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This work quantified how cell velocity and effective power output are influenced by the rate of actin depolymerization, which is affected by myosin contraction. Model analysis shows that the cell migration velocity displays a biphasic dependence on the rate of actin depolymerization and myosin contraction.

Although metastasis is the leading cause of cancer deaths, it is quite rare at the cellular level. Only a rare subset of cancer cells (∼1 in 1.5 billion) can complete the entire metastatic cascade: invasion, intravasation, survival in the circulation, extravasation, and colonization (i.e. are metastasis competent). We propose that cells engaging a Polyaneuploid Cancer Cell (PACC) phenotype are metastasis competent. PACCs are enlarged, non-dividing cells with increased genomic content that form in response to stress. Single-cell tracking using time-lapse microscopy reveals that PACCs are more motile than nonPACCs. Additionally, PACCs exhibit increased capacity for environment-sensing and directional migration in chemotactic environments, predicting successful invasion. Magnetic Twisting Cytometry and Atomic Force Microscopy reveal that cells in the PACC state display hyper-elastic properties like increased peripheral deformability and maintained peri-nuclear cortical integrity that predict successful intravasation and extravasation. Furthermore, four orthogonal methods reveal that PACCs have increased expression of Vimentin, a known hyper-elastic biomolecule. Lastly, anoikis-resistance assays and detection of PACCs in the blood of a patient with metastatic castrate-resistant prostate cancer using a selection- free circulating tumor cell detection platform reveal that PACCs are capable of surviving in the circulation. Taken together with the knowledge that PACCs are capable of eventual depolyploidization and progeny formation (as a potential route to colonization), these data support PACCs as candidate metastasis-competent cells worthy of further analysis.

Active fluid transport across epithelial monolayers is emerging as a major driving force of tissue morphogenesis in a variety of healthy and diseased systems, as well as during embryonic development. Cells use directional transport of ions and osmotic gradients to drive fluid flow across the cell surface, in the process also building up fluid pressure. The basic physics of this process is described by the osmotic engine model, which also underlies actin-independent cell migration. Recently, the trans-epithelial fluid flux and the hydraulic pressure gradient have been explicitly measured for a variety of cellular and tissue model systems across various species. For the kidney, it was shown that tubular epithelial cells behave as active mechanical fluid pumps: the trans-epithelial fluid flux depends on the hydraulic pressure difference across the epithelial layer. When a stall pressure is reached, the fluid flux vanishes. Hydraulic forces generated from active fluid pumping are important in tissue morphogenesis and homeostasis, and could also underlie multiple morphogenic events seen in other developmental contexts. In this review, we highlight findings that examined the role of trans-epithelial fluid flux and hydraulic pressure gradient in driving tissue-scale morphogenesis. We also review organ pathophysiology due to impaired fluid pumping and the loss of hydraulic pressure sensing at the cellular scale. Finally, we draw an analogy between cellular fluidic pumps and a connected network of water pumps in a city. The dynamics of fluid transport in an active and adaptive network is determined globally at the systemic level, and transport in such a network is best when each pump is operating at its optimal efficiency.

Migration of meiosis-I (MI) spindle from the cell center to a sub-cortical location is a critical step for mouse oocytes to undergo asymmetric meiotic cell division. In this study, we investigate the mechanism by which formin-2 (FMN2) orchestrates the initial movement of MI spindle. By defining protein domains responsible for targeting FMN2, we show that spindle-periphery localized FMN2 is required for spindle migration. The spindle-peripheral FMN2 nucleates short actin bundles from vesicles derived likely from the endoplasmic reticulum (ER) and concentrated in a layer outside the spindle. This layer is in turn surrounded by mitochondria. A model based on polymerizing actin filaments pushing against mitochondria, thus generating a counter force on the spindle, demonstrated an inherent ability of this system to break symmetry and evolve directional spindle motion. The model is further supported through experiments involving spatially biasing actin nucleation via optogenetics and disruption of mitochondrial distribution and dynamics., Mammalian oocytes divide asymmetrically during meiotic maturation. Here, the authors show that spindle movement away from oocyte center depends on actin filaments nucleated from the spindle periphery pushing against surrounding mitochondria, which polarizes spontaneously to produce directional spindle motion.

