Homeostasis of cytoplasmic crowding by cell wall fluidization and ribosomal counterions.

In bacteria, algae, fungi, and plant cells, the wall must expand in concert with cytoplasmic biomass production, otherwise cells would experience toxic molecular crowding1,2 or lyse. But how cells achieve expansion of this complex biomaterial in coordination with biosynthesis of macromolecules in the cytoplasm remains unexplained3, although recent works have revealed that these processes are indeed coupled4,5. Here, we report a striking increase of turgor pressure with growth rate in E. coli, suggesting that the speed of cell wall expansion is controlled via turgor. Remarkably, despite this increase in turgor pressure, cellular biomass density remains constant across a wide range of growth rates. By contrast, perturbations of turgor pressure that deviate from this scaling directly alter biomass density. A mathematical model based on cell wall fluidization by cell wall endopeptidases not only explains these apparently confounding observations but makes surprising quantitative predictions that we validated experimentally. The picture that emerges is that turgor pressure is directly controlled via counterions of ribosomal RNA. Elegantly, the coupling between rRNA and turgor pressure simultaneously coordinates cell wall expansion across a wide range of growth rates and exerts homeostatic feedback control on biomass density. This mechanism may regulate cell wall biosynthesis from microbes to plants and has important implications for the mechanism of action of antibiotics6.

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.

The discovery of tau protein.

In January of this year I received an unexpected request from George Bloom to contribute an historical perspective on "the discovery of tau protein," an event that occurred roughly 50 years ago. My first thought was that it could not have been that long ago, as the memories of what was my first independent scientific discovery are still fresh in my mind today. But 50 years is half a century and, as I thought about the events, I realized how much the practice of science has changed.

Membrane potential mediates an ancient mechano-transduction mechanism for multi-cellular homeostasis.

Membrane potential is a property of all living cells1. However, its physiological role in non-excitable cells is poorly understood. Resting membrane potential is typically considered fixed for a given cell type and under tight homeostatic control2, akin to body temperature in mammals. Contrary to this widely accepted paradigm, we found that membrane potential is a dynamic property that directly reflects tissue density and mechanical forces acting on the cell. Serving as a quasi-instantaneous, global readout of density and mechanical pressure, membrane potential is integrated with signal transduction networks by affecting the conformation and clustering of proteins in the membrane3,4, as well as the transmembrane flux of key signaling ions5,6. Indeed, we show that important mechano-sensing pathways, YAP, Jnk and p387-121314, are directly controlled by membrane potential. We further show that mechano-transduction via membrane potential plays a critical role in the homeostasis of epithelial tissues, setting tissue density by controlling proliferation and cell extrusion of cells. Moreover, a wave of depolarization triggered by mechanical stretch enhances the speed of wound healing. Mechano-transduction via membrane potential likely constitutes an ancient homeostatic mechanism in multi-cellular organisms, potentially serving as a steppingstone for the evolution of excitable tissues and neuronal mechano-sensing. The breakdown of membrane potential mediated homeostatic regulation may contribute to tumor growth.

A universal mechanism of biomass density homeostasis via ribosomal counterions.

In all growing cells, the cell envelope must expand in concert with cytoplasmic biomass to prevent lysis or molecular crowding. The complex cell wall of microbes and plants makes this challenge especially daunting and it unclear how cells achieve this coordination. Here, we uncover a striking linear increase of cytoplasmic pressure with growth rate in E. coli. Remarkably, despite this increase in turgor pressure with growth rate, cellular biomass density was constant across a wide range of growth rates. In contrast, perturbing pressure away from this scaling directly affected biomass density. A mathematical model, in which endopeptidase-mediated cell wall fluidization enables turgor pressure to set the pace of cellular volume expansion, not only explains these confounding observations, but makes several surprising quantitative predictions that we validated experimentally. The picture that emerges is that changes in turgor pressure across growth rates are mediated by counterions of ribosomal RNA. Profoundly, the coupling between rRNA and cytoplasmic pressure simultaneously coordinates cell wall expansion across growth rates and exerts homeostatic feedback control on biomass density. Because ribosome content universally scales with growth rate in fast growing cells, this universal mechanism may control cell wall biosynthesis in microbes and plants and drive the expansion of ribosome-addicted tumors that can exert substantial mechanical forces on their environment.

