crops is limited by knowledge gaps in physical traits of guard cell (GC) and subsidiary cell (SC). We employed infrared gas exchange analysis and a stomatal assay to explore the relationship between GC/SC sizes and stomatal kinetics across diverse drought conditions in two C3 (wheat and barley) and three C4 (maize, sorghum and foxtail millet) upland Poaceae crops. Through statistical analyses, we proposed a GCSC-τ model to demonstrate how morphological differences affect stomatal kinetics in C4 Poaceae crops. Our findings reveal that morphological variations specifically correlate with stomatal kinetics in C4 Poaceae crops, but not in C3 ones. Subsequent modelling and experimental validation provide further evidence that GC/SC sizes significantly impact stomatal kinetics, which affects stomatal responses to different drought conditions and thereby WUE in C4 Poaceae crops. These findings emphasize the crucial advantage of GC/SC morphological characteristics and stomatal kinetics for the drought adaptability of C4 Poaceae crops, highlighting their potential as future climate-resilient crops.

Functional expression of the proton sensors ASIC1a, TMEM206, and OGR1 together with BKCa channels is associated with cell volume changes and cell death under strongly acidic conditions in DAOY medulloblastoma cells.

Fast growing solid tumors are frequently surrounded by an acidic microenvironment. Tumor cells employ a variety of mechanisms to survive and proliferate under these harsh conditions. In that regard, acid-sensitive membrane receptors constitute a particularly interesting target, since they can affect cellular functions through ion flow and second messenger cascades. Our knowledge of these processes remains sparse, however, especially regarding medulloblastoma, the most common pediatric CNS malignancy. In this study, using RT-qPCR, whole-cell patch clamp, and Ca2+-imaging, we uncovered several ion channels and a G protein-coupled receptor, which were regulated directly or indirectly by low extracellular pH in DAOY and UW228 medulloblastoma cells. Acidification directly activated acid-sensing ion channel 1a (ASIC1a), the proton-activated Cl- channel (PAC, ASOR, or TMEM206), and the proton-activated G protein-coupled receptor OGR1. The resulting Ca2+ signal secondarily activated the large conductance calcium-activated potassium channel (BKCa). Our analyses uncover a complex relationship of these transmembrane proteins in DAOY cells that resulted in cell volume changes and induced cell death under strongly acidic conditions. Collectively, our results suggest that these ion channels in concert with OGR1 may shape the growth and evolution of medulloblastoma cells in their acidic microenvironment.

The volume of healthy red blood cells is optimal for advective oxygen transport in arterioles.

Red blood cells (RBCs) are vital for transporting oxygen from the lungs to the body's tissues through the intricate circulatory system. They achieve this by binding and releasing oxygen molecules to the abundant hemoglobin within their cytosol. The volume of RBCs affects the amount of oxygen they can carry, yet whether this volume is optimal for transporting oxygen through the circulatory system remains an open question. This study explores, through high-fidelity numerical simulations, the impact of RBC volume on advective oxygen transport efficiency through arterioles, which form the area of greatest flow resistance in the circulatory system. The results show that, strikingly, RBCs with volumes similar to those found in vivo are most efficient to transport oxygen through arterioles. The flow resistance is related to the cell-free layer thickness, which is influenced by the shape and the motion of the RBCs: at low volumes, RBCs deform and fold, while at high volumes, RBCs collide and follow more diffuse trajectories. In contrast, RBCs with a healthy volume maximize the cell-free layer thickness, resulting in a more efficient advective transport of oxygen.

Cell size and xylem differentiation regulating genes from Salicornia europaea contribute to plant salt tolerance.