Data de publicació electrònica: 02-11-2022 Cells respond to physical stimuli, such as stiffness1, fluid shear stress2 and hydraulic pressure3,4. Extracellular fluid viscosity is a key physical cue that varies under physiological and pathological conditions, such as cancer5. However, its influence on cancer biology and the mechanism by which cells sense and respond to changes in viscosity are unknown. Here we demonstrate that elevated viscosity counterintuitively increases the motility of various cell types on two-dimensional surfaces and in confinement, and increases cell dissemination from three-dimensional tumour spheroids. Increased mechanical loading imposed by elevated viscosity induces an actin-related protein 2/3 (ARP2/3)-complex-dependent dense actin network, which enhances Na+/H+ exchanger 1 (NHE1) polarization through its actin-binding partner ezrin. NHE1 promotes cell swelling and increased membrane tension, which, in turn, activates transient receptor potential cation vanilloid 4 (TRPV4) and mediates calcium influx, leading to increased RHOA-dependent cell contractility. The coordinated action of actin remodelling/dynamics, NHE1-mediated swelling and RHOA-based contractility facilitates enhanced motility at elevated viscosities. Breast cancer cells pre-exposed to elevated viscosity acquire TRPV4-dependent mechanical memory through transcriptional control of the Hippo pathway, leading to increased migration in zebrafish, extravasation in chick embryos and lung colonization in mice. Cumulatively, extracellular viscosity is a physical cue that regulates both short- and long-term cellular processes with pathophysiological relevance to cancer biology. This work was supported in part by R01 CA257647 (to K.K. and D.M.G.), R01 GM134542 (to S.X.S. and K.K.), NSF 2045715 (to Y.L.), R01 AR071976 (to C.-M.F. and J.T.), R01 AR072644 (to C.-M.F. and J.T.) and R01 CA054358 (to A.P.F.), the Spanish Ministry of Science, Education and Universities through grants RTI2018 099718-B-100 (to M.A.V.) and an institutional “Maria de Maeztu” Programme for Units of Excellence in R&D and FEDER funds (to M.A.V.), and postdoctoral fellowships from the Fonds de recherche du Quebec—Nature et technologies and the Natural Sciences and Engineering Research Council of Canada (to A.K.). The opinions, findings and conclusions, or recommendations expressed are those of the authors and do not necessarily reflect the views of any of the funding agencies.

Metastasis is responsible for 90% of cancer deaths, yet on a cellular level, successful metastasis is rare. Barriers to metastasis exist at every step of the metastatic cascade (invasion, intravasation, survival in the circulation, extravasation, and colonization.) Modeling predicts that only one of every 1.5 billion circulating tumor cells results in clinically-detectable metastasis. Identification of the rare subset of metastasis-competent cells is the most crucial goal of cancer research. Polyanuploid Cancer Cells (PACCs) are physically enlarged, treatment-resistant cells with increased genomic content that form in response to stress. After stress-induction, PACCs exist in a non-proliferative state for many weeks before undergoing eventual depolyploidization to produce progeny of “typical” cancer cell size, morphology, and nuclear content. PACCs can be identified in both primary and metastatic patient tumor tissues. As such, we propose Polyaneuploid Cancer Cells (PACCs) are metastasis-competent cells. Single-cell tracking reveals that PACCs are more motile than nonPACCs. Additionally, we observe that PACCs exhibit increased directional migration in 2D chemotactic environments. Optical-tracking of spontaneous bead motion reveals that PACCs demonstrate increased cytoskeletal rearrangement, an observation that aligns with increased environment-sensing and directional motility. In total, this predicts successful invasion. Analyses of cellular deformability using Magnetic Twisting Cytometry and Atomic Force Microscopy jointly reveal that cells in the PACC state display hyper-elastic properties. Among these include increased peripheral deformability and maintained peri-nuclear cortical integrity, both of which predict successful intravasation and extravasation. Functional deformability of PACCs navigating through narrow channels in a chemotactic environment was confirmed using a custom microfluids device. RTqPCR, NanoString mRNA quantification, Western blot, and immunofluorescent imaging reveal that PACCs highly overexpress Vimentin, a cytoskeletal component known to confer hyper-elasticity. Notably, there is no correlation between Vimentin content and motility dynamics in PACCs, indicating that the role of Vimentin in PACCs may primarily drive increased hyper-elasticity rather than increased motility. Anoikis-resistance assays and detection of PACCs in the blood of a patient with metastatic prostate cancer using a selection-free circulating tumor cell detection platform reveal that PACCs are capable of surviving in the circulation. Our work to date reports that PACCs demonstrate increased motility, environment-sensing, hyper-elasticity, and anoikis-resistance. Taken together with the knowledge that PACCs exist in a treatment-resistant state and are capable of eventual depolyploidization (as a potential route to successful colony formation), this data suggests PACCs are a candidate rare metastasis-competent cell type worthy of further analysis. Citation Format: Mikaela M. Mallin, Nicholas Kim, Mohammad Ikbal Choudhury, SeJong Lee, Steven S. An, Sean X. Sun, Konstantinos Konstantopoulos, Sarah R. Amend, Kenneth J. Pienta. Polyaneuploid Cancer Cells (PACCs) as metastasis-competent cells [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A017.