Dorsal lip maturation and initial archenteron extension depend on Wnt11 family ligands.

Wnt11 family proteins are ligands that activate a type of Dishevelled-mediated, non-canonical Wnt signaling pathway. Loss of function causes defects in gastrulation and/or anterior-posterior axis extension in all vertebrates. Non-mammalian vertebrate genomes encode two Wnt11 family proteins whose distinct functions have been unclear. We knocked down Wnt11b and Wnt11, separately and together, in Xenopus laevis. Single morphants exhibited very similar phenotypes of delayed blastopore closure, but they had different phenotypes during the tailbud period. In response to their very similar gastrulation phenotypes, we chose to characterize dual morphants. Using dark field illuminated time-lapse imaging and kymograph analysis, we identified a failure of dorsal blastopore lip maturation that correlated with slower blastopore closure and failure to internalize the endoderm at the dorsal blastopore lip. We connected these externally visible phenotypes to cellular events in the internal tissues by imaging intact fixed embryos stained for anillin and microtubules. We found that the initial extension of the archenteron is correlated with blastopore lip maturation, and archenteron extension is dramatically disrupted by decreased Wnt11 family signaling. We were aided in our interpretation of the immunofluorescence by the novel, membrane proximal location of the cleavage furrow protein anillin in the epithelium of the blastopore lip and early archenteron.

Inverse Lansing Effect: Maternal Age and Provisioning Affecting Daughters' Longevity and Male Offspring Production.

AbstractMaternal age effects on offspring life history are known in a variety of organisms, with offspring of older mothers typically having lower life expectancy (the Lansing effect). However, there is no consensus on the generality and mechanisms of this pattern. We tested predictions of the Lansing effect in several Daphnia magna clones and observed clone-specific magnitude and direction of the maternal age effect on offspring longevity. We also report ambidirectional, genotype-specific effects of maternal age on the propensity of daughters to produce male offspring. Focusing on two clones with contrasting life histories, we demonstrate that maternal age effects can be explained by lipid provisioning of embryos by mothers of different ages. Individuals from a single-generation maternal age reversal treatment showed intermediate life span and intermediate lipid content at birth. In the clone characterized by the "inverse Lansing effect," neonates produced by older mothers showed higher mitochondrial membrane potential in neural tissues than their counterparts born to younger mothers. We conclude that an inverse Lansing effect is possible and hypothesize that it may be caused by age-specific maternal lipid provisioning creating a calorically restricted environment during embryonic development, which in turn reduces fecundity and increases life span in offspring.

The uniformity and stability of cellular mass density in mammalian cell culture.

Cell dry mass is principally determined by the sum of biosynthesis and degradation. Measurable change in dry mass occurs on a time scale of hours. By contrast, cell volume can change in minutes by altering the osmotic conditions. How changes in dry mass and volume are coupled is a fundamental question in cell size control. If cell volume were proportional to cell dry mass during growth, the cell would always maintain the same cellular mass density, defined as cell dry mass dividing by cell volume. The accuracy and stability against perturbation of this proportionality has never been stringently tested. Normalized Raman Imaging (NoRI), can measure both protein and lipid dry mass density directly. Using this new technique, we have been able to investigate the stability of mass density in response to pharmaceutical and physiological perturbations in three cultured mammalian cell lines. We find a remarkably narrow mass density distribution within cells, that is, significantly tighter than the variability of mass or volume distribution. The measured mass density is independent of the cell cycle. We find that mass density can be modulated directly by extracellular osmolytes or by disruptions of the cytoskeleton. Yet, mass density is surprisingly resistant to pharmacological perturbations of protein synthesis or protein degradation, suggesting there must be some form of feedback control to maintain the homeostasis of mass density when mass is altered. By contrast, physiological perturbations such as starvation or senescence induce significant shifts in mass density. We have begun to shed light on how and why cell mass density remains fixed against some perturbations and yet is sensitive during transitions in physiological state.

Protein and lipid mass concentration measurement in tissues by stimulated Raman scattering microscopy.