Cell wall is involved in plant growth and plays pivotal roles in plant adaptation to environmental stresses. Cell wall remodelling may be crucial to salt adaptation in the euhalophyte Salicornia europaea. However, the mechanism underlying this process is still unclear. Here, full-length transcriptome indicated cell wall-related genes were comprehensively regulated under salinity. The morphology and cell wall components in S. europaea shoot were largely modified under salinity. Through the weighted gene co-expression network analysis, SeXTH2 encoding xyloglucan endotransglucosylase/hydrolases, and two SeLACs encoding laccases were focused. Meanwhile, SeEXPB was focused according to expansin activity and the expression profiling. Function analysis in Arabidopsis validated the functions of these genes in enhancing salt tolerance. SeXTH2 and SeEXPB overexpression led to larger cells and leaves with hemicellulose and pectin content alteration. SeLAC1 and SeLAC2 overexpression led to more xylem vessels, increased secondary cell wall thickness and lignin content. Notably, SeXTH2 transgenic rice exhibited enhanced salt tolerance and higher grain yield. Altogether, these genes may function in the succulence and lignification process in S. europaea. This work throws light on the regulatory mechanism of cell wall remodelling in S. europaea under salinity and provides potential strategies for improving crop salt tolerance and yields.

Decoupling cell size homeostasis in diatoms from the geometrical constraints of the silica cell wall.

Unicellular organisms are known to exert tight control over their cell size. In the case of diatoms, abundant eukaryotic microalgae, two opposing notions are widely accepted. On the one hand, the rigid silica cell wall that forms inside the parental cell is thought to enforce geometrical reduction of the cell size. On the other hand, numerous exceptions cast doubt on the generality of this model. Here, we monitored clonal cultures of the diatom Stephanopyxis turris for up to 2 yr, recording the sizes of thousands of cells, in order to follow the distribution of cell sizes in the population. Our results show that S. turris cultures above a certain size threshold undergo a gradual size reduction, in accordance with the postulated geometrical driving force. However, once the cell size reaches a lower threshold, it fluctuates around a constant size using the inherent elasticity of cell wall elements. These results reconcile the disparate observations on cell size regulation in diatoms by showing two distinct behaviors, reduction and homeostasis. The geometrical size reduction is the dominant driving force for large cells, but smaller cells have the flexibility to re-adjust the size of their new cell walls.

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.

Plant cell size: Links to cell cycle, differentiation and ploidy.

Cell size affects many processes, including exchange of nutrients and external signals, cell division and tissue mechanics. Across eukaryotes, cells have evolved mechanisms that assess their own size to inform processes such as cell cycle progression or gene expression. Here, we review recent progress in understanding plant cell size regulation and its implications, relating these findings to work in other eukaryotes. Highlights include use of DNA contents as reference point to control the cell cycle in shoot meristems, a size-dependent cell fate decision during stomatal development and insights into the interconnection between ploidy, cell size and cell wall mechanics.

Streamlined measurement of chimeric antigen receptor T-cell concentration, size, viability and two-color phenotyping during manufacturing.

BACKGROUND AIMS: The successful development of CD19-targeted chimeric antigen receptor (CAR) T-cell therapies has led to an exponential increase in the number of patients recieving treatment and the advancement of novel CAR T products. Therefore, there is a strong need to develop streamlined platforms that allow rapid, cost-effective, and accurate measurement of the key characteristics of CAR T cells during manufacturing (i.e., cell number, cell size, viability, and basic phenotype). METHODS: In this study, we compared the novel benchtop cell analyzer Moxi GO II (ORFLO Technologies), which enables simultaneous evaluation of all the aforementioned parameters, with current gold standards in the field: the Multisizer Coulter Counter (cell counter) and the BD LSRFortessa (flow cytometer). RESULTS: Our results demonstrated that the Moxi GO II can accurately measure cell number and cell size (i.e., cell volume) while simultaneously assessing simple two-color flow cytometry parameters, such as CAR T-cell viability and CD4 or CAR expression. CONCLUSIONS: These measurements are comparable with those of gold standard instruments, demonstrating that the Moxi GO II is a promising platform for quickly monitoring CAR T-cell growth and phenotype in research-grade and clinical samples.

Unraveling power-law scaling through exponential cell division dynamics.