A method of microscale pressure measurement based on immiscible fluid/fluid interface is proposed. This method utilizes observed curvature changes in a fluid/fluid interface, and can accurately report hydraulic pressure in fluids at length scales of 10 microns. The method is especially suited for measuring fluid pressure in micro-scale biological samples. Using this method, we probe fluid pressure build up in epithelial domes, murine mammary gland organoids embedded in hydrogel, and lumen pressure in the developing mouse embryo. Results reveal that the pressure developed across epithelial barriers is on the order of 100~300 Pa, and is modulated by ion channel activity.

Significance Cell migration requires energy, but the metabolic cost of migration has not been quantitatively explored in detail. Here, we use a 2-phase model of the cell cytoplasm to compute cell velocities and energy efficiencies during cell movement. This model predicts that actin polymerization-driven migration is very inefficient in high-hydraulic-resistance environments. Instead, cells can adopt the water-driven mechanism. Therefore, the energetics and mechanical efficiencies of cell movement are predicted to depend on the physical environment., In this work, we explore fundamental energy requirements during mammalian cell movement. Starting with the conservation of mass and momentum for the cell cytosol and the actin-network phase, we develop useful identities that compute dissipated energies during extensions of the cell boundary. We analyze 2 complementary mechanisms of cell movement: actin-driven and water-driven. The former mechanism occurs on 2-dimensional cell-culture substrate without appreciable external hydraulic resistance, while the latter mechanism is prominent in confined channels where external hydraulic resistance is high. By considering various forms of energy input and dissipation, we find that the water-driven cell-migration mechanism is inefficient and requires more energy. However, in environments with sufficiently high hydraulic resistance, the efficiency of actin-polymerization-driven cell migration decreases considerably, and the water-based mechanism becomes more efficient. Hence, the most efficient way for cells to move depends on the physical environment. This work can be extended to higher dimensions and has implication for understanding energetics of morphogenesis in early embryonic development and cancer-cell metastasis and provides a physical basis for understanding changing metabolic requirements for cell movement in different conditions.

The nucleus is a significant obstacle that impedes migration of cells through confining microenvironments. Mistriotis et al. propose a conceptual model in which confinement-induced RhoA/myosin-II activation promotes nuclear volume expansion, nuclear envelope blebbing, and rupture by triggering passive nuclear influx from the cell posterior, ultimately leading to reduced cell motility., Cells migrate in vivo through complex confining microenvironments, which induce significant nuclear deformation that may lead to nuclear blebbing and nuclear envelope rupture. While actomyosin contractility has been implicated in regulating nuclear envelope integrity, the exact mechanism remains unknown. Here, we argue that confinement-induced activation of RhoA/myosin-II contractility, coupled with LINC complex-dependent nuclear anchoring at the cell posterior, locally increases cytoplasmic pressure and promotes passive influx of cytoplasmic constituents into the nucleus without altering nuclear efflux. Elevated nuclear influx is accompanied by nuclear volume expansion, blebbing, and rupture, ultimately resulting in reduced cell motility. Moreover, inhibition of nuclear efflux is sufficient to increase nuclear volume and blebbing on two-dimensional surfaces, and acts synergistically with RhoA/myosin-II contractility to further augment blebbing in confinement. Cumulatively, confinement regulates nuclear size, nuclear integrity, and cell motility by perturbing nuclear flux homeostasis via a RhoA-dependent pathway.

Skeletal muscles can regenerate throughout life time from resident Pax7-expressing (Pax7+) muscle stem cells (MuSCs)1–3. Pax7+ MuSCs are normally quiescent and localized at a niche in which they are attached to the extracellular matrix basally and compressed against the myofiber apically3–5. Upon muscle injury, MuSCs lose apical contact with the myofiber and re-enter cell cycle to initiate regeneration. Prior studies on the physical niche of MuSCs focused on basal elasticity6,7, and significance of the apical force exerted on MuSCs remains unaddressed. Here we simulate MuSCs’ mechanical environment in vivo by applying physical compression to MuSCs’ apical surface. We demonstrate that compression drives activated MuSCs back to a quiescent stem cell state, even when seeded on different basal elasticities. By mathematical modeling and manipulating cell tension, we conclude that low overall tension combined with high edge tension generated by compression lead to MuSC quiescence. We further show that apical compression results in up-regulation of Notch downstream genes, accompanied by increased levels of nuclear Notch. The compression-induced nuclear Notch is ligand-independent, as it does not require the canonical S2-cleavage of Notch by ADAM10/17. Our results fill the knowledge gap on the role of apical tension for MuSC fate. Implications to how stem cell fate and activity are interlocked with the mechanical integrity of its resident tissue are discussed.