Cell mass and chemical composition are important aggregate cellular properties that are especially relevant to physiological processes, such as growth control and tissue homeostasis. Despite their importance, it has been difficult to measure these features quantitatively at the individual cell level in intact tissue. Here, we introduce normalized Raman imaging (NoRI), a stimulated Raman scattering (SRS) microscopy method that provides the local concentrations of protein, lipid, and water from live or fixed tissue samples with high spatial resolution. Using NoRI, we demonstrate that protein, lipid, and water concentrations at the single cell are maintained in a tight range in cells under the same physiological conditions and are altered in different physiological states, such as cell cycle stages, attachment to substrates of different stiffness, or by entering senescence. In animal tissues, protein and lipid concentration varies with cell types, yet an unexpected cell-to-cell heterogeneity was found in cerebellar Purkinje cells. The protein and lipid concentration profile provides means to quantitatively compare disease-related pathology, as demonstrated using models of Alzheimer's disease. This demonstration shows that NoRI is a broadly applicable technique for probing the biological regulation of protein mass, lipid mass, and water mass for studies of cellular and tissue growth, homeostasis, and disease.

Computational Models for Clinical Applications in Personalized Medicine-Guidelines and Recommendations for Data Integration and Model Validation.

The future development of personalized medicine depends on a vast exchange of data from different sources, as well as harmonized integrative analysis of large-scale clinical health and sample data. Computational-modelling approaches play a key role in the analysis of the underlying molecular processes and pathways that characterize human biology, but they also lead to a more profound understanding of the mechanisms and factors that drive diseases; hence, they allow personalized treatment strategies that are guided by central clinical questions. However, despite the growing popularity of computational-modelling approaches in different stakeholder communities, there are still many hurdles to overcome for their clinical routine implementation in the future. Especially the integration of heterogeneous data from multiple sources and types are challenging tasks that require clear guidelines that also have to comply with high ethical and legal standards. Here, we discuss the most relevant computational models for personalized medicine in detail that can be considered as best-practice guidelines for application in clinical care. We define specific challenges and provide applicable guidelines and recommendations for study design, data acquisition, and operation as well as for model validation and clinical translation and other research areas.

Intelligent high-throughput intervention testing platform in Daphnia.

We present a novel platform for testing the effects of interventions on the life- and healthspan of a short-lived freshwater organism with complex behavior and physiology-the planktonic crustacean Daphnia magna. Within this platform, dozens of complex behavioral features of both routine motion and response to stimuli are continuously quantified over large synchronized cohorts via an automated phenotyping pipeline. We build predictive machine-learning models calibrated using chronological age and extrapolate onto phenotypic age. We further apply the model to estimate the phenotypic age under pharmacological perturbation. Our platform provides a scalable framework for drug screening and characterization in both life-long and instant assays as illustrated using a long-term dose-response profile of metformin and a short-term assay of well-studied substances such as caffeine and alcohol.

The nonredundant nature of the Axin2 regulatory network in the canonical Wnt signaling pathway.

Axin is one of two essential scaffolds in the canonical Wnt pathway that converts signals at the plasma membrane to signals inhibiting the degradation of ß-catenin, leading to its accumulation and specific gene activation. In vertebrates, there are two forms of Axin, Axin1 and Axin2, which are similar at the protein level and genetically redundant. We show here that differential regulation of the two genes on the transcriptional and proteostatic level confers differential responsiveness that can be used in tissue-specific regulation. Such subtle features may distinguish other redundant gene pairs that are commonly found in vertebrates through gene knockout experiments.

Lack of age-related respiratory changes in Daphnia.

Aging is a multifaceted process of accumulation of damage and waste in cells and tissues; age-related changes in mitochondria and in respiratory metabolism have the focus of aging research for decades. Studies of aging in nematodes, flies and mammals all revealed age-related decline in respiratory functions, with somewhat controversial causative role. Here we investigated age-related changes in respiration rates, lactate/pyruvate ratio, a commonly used proxy for NADH/NAD+ balance, and mitochondrial membrane potential in 4 genotypes of an emerging model organism for aging research, a cyclic parthenogen Daphnia magna. We show that total body weight-adjusted respiration rate decreased with age, although this decrease was small in magnitude and could be fully accounted for by the decrease in locomotion and feeding activity. Neither total respiration normalized by protein content, nor basal respiration rate measured in anaesthetized animals decreased with age. Lactate/pyruvate ratio and mitochondrial membrane potential (∆Ψmt) showed no age-related changes, with possible exceptions of ∆Ψmt in epipodites (excretory and gas exchange organs) in which ∆Ψmt decreased with age and in the optical lobe of the brain, in which ∆Ψmt showed a maximum at middle age. We conclude that actuarial senescence in Daphnia is not caused by a decline in respiratory metabolism and discuss possible mechanisms of maintaining mitochondrial healthspan throughout the lifespan.

