Lamins: The backbone of the nucleocytoskeleton interface.

The nuclear lamina (NL) is a crucial component of the inner nuclear membrane (INM) and consists of lamin filaments and associated proteins. Lamins are type V intermediate filament proteins essential for maintaining the integrity and mechanical properties of the nucleus. In human cells, 'B-type' lamins (lamin B1 and lamin B2) are ubiquitously expressed, while 'A-type' lamins (lamin A, lamin C, and minor isoforms) are expressed in a tissue- and development-specific manner. Lamins homopolymerize to form filaments that localize primarily near the INM, but A-type lamins also localize to and function in the nucleoplasm. Lamins play central roles in the assembly, structure, positioning, and mechanics of the nucleus, modulating cell signaling and influencing development, differentiation, and other activities. This review highlights recent findings on the structure and regulation of lamin filaments, providing insights into their multifaceted functions, including their role as "mechanosensors", delving into the emerging significance of lamin filaments as vital links between cytoskeletal and nuclear structures, chromatin organization, and the genome.

Differential effects of aneuploidy on growth and differentiation in human intestinal stem cells.

Aneuploidy, a near ubiquitous genetic feature of tumors, is a context-dependent driver of cancer evolution; however, the mechanistic basis of this role remains unclear. Here, by inducing heterogeneous aneuploidy in non-transformed human colon organoids (colonoids), we investigate how the effects of aneuploidy on cell growth and differentiation may promote malignant transformation. Single-cell RNA sequencing reveals that the gene expression signature across over 100 unique aneuploid karyotypes is enriched with p53 responsive genes. The primary driver of p53 activation is karyotype complexity. Complex aneuploid cells with multiple unbalanced chromosomes activate p53 and undergo G1 cell-cycle arrest, independent of DNA damage and without evidence of senescence. By contrast, simple aneuploid cells with 1-3 chromosomes gained or lost continue to proliferate, demonstrated by single cell tracking in colonoids. Notably, simple aneuploid cells exhibit impaired differentiation when niche factors are withdrawn. These findings suggest that while complex aneuploid cells are eliminated from the normal epithelium due to p53 activation, simple aneuploid cells can escape this checkpoint and may contribute to niche factor-independent growth of cancer-initiating cells.

Cytoskeletal activation of NHE1 regulates cell volume and DNA methylation.

The regulation of mammalian cell volume is crucial for maintaining key cellular processes. Cells can rapidly respond to osmotic and hydrostatic pressure imbalances during environmental challenges, generating fluxes of water and ions that alter volume within minutes. While the role of ion pump and leak in cell volume regulation has been well-established, the role of the actomyosin cytoskeleton and its substantial interplay with ion transporters are still unclear. In this work, we discover a system of cell volume regulation controlled by cytoskeletal activation of ion transporters. Under hypotonic shock, NIH-3T3 and MCF-10A display a 20% secondary volume increase (SVI) following the initial regulatory volume decrease. We show that SVI is initiated by Ca 2+ influx through stretch activated channel Piezo1 and subsequent actomyosin remodeling. Rather than contracting cells, actomyosin triggers cell swelling by activating Na + -H + exchanger 1 (NHE1) through their co-binding partner ezrin. Cytoskeletal activation of NHE1 can be similarly triggered by mechanical stretch and attenuated by soft substrates. This mechanism is absent in certain cancer cell lines such as HT1080 and MDA-MB-231, where volume regulation is dominated by intrinsic response of ion transporters. Moreover, cytoskeletal activation of NHE1 during SVI induces nuclear deformation, leading to DNA demethylation and a significant, immediate transcriptomic response in 3T3 cells, a phenomenon that is absent in HT1080 cells. Overall, our findings reveal the central role of Ca 2+ and actomyosin-mediated mechanosensation in the regulation of ion transport, cell volume, DNA methylation, and transcriptomics.

Mechano-induced homotypic patterned domain formation by monocytes.

Matrix stiffness and corresponding mechano-signaling play indispensable roles in cellular phenotypes and functions. How tissue stiffness influences the behavior of monocytes, a major circulating leukocyte of the innate system, and how it may promote the emergence of collective cell behavior is less understood. Here, using tunable collagen-coated hydrogels of physiological stiffness, we show that human primary monocytes undergo a dynamic local phase separation to form highly patterned multicellular multi-layered domains on soft matrix. Local activation of the ß2 integrin initiates inter-cellular adhesion, while global soluble inhibitory factors maintain the steady-state domain pattern over days. Patterned domain formation generated by monocytes is unique among other key immune cells, including macrophages, B cells, T cells, and NK cells. While inhibiting their phagocytic capability, domain formation promotes monocytes' survival. We develop a computational model based on the Cahn-Hilliard equation, which includes combined local activation and global inhibition mechanisms of intercellular adhesion suggested by our experiments, and provides experimentally validated predictions of the role of seeding density and both chemotactic and random cell migration on pattern formation.