Multicellular organization with precise spatial definition is an essential step in a wide range of biological processes, including morphogenesis, development, and healing. Gradients and patterns of chemoattractants are well-described guides of multicellular organization, but the influences of three-dimensional geometry of soft hydrogels on multicellular organization are less well defined. Here, we report the discovery of a new mode of self-organization of endothelial cells in ring-like patterns on the perimeters of hydrogel microwells that is independent of protein or chemical patterning and is driven only by geometry and substrate stiffness. We observe quantitatively striking influences of both the microwell aspect ratio (ε = perimeter/depth) and the hydrogel modulus. We systematically investigate the physical factors of cells and substrates that drive this multicellular behavior and present a mathematical model that explains the multicellular organization based upon balancing extracellular and cytoskeletal forces. These forces are determined in part by substrate stiffness, geometry, and cell density. The force balance model predicts the direction and distance of translational cell migration based on the dynamic interaction between tangential cytoskeletal tension and cell-cell and cell-substrate adhesion. We further show that the experimental observations can be leveraged to drive customized multicellular self-organization. Our observation of this multicellular behavior demonstrates the importance of the combinatorial effects of geometry and stiffness in complex biological processes. It also provides a new methodology for direction of cell organization that may facilitate the engineering of bionics and integrated model organoid systems.

The role of mechanical forces driving kidney epithelial fluid transport and morphogenesis in kidney diseases is unclear. Here, using a microfluidic platform to recapitulate fluid transport activity of kidney cells, we report that renal epithelial cells can actively generate hydraulic pressure gradients across the epithelium. The fluidic flux declines with increasing hydraulic pressure until a stall pressure, in a manner similar to mechanical fluid pumps. For normal human kidney cells, the fluidic flux is from apical to basal, and the pressure is higher on the basal side. For human Autosomal Dominant Polycystic Kidney Disease cells, the fluidic flux is reversed from basal to apical. Molecular and proteomic studies reveal that renal epithelial cells are sensitive to hydraulic pressure gradients, changing gene expression profiles and spatial arrangements of ion exchangers and the cytoskeleton in different pressure conditions. These results implicate mechanical force and hydraulic pressure as important variables during kidney function and morphological change, and provide insights into pathophysiological mechanisms underlying the development and transduction of hydraulic pressure gradients.

In eukaryotes, the cell volume is observed to be strongly correlated with the nuclear volume. The slope of this correlation depends on the cell type, growth condition, and the physical environment of the cell. We develop a computational model of cell growth and proteome increase, incorporating the kinetics of amino acid import, protein/ribosome synthesis and degradation, and active transport of proteins between the cytoplasm and the nucleoplasm. We also include a simple model of ribosome biogenesis and assembly. Results show that the cell volume is tightly correlated with the nuclear volume, and the cytoplasm-nucleoplasm transport rates strongly influences the cell growth rate as well as the cytoplasm/nucleoplasm ratio. Ribosome assembly and the ratio of ribosomal proteins to mature ribosomes also influence the cell volume and the cell growth rate. We find that in order to regulate the cell growth rate and the cytoplasm/nucleoplasm ratio, the cell must optimally control groups of kinetic parameters together, which could explain the quantitative roles of canonical growth pathways. Finally, using an extension of our model and single cell RNAseq data, it is possible to construct a detailed proteome distribution, provided that a quantitative cell division mechanism is known.Author summaryWe develop computational model of cell proteome increase and cell growth to compute the cell volume to nuclear volume ratio. The model incorporates essential kinetics of protein and ribosome synthesis/degradation, and their transport across the nuclear envelope. The model also incorporates ribosome biogenesis and assembly. The model identifies the most important parameters in determining the cell growth rate and the nucleoplasm/cytoplasm ratio, and provides a computational starting point to construct the cell proteome based on the RNAseq data.