Phylogenomic analyses of the genus Drosophila reveals genomic signals of climate adaptation.

Many Drosophila species differ widely in their distributions and climate niches, making them excellent subjects for evolutionary genomic studies. Here, we have developed a database of high-quality assemblies for 46 Drosophila species and one closely related Zaprionus. Fifteen of the genomes were newly sequenced, and 20 were improved with additional sequencing. New or improved annotations were generated for all 47 species, assisted by new transcriptomes for 19. Phylogenomic analyses of these data resolved several previously ambiguous relationships, especially in the melanogaster species group. However, it also revealed significant phylogenetic incongruence among genes, mainly in the form of incomplete lineage sorting in the subgenus Sophophora but also including asymmetric introgression in the subgenus Drosophila. Using the phylogeny as a framework and taking into account these incongruences, we then screened the data for genome-wide signals of adaptation to different climatic niches. First, phylostratigraphy revealed relatively high rates of recent novel gene gain in three temperate pseudoobscura and five desert-adapted cactophilic mulleri subgroup species. Second, we found differing ratios of nonsynonymous to synonymous substitutions in several hundred orthologues between climate generalists and specialists, with trends for significantly higher ratios for those in tropical and lower ratios for those in temperate-continental specialists respectively than those in the climate generalists. Finally, resequencing natural populations of 13 species revealed tropics-restricted species generally had smaller population sizes, lower genome diversity and more deleterious mutations than the more widespread species. We conclude that adaptation to different climates in the genus Drosophila has been associated with large-scale and multifaceted genomic changes.

Identification of kinases activated by multiple pro-angiogenic growth factors.

Antiangiogenic therapy began as an effort to inhibit VEGF signaling, which was thought to be the sole factor driving tumor angiogenesis. It has become clear that there are more pro-angiogenic growth factors that can substitute for VEGF during tumor vascularization. This has led to the development of multi-kinase inhibitors which simultaneously target multiple growth factor receptors. These inhibitors perform better than monotherapies yet to date no multi-kinase inhibitor targets all receptors known to be involved in pro-angiogenic signaling and resistance inevitably occurs. Given the large number of pro-angiogenic growth factors identified, it may be impossible to simultaneously target all pro-angiogenic growth factor receptors. Here we search for kinase targets, some which may be intracellularly localized, that are critical in endothelial cell proliferation irrespective of the growth factor used. We develop a quantitative endothelial cell proliferation assay and combine it with "kinome regression" or KIR, a recently developed method capable of identifying kinases that influence a quantitative phenotype. We report the kinases implicated by KIR and provide orthogonal evidence of their importance in endothelial cell proliferation. Our approach may point to a new strategy to develop a more complete anti-angiogenic blockade.

Noncanonical open reading frames encode functional proteins essential for cancer cell survival.

Although genomic analyses predict many noncanonical open reading frames (ORFs) in the human genome, it is unclear whether they encode biologically active proteins. Here we experimentally interrogated 553 candidates selected from noncanonical ORF datasets. Of these, 57 induced viability defects when knocked out in human cancer cell lines. Following ectopic expression, 257 showed evidence of protein expression and 401 induced gene expression changes. Clustered regularly interspaced short palindromic repeat (CRISPR) tiling and start codon mutagenesis indicated that their biological effects required translation as opposed to RNA-mediated effects. We found that one of these ORFs, G029442-renamed glycine-rich extracellular protein-1 (GREP1)-encodes a secreted protein highly expressed in breast cancer, and its knockout in 263 cancer cell lines showed preferential essentiality in breast cancer-derived lines. The secretome of GREP1-expressing cells has an increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth-inhibitory effect of GREP1 knockout. Our experiments suggest that noncanonical ORFs can express biologically active proteins that are potential therapeutic targets.

What makes the cell cycle tick? a celebration of the awesome power of biochemistry and the frog egg.