The influence of polarized membrane ion carriers and extracellular electrical/pH gradients on cell ionic homeostasis and locomotion.

Anisotropic environmental signals or polarized membrane ion/solute carriers can generate spatially-varying intracellular gradients, leading to polarized cell dynamics. For example, directional migration of neutrophils, galvanotaxis of glioblastoma, and water flux in kidney cells, all result from the polarized distribution of membrane ion carriers and other intracellular components. The underlying physical mechanisms behind how polarized ion carriers interact with environmental signals are not well studied. Here, we use a physiologically-relevant, physics-based mathematical model to reveal how ion carriers generate intracellular ionic and voltage gradients. The model is able to discern the contribution of individual ion carriers to the intracellular pH gradient, electric potential, and water flux. We discover that an extracellular pH gradient leads to an intracellular pH gradient via chloride-bicarbonate exchangers, whereas an extracellular electric field leads to an intracellular electric potential gradient via passive potassium channels. In addition, the mechanical-biochemical coupling can modulate actin distribution and flow, and create biphasic dependence of the cell speed on water flux. Moreover, we find that F-actin interaction with NHE alone can generate cell movement, even when other ion carriers are not polarized. Taken together, the model shows the importance of cell ion dynamics in modulating cell migration and cytoskeletal dynamics.

Mechanics of cell-cell junctions.

Tissue cells in epithelial or endothelial monolayers are connected through cell-cell junctions, which are stabilized by transmembrane E-cadherin bonds and intracellular actin filaments. These bonds and junctions play a crucial role in maintaining the barrier function of epithelia and endothelia and are believed to transmit forces between cells. Additionally, E-cadherin bonds can impact the shape of cell-cell junctions. In this study, we develop a continuum mechanical model of the cell-cell junction by explicitly incorporating the cell membrane, distributions of E-cadherin bonds, cytoplasmic fluid pressure, and F-actin dynamics. The static force-balanced version of the model is able to analyze the influences of cell cortical tension, actin dynamics, and cytoplasmic pressure on the junction shape and E-cadherin bonds. Furthermore, an extended model that incorporates fluid flow, across the cell boundary as well as around the cell, is also examined. This model can couple cell-shape changes with cell cortical tension and fluid flow, and predicts the additional effect of fluid motion on cell-cell junction mechanics. Taken together, our models serve as an intermediate link between molecular-scale models of cell-junction molecules and cell-scale models of tissue and epithelia.

Cells in the polyaneuploid cancer cell (PACC) state have increased metastatic potential.

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. Cells in the PACC state are enlarged, endocycling (i.e. non-dividing) cells with increased genomic content that form in response to stress. Single-cell tracking using time lapse microscopy reveals that PACC state cells have increased motility. Additionally, cells in the PACC state 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 cells in the PACC state have increased expression of vimentin, a hyper-elastic biomolecule known to modulate biomechanical properties and induce mesenchymal-like motility. Taken together, these data indicate that cells in the PACC state have increased metastatic potential and are worthy of further in vivo analysis.

On the role of myosin-induced actin depolymerization during cell migration.

Mammalian cell migration in open spaces requires F-actin polymerization and myosin contraction. While many studies have focused on myosin's coupling to focal adhesion and stress fibers, the indirect effect of myosin contraction on cell migration through actin depolymerization is not well studied. In this work, we quantified how cell velocity and effective power output are influenced by the rate of actin depolymerization, which is affected by myosin contraction. In addition, we derived scaling laws to provide physical insights into cell migration. Model analysis shows that the cell migration velocity displays a biphasic dependence on the rate of actin depolymerization and myosin contraction. Our model further predicts that the effective cell energy output depends not only on the cell velocity but also on myosin contractility. The work has implications on in vivo processes such as immune response and cancer metastasis, where cells overcome barriers imposed by the physical environment.

Mechanical compression creates a quiescent muscle stem cell niche.

Tissue stem cell niches are regulated by their mechanical environment, notably the extracellular matrix (ECM). Skeletal muscles consist of bundled myofibers for force transmission. Within this macroscopic architecture, quiescent Pax7-expressing (Pax7+) muscle stem cells (MuSCs) are compressed between ECM basally and myofiber apically. Muscle injury causes MuSCs to lose apical compression from the myofiber and re-enter the cell cycle for regeneration. While ECM elasticities have been shown to affect MuSC's renewal, the significance of apical compression remains unknown. To investigate the role of apical compression, we simulate the MuSCs' in vivo mechanical environment by applying physical compression to MuSCs' apical surface. We demonstrate that compression drives activated MuSCs back to a quiescent stem cell state, regardless of basal elasticities and chemistries. By mathematical modeling and cell tension manipulation, we conclude that low overall tension combined with high axial tension generated by compression leads to MuSCs' stemness and quiescence. Unexpectedly, we discovered that apical compression results in up-regulation of Notch downstream genes, accompanied by the increased levels of nuclear Notch1&3 in a Delta ligand (Dll) and ADAM10/17 independent manner. Our results fill a knowledge gap on the role of apical compression for MuSC fate and have implications to stem cells in other tissues.