Using a novel microfluidic platform to recapitulate fluid transport activity of kidney cells, we report that renal epithelial cells can actively generate hydraulic pressure gradients across the epithelium. The fluidic flux declines with increasing hydraulic pressure until a stall pressure, at which the flux vanishes--in a manner similar to mechanical fluidic pumps. The developed pressure gradient translates to a force of 50-100 nanoNewtons per cell. For normal human kidney cells, the fluidic flux is from apical to basal, and the pressure is higher on the basal side. For human Autosomal Dominant Polycystic Kidney Disease (ADPKD) cells, the fluidic flux is reversed from basal to apical with a higher stall pressure. Molecular studies and proteomic analysis reveal that renal epithelial cells are sensitive to hydraulic pressure gradients, developing different expression profiles and spatial arrangements of ion exchangers and the cytoskeleton in different pressure conditions. These results, together with data from osmotic and pharmacological perturbations of fluidic pumping, implicate mechanical force and hydraulic pressure as important variables during morphological changes in epithelial tubules, and provide further insights into pathophysiological mechanisms underlying the development of high luminal pressure within renal cysts.

Using a microfluidic method, it was found that YAP and TAZ are novel regulators of single-cell size and act independently of mTOR. YAP also influences cell cytoplasmic pressure and acts together with cytoskeletal tension to influence cell cycle progression., How mammalian cells regulate their physical size is currently poorly understood, in part due to the difficulty in accurately quantifying cell volume in a high-throughput manner. Here, using the fluorescence exclusion method, we demonstrate that the mechanosensitive transcriptional regulators YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) are regulators of single-cell volume. The role of YAP/TAZ in volume regulation must go beyond its influence on total cell cycle duration or cell shape to explain the observed changes in volume. Moreover, for our experimental conditions, volume regulation by YAP/TAZ is independent of mTOR. Instead, we find that YAP/TAZ directly impacts the cell division volume, and YAP is involved in regulating intracellular cytoplasmic pressure. Based on the idea that YAP/TAZ is a mechanosensor, we find that inhibiting myosin assembly and cell tension slows cell cycle progression from G1 to S. These results suggest that YAP/TAZ may be modulating cell volume in combination with cytoskeletal tension during cell cycle progression.

Aneuploidy, which refers to unbalanced chromosome numbers, represents a class of genetic variation that is associated with cancer, birth defects and eukaryotic micro-organisms1–4. Whereas it is known that each aneuploid chromosome stoichiometry can give rise to a distinct pattern of gene expression and phenotypic profile4,5, it remains a fundamental question as to whether there are common cellular defects that are associated with aneuploidy. Here we show the existence in budding yeast of a common aneuploidy gene-expression signature that is suggestive of hypo-osmotic stress, using a strategy that enables the observation of common transcriptome changes of aneuploidy by averaging out karyotype-specific dosage effects in aneuploid yeast-cell populations with random and diverse chromosome stoichiometry. Consistently, aneuploid yeast exhibited increased plasma-membrane stress that led to impaired endocytosis, and this defect was also observed in aneuploid human cells. Thermodynamic modelling showed that hypo-osmotic-like stress is a general outcome of the proteome imbalance that is caused by aneuploidy, and also predicted a relationship between ploidy and cell size that was observed in yeast and aneuploid cancer cells. A genome-wide screen uncovered a general dependency of aneuploid cells on a pathway of ubiquitin-mediated endocytic recycling of nutrient transporters. Loss of this pathway, coupled with the endocytic defect inherent to aneuploidy, leads to a marked alteration of intracellular nutrient homeostasis. A common aneuploidy gene-expression signature is identified in yeast that is suggestive of hypo-osmotic stress, and which leads to cells that exhibit increased plasma-membrane stress and impaired endocytosis.

Extracellular matrices in animal tissue are hydrogels mostly made of collagen. In these matrices, collagen fibers are hierarchically assembled and cross-linked to form a porous and elastic material, through which migrating cells can move by either pushing through open matrix pores, or by actively digesting collagen fibers. The influence of matrix mechanical properties on cell behavior is well studied. Less attention has been focused on hydraulic properties of extracellular matrices, and how hydrodynamic flows in these porous hydrogels are influenced by matrix composition and architecture. Here we study the response of collagen hydrogels using rapid changes in the hydraulic pressure within a microfluidic device, and analyze the data using a poroelastic theory. Major poroelastic parameters can be obtained in a single experiment. Results show that depending on the density, porosity, and the degree of geometric confinement, moving micron-sized objects such as cells can experience substantially increased hydraulic resistance (by as much as 10(6) times) when compared to 2D environments. Therefore, in addition to properties such as mechanical stiffness, the fluidic environment of the cell is also likely to impact cell behavior.