The cell cycle, a 19th century discovery of cytologists, only achieved a satisfactory biochemical explanation in the last 20 years of the 20th century. This personal retrospective focuses on how biochemical studies of the frog egg helped identify the cyclin-based mitotic oscillator and how this approach quickly merged with genetic studies in yeast to establish the basic mechanism of the eukaryotic cell division cycle. The key feature that made this a cyclic process was regulated protein degradation, mediated by ubiquitin, catalyzed by a massive enzyme machine, called the Anaphase Promoting Complex.

Computationally enhanced quantitative phase microscopy reveals autonomous oscillations in mammalian cell growth.

The fine balance of growth and division is a fundamental property of the physiology of cells, and one of the least understood. Its study has been thwarted by difficulties in the accurate measurement of cell size and the even greater challenges of measuring growth of a single cell over time. We address these limitations by demonstrating a computationally enhanced methodology for quantitative phase microscopy for adherent cells, using improved image processing algorithms and automated cell-tracking software. Accuracy has been improved more than twofold and this improvement is sufficient to establish the dynamics of cell growth and adherence to simple growth laws. It is also sufficient to reveal unknown features of cell growth, previously unmeasurable. With these methodological and analytical improvements, in several cell lines we document a remarkable oscillation in growth rate, occurring throughout the cell cycle, coupled to cell division or birth yet independent of cell cycle progression. We expect that further exploration with this advanced tool will provide a better understanding of growth rate regulation in mammalian cells.

Towards standardization guidelines for in silico approaches in personalized medicine.

Despite the ever-progressing technological advances in producing data in health and clinical research, the generation of new knowledge for medical benefits through advanced analytics still lags behind its full potential. Reasons for this obstacle are the inherent heterogeneity of data sources and the lack of broadly accepted standards. Further hurdles are associated with legal and ethical issues surrounding the use of personal/patient data across disciplines and borders. Consequently, there is a need for broadly applicable standards compliant with legal and ethical regulations that allow interpretation of heterogeneous health data through in silico methodologies to advance personalized medicine. To tackle these standardization challenges, the Horizon2020 Coordinating and Support Action EU-STANDS4PM initiated an EU-wide mapping process to evaluate strategies for data integration and data-driven in silico modelling approaches to develop standards, recommendations and guidelines for personalized medicine. A first step towards this goal is a broad stakeholder consultation process initiated by an EU-STANDS4PM workshop at the annual COMBINE meeting (COMBINE 2019 workshop report in same issue). This forum analysed the status quo of data and model standards and reflected on possibilities as well as challenges for cross-domain data integration to facilitate in silico modelling approaches for personalized medicine.

Single-molecule dynamics of Dishevelled at the plasma membrane and Wnt pathway activation.

Dvl (Dishevelled) is one of several essential nonenzymatic components of the Wnt signaling pathway. In most current models, Dvl forms complexes with Wnt ligand receptors, Fzd and LRP5/6 at the plasma membrane, which then recruits the destruction complex, eventually leading to inactivation of ß-catenin degradation. Although this model is widespread, direct evidence for the individual steps is lacking. In this study, we tagged mEGFP to C terminus of dishevelled2 gene using CRISPR/Cas9-induced homologous recombination and observed its dynamics directly at the single-molecule level with total internal reflection fluorescence (TIRF) microscopy. We focused on two questions: 1) What is the native size and what are the dynamic features of membrane-bound Dvl complexes during Wnt pathway activation? 2) What controls the behavior of these complexes? We found that membrane-bound Dvl2 is predominantly monomer in the absence of Wnt (observed mean size 1.1). Wnt3a stimulation leads to an increase in the total concentration of membrane-bound Dvl2 from 0.12/µm2 to 0.54/µm2 Wnt3a also leads to increased oligomerization which raises the weighted mean size of Dvl2 complexes to 1.5, with 56.1% of Dvl still as monomers. The driving force for Dvl2 oligomerization is the increased concentration of membrane Dvl2 caused by increased affinity of Dvl2 for Fzd, which is independent of LRP5/6. The oligomerized Dvl2 complexes have increased dwell time, 2 ∼ 3 min, compared to less than 1 s for monomeric Dvl2. These properties make Dvl a unique scaffold, dynamically changing its state of assembly and stability at the membrane in response to Wnt ligands.