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.

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.

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.

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.

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.

Tissue-specific compensatory mechanisms maintain tissue architecture and body size independent of cell size in polyploid zebrafish.

Genome duplications and ploidy transitions have occurred in nearly every major taxon of eukaryotes, but they are far more common in plants than in animals. Due to the conservation of the nuclear:cytoplasmic volume ratio increased DNA content results in larger cells. In plants, polyploid organisms are larger than diploids as cell number remains relatively constant. Conversely, vertebrate body size does not correlate with cell size and ploidy as vertebrates compensate for increased cell size to maintain tissue architecture and body size. This has historically been explained by a simple reduction in cell number that matches the increase in cell size maintaining body size as ploidy increases, but here we show that the compensatory mechanisms that maintain body size in triploid zebrafish are tissue-specific: A) erythrocytes respond in the classical pattern with a reduced number of larger erythrocytes in circulation, B) muscle, a tissue comprised of polynucleated muscle fibers, compensates by reducing the number of larger nuclei such that myofiber and myotome size in unaffected by ploidy, and C) vascular tissue compensates by thickening blood vessel walls, possibly at the expense of luminal diameter. Understanding the physiological implications of ploidy on tissue function requires a detailed description of the specific mechanisms of morphological compensation occurring in each tissue to understand how ploidy changes affect development and physiology.

Smoking is associated with increased eryptosis, suicidal erythrocyte death, in a large population-based cohort.

Smoking has multiple detrimental effects on health, and is a major preventable cause of premature death and chronic disease. Despite the well-described effect of inhaled substances from tobacco smoke on cell toxicity, the association between smoking and suicidal erythrocyte death, termed eryptosis, is virtually unknown. Therefore, the blood samples of 2023 participants of the German National Cohort Study (NAKO) were analyzed using flow cytometry analysis to determine eryptosis from fluorescent annexin V-FITC-binding to phosphatidylserine-exposing erythrocytes. Blood analyses were complemented by the measurement of hematologic parameters including red blood cell count, hematocrit, hemoglobin, mean corpuscular cell volume (MCV) and mean corpuscular hemoglobin (MCH). Eryptosis was higher in smokers than in non- and ex-smokers, and positively associated with the number of cigarettes smoked daily (r = 0.08, 95% CI [0.03, 0.12]). Interestingly, despite increased eryptosis, smokers had higher red blood cell indices than non-smokers. To conclude, smokers were characterized by higher eryptosis than non-smokers, without showing any obvious detrimental effect on classic hematological parameters.

Multicompartmental coacervate-based protocell by spontaneous droplet evaporation.

Hierarchical compartmentalization, a hallmark of both primitive and modern cells, enables the concentration and isolation of biomolecules, and facilitates spatial organization of biochemical reactions. Coacervate-based compartments can sequester and recruit a large variety of molecules, making it an attractive protocell model. In this work, we report the spontaneous formation of core-shell cell-sized coacervate-based compartments driven by spontaneous evaporation of a sessile droplet on a thin-oil-coated substrate. Our analysis reveals that such far-from-equilibrium architectures arise from multiple, coupled segregative and associative liquid-liquid phase separation, and are stabilized by stagnation points within the evaporating droplet. The formation of stagnation points results from convective capillary flows induced by the maximum evaporation rate at the liquid-liquid-air contact line. This work provides valuable insights into the spontaneous formation and maintenance of hierarchical compartments under non-equilibrium conditions, offering a glimpse into the real-life scenario.

Chromosome Division in Early Embryos-Is Everything under Control? And Is the Cell Size Important?

Chromosome segregation in female germ cells and early embryonic blastomeres is known to be highly prone to errors. The resulting aneuploidy is therefore the most frequent cause of termination of early development and embryo loss in mammals. And in specific cases, when the aneuploidy is actually compatible with embryonic and fetal development, it leads to severe developmental disorders. The main surveillance mechanism, which is essential for the fidelity of chromosome segregation, is the Spindle Assembly Checkpoint (SAC). And although all eukaryotic cells carry genes required for SAC, it is not clear whether this pathway is active in all cell types, including blastomeres of early embryos. In this review, we will summarize and discuss the recent progress in our understanding of the mechanisms controlling chromosome segregation and how they might work in embryos and mammalian embryos in particular. Our conclusion from the current literature is that the early mammalian embryos show limited capabilities to react to chromosome segregation defects, which might, at least partially, explain the widespread problem of aneuploidy during the early development in mammals.

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.

Mathematical modeling of adipocyte size distributions: Identifiability and parameter estimation from rat data.