Hydraulic resistance induces cell phenotype oscillations in confinement via the coupled dynamics of actin, myosin, and calcium., Cells penetrating into confinement undergo mesenchymal-to-amoeboid transition. The topographical features of the microenvironment expose cells to different hydraulic resistance levels. How cells respond to hydraulic resistance is unknown. We show that the cell phenotype shifts from amoeboid to mesenchymal upon increasing resistance. By combining automated morphological tracking and wavelet analysis along with fluorescence recovery after photobleaching (FRAP), we found an oscillatory phenotypic transition that cycles from blebbing to short, medium, and long actin network formation, and back to blebbing. Elevated hydraulic resistance promotes focal adhesion maturation and long actin filaments, thereby reducing the period required for amoeboid-to-mesenchymal transition. The period becomes independent of resistance upon blocking the mechanosensor TRPM7. Mathematical modeling links intracellular calcium oscillations with actomyosin turnover and force generation and recapitulates experimental data. We identify hydraulic resistance as a critical physical cue controlling cell phenotype and present an approach for connecting fluorescent signal fluctuations to morphological oscillations.

Mesoscale molecular assemblies on the cell surface, such as cilia and filopodia, integrate information, control transport and amplify signals. Synthetic devices mimicking these structures could sensitively monitor these cellular functions and direct new ones. The challenges in creating such devices, however are that they must be integrated with cells in a precise kinetically controlled process and a device's structure and its precisely structured cell interface must then be maintained during active cellular function. Here we report the ability to integrate synthetic micro-scale filaments, DNA nanotubes, into a cell's architecture by anchoring them by their ends to specific receptors on the surfaces of mammalian cells. These filaments can act as shear stress meters: how anchored nanotubes bend at the cell surface quantitatively indicates the magnitude of shear stresses between 0-2 dyn per cm2, a regime important for cell signaling. Nanotubes can also grow while anchored to cells, thus acting as dynamic components of cells. This approach to cell surface engineering, in which synthetic biomolecular assemblies are organized within existing cellular architecture, could make it possible to build new types of sensors, machines and scaffolds that can interface with, control and measure properties of cells., 20 pages, 5 figures

All mammalian cells live in the aqueous medium, yet for many cell biologists, water is a passive arena in which proteins are the leading players that carry out essential biological functions. Recent studies, as well as decades of previous work, have accumulated evidence to show that this is not the complete picture. Active fluxes of water and solutes of water can play essential roles during cell shape changes, cell motility and tissue function, and can generate significant mechanical forces. Moreover, the extracellular resistance to water flow, known as the hydraulic resistance, and external hydraulic pressures are important mechanical modulators of cell polarization and motility. For the cell to maintain a consistent chemical environment in the cytoplasm, there must exist an intricate molecular system that actively controls the cell water content as well as the cytoplasmic ionic content. This system is difficult to study and poorly understood, but ramifications of which may impact all aspects of cell biology from growth to metabolism to development. In this Review, we describe how mammalian cells maintain the cytoplasmic water content and how water flows across the cell surface to drive cell movement. The roles of mechanical forces and hydraulic pressure during water movement are explored.

Cells are remarkable machines capable of performing an exquisite range of functions, many of which depend crucially on the activity of molecular motors that generate forces. Recent experiments have shown that intracellular random movements are not solely thermal in nature but also arise from stochasticity in the forces from these molecular motors. Here we consider the effects of these nonthermal random forces. We show that stochastic motor force not only enhances diffusion but also leads to size-dependent transport of objects that depends on the local density of the cytoskeletal filaments on which motors operate. As a consequence, we find that objects that are larger than the mesh size of the cytoskeleton should be attracted to regions of high cytoskeletal density, while objects that are smaller than the mesh size will preferentially avoid these regions. These results suggest a mechanism for size-based organelle positioning and also suggest that motor-driven random forces can additionally enhance motor-driven transport.

Growing interest in renewable energy continues to motivate new work on microbial biohydrogen production and in particular utilizing Escherichia coli a well‐studied, facultative anaerobe. Here we characterize, for the first time the H2 production rate and capacity, of E coli isolates from the 50 000th generation of the Long‐Term Evolution Experiment. Under these reaction conditions, peak production rates near or above 5 mL per hour for 100 mL of LB media was established for the ancestral strains and batch efficiencies between 0.15 and 0.22 mL H2 produced per 1 mL lysogeny broth (LB) media were achieved. All 11 isolates studied, which had been aerobically cultured in minimal media since 1988, exhibited a decreased H2 production rate or capacity with many strains unable to grow under anaerobic conditions at all. The genomes of these strains have been sequenced and a preliminary analysis of the correlations between genotype and phenotype shows that mutations in gene ydjO are exclusively observed in the two isolates which produce H2, potentially suggesting a role for this gene in the maintenance of wild type metabolic pathways in the context of diverse mutational backgrounds. These results provide hints towards uncovering new genetic targets for the pursuit of bacterial strains with increased capacity for H2 production as well as a case study in speciation and the control of phenotypic switching.