A primary objective of biology is the development of universal laws that define how organic form develops and how it evolves as a function of size, both ontogenetically and across evolutionary time. Scaling theory has been essential in reaching this goal by giving a complete perspective point, particularly in illuminating the fundamental biological features produced within scaling exponents defining families of equations. Nonetheless, the theoretical basis of the allometric equation within scaling theory are inadequately explained, particularly when it comes to establishing links between micro-level processes at the cellular level and macro-level phenomena. We proposed an unlimited cell bipartition, resulting in an exponential growth in cell numbers during an individual's lifespan, to bridge this conceptual gap between cellular processes and allometric scaling. The power-law scaling between body mass and organ weight was produced by the synchronous exponential increments and the allometric exponent is rate of logarithmic cell proliferation rate. Substituting organ weight for erythrocyte weight aided in the development of a power-law scaling relationship between body mass and metabolic rate. Furthermore, it is critical to understand how cell size affects the exponent in power-law scaling. We find that a bigger exponent will result from an increase in the average weight of organ cells or a decrease in the average weight of all cells. Furthermore, cell proliferation dynamics showed a complex exponential scaling between body mass and longevity, defying the previously reported power-law scaling. We discovered a quadratic link between longevity and logarithmic body mass. Notably, all of the parameters included in these relationships are explained by indices linked to cell division and embryonic development. This research adds to our understanding of the complex interaction between cellular processes and overarching scaling phenomena in biology.

Subscaling of a cytosolic RNA binding protein governs cell size homeostasis in the multiple fission alga Chlamydomonas.

Coordination of growth and division in eukaryotic cells is essential for populations of proliferating cells to maintain size homeostasis, but the underlying mechanisms that govern cell size have only been investigated in a few taxa. The green alga Chlamydomonas reinhardtii (Chlamydomonas) proliferates using a multiple fission cell cycle that involves a long G1 phase followed by a rapid series of successive S and M phases (S/M) that produces 2n daughter cells. Two control points show cell-size dependence: the Commitment control point in mid-G1 phase requires the attainment of a minimum size to enable at least one mitotic division during S/M, and the S/M control point where mother cell size governs cell division number (n), ensuring that daughter distributions are uniform. tny1 mutants pass Commitment at a smaller size than wild type and undergo extra divisions during S/M phase to produce small daughters, indicating that TNY1 functions to inhibit size-dependent cell cycle progression. TNY1 encodes a cytosolic hnRNP A-related RNA binding protein and is produced once per cell cycle during S/M phase where it is apportioned to daughter cells, and then remains at constant absolute abundance as cells grow, a property known as subscaling. Altering the dosage of TNY1 in heterozygous diploids or through mis-expression increased Commitment cell size and daughter cell size, indicating that TNY1 is a limiting factor for both size control points. Epistasis placed TNY1 function upstream of the retinoblastoma tumor suppressor complex (RBC) and one of its regulators, Cyclin-Dependent Kinase G1 (CDKG1). Moreover, CDKG1 protein and mRNA were found to over-accumulate in tny1 cells suggesting that CDKG1 may be a direct target of repression by TNY1. Our data expand the potential roles of subscaling proteins outside the nucleus and imply a control mechanism that ties TNY1 accumulation to pre-division mother cell size.

Rice bundle sheath cell shape is regulated by the timing of light exposure during leaf development.

Plant leaves contain multiple cell types which achieve distinct characteristics whilst still coordinating development within the leaf. The bundle sheath possesses larger individual cells and lower chloroplast content than the adjacent mesophyll, but how this morphology is achieved remains unknown. To identify regulatory mechanisms determining bundle sheath cell morphology we tested the effects of perturbing environmental (light) and endogenous signals (hormones) during leaf development of Oryza sativa (rice). Total chloroplast area in bundle sheath cells was found to increase with cell size as in the mesophyll but did not maintain a 'set-point' relationship, with the longest bundle sheath cells demonstrating the lowest chloroplast content. Application of exogenous cytokinin and gibberellin significantly altered the relationship between cell size and chloroplast biosynthesis in the bundle sheath, increasing chloroplast content of the longest cells. Delayed exposure to light reduced the mean length of bundle sheath cells but increased corresponding leaf length, whereas premature light reduced final leaf length but did not affect bundle sheath cells. This suggests that the plant hormones cytokinin and gibberellin are regulators of the bundle sheath cell-chloroplast relationship and that final bundle sheath length may potentially be affected by light-mediated control of exit from the cell cycle.