Fat cells, called adipocytes, are designed to regulate energy homeostasis by storing energy in the form of lipids. Adipocyte size distribution is assumed to play a role in the development of obesity-related diseases. These cells that do not have a characteristic size, indeed a bimodal size distribution is observed in adipose tissue. We propose a model based on a partial differential equation to describe adipocyte size distribution. The model includes a description of the lipid fluxes and the cell size fluctuations and using a formulation of a stationary solution fast computation of bimodal distribution is achieved. We investigate the parameter identifiability and estimate parameter values with CMA-ES algorithm. We first validate the procedure on synthetic data, then we estimate parameter values with experimental data of 32 rats. We discuss the estimated parameter values and their variability within the population, as well as the relation between estimated values and their biological significance. Finally, a sensitivity analysis is performed to specify the influence of parameters on cell size distribution and explain the differences between the model and the measurements. The proposed framework enables the characterization of adipocyte size distribution with four parameters and can be easily adapted to measurements of cell size distribution in different health conditions.

Control of successive unequal cell divisions by neural cell fate regulators determines embryonic neuroblast cell size.

Asymmetric cell divisions often generate daughter cells of unequal size in addition to different fates. In some contexts, daughter cell size asymmetry is thought to be a key input to specific binary cell fate decisions. An alternative possibility is that unequal division is a mechanism by which a variety of cells of different sizes are generated during embryonic development. We show here that two unequal cell divisions precede neuroblast formation in the C lineage of Caenorhabditis elegans. The equalisation of these divisions in a pig-1/MELK mutant background has little effect on neuroblast specification. Instead, we demonstrate that let-19/MDT13 is a regulator of the proneural basic helix-loop-helix transcription factor hlh-14/ASCL1 and find that both are required to concomitantly regulate the acquisition of neuroblast identity and neuroblast cell size. Thus, embryonic neuroblast cell size in this lineage is progressively regulated in parallel with identity by key neural cell fate regulators. We propose that key cell fate determinants have a previously unappreciated function in regulating unequal cleavage, and therefore cell size, of the progenitor cells whose daughter cell fates they then go on to specify.

Influence of cell volume on the gene transcription rate.

Cells vary in volume throughout their life cycle and in many other circumstances, while their genome remains identical. Hence, the RNA production factory must adapt to changing needs, while maintaining the same production lines. This paradox is resolved by different mechanisms in distinct cells and circumstances. RNA polymerases have evolved to cope with the particular circumstances of each case and the different characteristics of the several RNA molecule types, especially their stabilities. Here we review current knowledge on these issues. We focus on the yeast Saccharomyces cerevisiae, where many of the studies have been performed, although we compare and discuss the results obtained in other eukaryotes and propose several ideas and questions to be tested and solved in the future. TAKE AWAY.

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.

Adipocyte size, adipose tissue calories, and circulating adipokines, before and after diet-induced weight loss in humans.

OBJECTIVE: Adipose tissue (AT) contains a bimodal population of large and small adipocytes. Changes in fat cell size (FCS) distribution and AT caloric density (kcal/g) with weight loss are unclear. We aimed to evaluate changes in FCS and AT calories in weight loss and determine associations with anthropometrics. MATERIALS AND METHODS: Healthy adults (6 men/4 women; age 33 ± 11 years; BMI 35 ± 6 kg/m2) underwent DXA and subcutaneous abdominal/thigh fat biopsies, before and after 6 weeks of caloric restriction. AT calories (bomb calorimetry) and hormones (adiponectin, leptin, FGF21) were measured. RESULTS: Abdominal large cell diameter (LCD; Δ = -13.2 µm, p = 0.01) and nadir (Δ = -7.3 µm, p = 0.03) decreased. In repeated measures correlations (rrm), abdominal and thigh LCD and nadir were associated with fat mass (FM) loss (rrm = 0.68; rrm = 0.63; rrm = 0.66; rrm = 0.62, p's < 0.05, respectively) and waist circumference decrease (rrm = 0.70; rrm = 0.60, p's ≤ 0.05). Small cell percentage did not change and was not associated with FM changes. Abdominal AT calories were unchanged with weight loss. Change in leptin was associated with change in abdominal LCD (rrm = 0.77, p = 0.01). CONCLUSIONS: Caloric restriction reduces adipocyte LCD and nadir. These changes are associated with FM loss. Larger fat cells should be considered as phenotypic targets for weight loss. CLINICAL TRIALS REGISTRATION: clinicaltrials.gov identifier: NCT00687115, May 29, 2008.

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'.

Physiology of the volume-sensitive/regulatory anion channel VSOR/VRAC. Part 1: from its discovery and phenotype characterization to the molecular entity identification.

The volume-sensitive outwardly rectifying or volume-regulated anion channel, VSOR/VRAC, which was discovered in 1988, is expressed in most vertebrate cell types and is essentially involved in cell volume regulation after swelling and in the induction of cell death. This series of review articles describes what is already known and what remains to be uncovered about the functional and molecular properties as well as the physiological and pathophysiological roles of VSOR/VRAC. This Part 1 review article describes, from the physiological standpoint, first its discovery and significance in cell volume regulation, second its phenotypical properties, and third its molecular identification. Although the pore-forming core molecules and the volume-sensing subcomponent of VSOR/VRAC were identified as LRRC8 members and TRPM7 in 2014 and 2021, respectively, it is stressed that the identification of the molecular entity of VSOR/VRAC is still not complete enough to explain the full set of phenotypical properties.

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.