Actin cytoskeletal asymmetry produces unbalanced hydrodynamic forces to drive spindle rotation during mouse meiotic division., Patterned cell divisions require a precisely oriented spindle that segregates chromosomes and determines the cytokinetic plane. In this study, we investigated how the meiotic spindle orients through an obligatory rotation during meiotic division in mouse oocytes. We show that spindle rotation occurs at the completion of chromosome segregation, whereby the separated chromosome clusters each define a cortical actomyosin domain that produces cytoplasmic streaming, resulting in hydrodynamic forces on the spindle. These forces are initially balanced but become unbalanced to drive spindle rotation. This force imbalance is associated with spontaneous symmetry breaking in the distribution of the Arp2/3 complex and myosin-II on the cortex, brought about by feedback loops comprising Ran guanosine triphosphatase signaling, Arp2/3 complex activity, and myosin-II contractility. The torque produced by the unbalanced hydrodynamic forces, coupled with a pivot point at the spindle midzone cortical contract, constitutes a unique mechanical system for meiotic spindle rotation.

The measurement of single cell size remains an obstacle towards a deeper understanding of cell growth control, tissue homeostasis, organogenesis, and a wide range of pathologies. Recent advances have placed a spotlight on the importance of cell volume in the regulation of fundamental cell signaling pathways including those known to orchestrate progression through the cell cycle. Here we provide our protocol for the Fluorescence Exclusion Method (FXm); references to the development of FXm; and a brief outlook on future advances in image analysis which may expand the range of problems studied utilizing FXm as well as lower the barrier to entry for groups interested in adding cell volume measurements into their experimental repertoire.

In vivo, cells can reside in diverse physical and biochemical environments. For example, epithelial cells typically live in a two-dimensional (2D) environment while metastatic cancer cells can move through dense three-dimensional (3D) matrices. These distinct environments impose different kinds of mechanical forces on cells, and thus potentially can influence the mechanism of cell migration. For example, cell movement on 2D flat surfaces is mostly driven by forces from focal adhesion and actin polymerization, while in confined geometries, it can be driven by water permeation. In this work, we utilize a two-phase model of the cellular cytoplasm, where the mechanics of the cytosol and the F-actin network are treated on an equal footing. Using conservation laws and simple force balance considerations, we are able to describe the contribution of water flux, actin polymerization and flow, and focal adhesions to cell migration in both 2D surfaces and in confined spaces. The theory shows how cell migration can seamlessly transition from a focal adhesion- and actin-based mechanism on 2D surfaces to a water-based mechanism in confined geometries.

Cell volume regulation is fundamentally important in phenomena such as cell growth, proliferation, tissue homeostasis, and embryogenesis. How the cell size is set, maintained, and changed over a cell's lifetime is not well understood. In this work we focus on how the volume of nonexcitable tissue cells is coupled to the cell membrane electrical potential and the concentrations of membrane-permeable ions in the cell environment. Specifically, we demonstrate that a sudden cell depolarization using the whole-cell patch clamp results in a 50% increase in cell volume, whereas hyperpolarization results in a slight volume decrease. We find that cell volume can be partially controlled by changing the chloride or the sodium/potassium concentrations in the extracellular environment while maintaining a constant external osmotic pressure. Depletion of external chloride leads to a volume decrease in suspended HN31 cells. Introducing cells to a high-potassium solution causes volume increase up to 50%. Cell volume is also influenced by cortical tension: actin depolymerization leads to cell volume increase. We present an electrophysiology model of water dynamics driven by changes in membrane potential and the concentrations of permeable ions in the cells surrounding. The model quantitatively predicts that the cell volume is directly proportional to the intracellular protein content.

Cell density organizes collective migration within an epithelium. Computational models predict the regulation of collective migration in an in vivo epithelium and demonstrate how commonly altered cellular properties can prime groups of cells to adopt migration patterns that may be harnessed in health or exploited in disease., The mechanisms underlying collective migration are important for understanding development, wound healing, and tumor invasion. Here we focus on cell density to determine its role in collective migration. Our findings show that increasing cell density, as might be seen in cancer, transforms groups from broad collectives to small, narrow streams. Conversely, diminishing cell density, as might occur at a wound front, leads to large, broad collectives with a distinct leader–follower structure. Simulations identify force-sensitive contractility as a mediator of how density affects collectives, and guided by this prediction, we find that the baseline state of contractility can enhance or reduce organization. Finally, we test predictions from these data in an in vivo epithelium by using genetic manipulations to drive collective motion between predicted migratory phases. This work demonstrates how commonly altered cellular properties can prime groups of cells to adopt migration patterns that may be harnessed in health or exploited in disease.

Measuring the physical size of the cell is valuable in understanding cell growth control. Current single-cell volume measurement methods for mammalian cells are labor-intensive, inflexible, and can cause cell damage. We introduce CTRL: Cell Topography Reconstruction Learner, a label-free technique incorporating Deep Learning and Fluorescence Exclusion for reconstructing cell topography and estimating mammalian cell volume from DIC microscopy images alone. The method achieves quantitative accuracy, requires minimal sample preparation, and applies to extensive biological and experimental conditions. Using this method, we observe a noticeable reduction in cell size fluctuations during cell cycle, which is consistent with the presence of a cell size checkpoint. (https://GitHub.com/sxslabjhu/CTRL)

Convolutional neural networks (ConvNets) have proven to be successful in both the classification and semantic segmentation of cell images. Here we establish a method for cell type classification utilizing images taken with a benchtop microscope directly from cell culture flasks, eliminating the need for a dedicated imaging platform. Significant flask-to-flask morphological heterogeneity was discovered and overcome to support network generalization to novel data. Cell density was found to be a prominent source of heterogeneity even when cells are not in contact. For the same cell types, expert classification was poor for single-cell images and better for multi-cell images, suggesting experts rely on the identification of characteristic phenotypes within subsets of each population. We also introduce Self-Label Clustering (SLC), an unsupervised clustering method relying on feature extraction from the hidden layers of a ConvNet, capable of cellular morphological phenotyping. This clustering approach is able to identify distinct morphological phenotypes within a cell type, some of which are observed to be cell density dependent. Finally, our cell classification algorithm was able to accurately identify cells in mixed populations, showing that ConvNet cell type classification can be a label-free alternative to traditional cell sorting and identification.

Convolutional neural networks (ConvNets) have been used for both classification and semantic segmentation of cellular images. Here we establish a method for cell type classification utilizing images taken on a benchtop microscope directly from cell culture flasks eliminating the need for a dedicated imaging platform. Significant flask-to-flask heterogeneity was discovered and overcome to support network generalization to novel data. Cell density was found to be a prominent source of heterogeneity even within the single-cell regime indicating the presence of morphological effects due to diffusion-mediated cell-cell interaction. Expert classification was poor for single-cell images and excellent for multi-cell images suggesting experts rely on the identification of characteristic phenotypes within subsets of each population and not ubiquitous identifiers. Finally we introduce Self-Label Clustering, an unsupervised clustering method relying on ConvNet feature extraction able to identify distinct morphological phenotypes within a cell type, some of which are observed to be cell density dependent.Author summaryK.Y., N.D.R., and S.X.S. designed experiments and computational analysis. K.Y. performed experiments and ConvNets design/training, K.Y., N.D.R and S.X.S wrote the paper.

How mammalian cells regulate their physical size is currently poorly understood, in part due to the difficulty of accurately quantifying cell volume in a high throughput manner. Here, using the fluorescence exclusion method, we demonstrate that the mechanosensitive transcriptional regulators YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) are novel regulators of single cell volume. We report that the role of YAP/TAZ in cell volume regulation must go beyond its influence on total cell cycle duration or the cell shape to explain the observed changes in volume. Moreover, for our experimental conditions, volume regulation by YAP/TAZ is independent of mTOR. Instead, we find YAP/TAZ directly impacts the cell division volume. Based on the idea that YAP/TAZ is a mechanosensor, we find that inhibiting the assembly of myosin and cell tension slows cell cycle progression from G1 to S. These results suggest that YAP/TAZ and the Hippo pathway may be modulating cell volume in combination with cytoskeletal tension during cell cycle progression.

Quantitative single cell measurements have shown that cell cycle duration (the time between cell divisions) for diverse cell types is a noisy variable. The underlying distribution is mean scalable with a universal shape for many cell types in a variety of environments. Here we show through both experiment and theory that increasing the amount of noise in the regulation of the cell cycle negatively impacts the growth rate but positively correlates with improved cellular response to fluctuating environments. Our findings suggest that even non-cooperative cells in exponential growth phase do not optimize fitness through growth rate alone, but also optimize adaptability to changing conditions. In a manner similar to genetic evolution, increasing the noise in biochemical processes correlates with improved response of the system to environmental changes., 5 pages, 4 figures