Extracellular fluid viscosity enhances cell migration and cancer dissemination.

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.

Polarized NHE1 and SWELL1 regulate migration direction, efficiency and metastasis.

Cell migration regulates diverse (patho)physiological processes, including cancer metastasis. According to the Osmotic Engine Model, polarization of NHE1 at the leading edge of confined cells facilitates water uptake, cell protrusion and motility. The physiological relevance of the Osmotic Engine Model and the identity of molecules mediating cell rear shrinkage remain elusive. Here, we demonstrate that NHE1 and SWELL1 preferentially polarize at the cell leading and trailing edges, respectively, mediate cell volume regulation, cell dissemination from spheroids and confined migration. SWELL1 polarization confers migration direction and efficiency, as predicted mathematically and determined experimentally via optogenetic spatiotemporal regulation. Optogenetic RhoA activation at the cell front triggers SWELL1 re-distribution and migration direction reversal in SWELL1-expressing, but not SWELL1-knockdown, cells. Efficient cell reversal also requires Cdc42, which controls NHE1 repolarization. Dual NHE1/SWELL1 knockdown inhibits breast cancer cell extravasation and metastasis in vivo, thereby illustrating the physiological significance of the Osmotic Engine Model.

The interplay between physical cues and mechanosensitive ion channels in cancer metastasis.

Physical cues have emerged as critical influencers of cell function during physiological processes, like development and organogenesis, and throughout pathological abnormalities, including cancer progression and fibrosis. While ion channels have been implicated in maintaining cellular homeostasis, their cell surface localization often places them among the first few molecules to sense external cues. Mechanosensitive ion channels (MICs) are especially important transducers of physical stimuli into biochemical signals. In this review, we describe how physical cues in the tumor microenvironment are sensed by MICs and contribute to cancer metastasis. First, we highlight mechanical perturbations, by both solid and fluid surroundings typically found in the tumor microenvironment and during critical stages of cancer cell dissemination from the primary tumor. Next, we describe how Piezo1/2 and transient receptor potential (TRP) channels respond to these physical cues to regulate cancer cell behavior during different stages of metastasis. We conclude by proposing alternative mechanisms of MIC activation that work in tandem with cytoskeletal components and other ion channels to bestow cells with the capacity to sense, respond and navigate through the surrounding microenvironment. Collectively, this review provides a perspective for devising treatment strategies against cancer by targeting MICs that sense aberrant physical characteristics during metastasis, the most lethal aspect of cancer.

Extracellular Hydraulic Resistance Enhances Cell Migration.

Cells migrating in vivo encounter microenvironments with varying physical properties. One such physical variable is the fluid viscosity surrounding the cell. Increased viscosity is expected to increase the hydraulic resistance experienced by the cell and decrease cell speed. The authors demonstrate that contrary to this expected result, cells migrate faster in high viscosity media on 2-dimensional substrates. Both actin dynamics and water dynamics driven by ion channel activity are examined. Results show that cells increase in area in high viscosity and actomyosin dynamics remain similar. Inhibiting ion channel fluxes in high viscosity media results in a large reduction in cell speed, suggesting that water flux contributes to the observed speed increase. Moreover, inhibiting actin-dependent vesicular trafficking that transports ion channels to the cell boundary changes ion channel spatial positioning and reduces cell speed in high viscosity media. Cells also display altered Ca2+ activity in high viscosity media, and when cytoplasmic Ca2+ is sequestered, cell speed reduction and altered ion channel positioning are observed. Taken together, it is found that the cytoplasmic actin-phase and water-phase are coupled to drive cell migration in high viscosity media, in agreement with physical modeling that also predicts the observed cell speedup in high viscosity environments.

Trans-epithelial fluid flow and mechanics of epithelial morphogenesis.

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.

Directing Multicellular Organization by Varying the Aspect Ratio of Soft Hydrogel Microwells.

Multicellular organization with precise spatial definition is essential to various biological processes, including morphogenesis, development, and healing in vascular and other tissues. Gradients and patterns of chemoattractants are well-described guides of multicellular organization, but the influences of 3D geometry of soft hydrogels are less well defined. Here, the discovery of a new mode of endothelial cell self-organization guided by combinatorial effects of stiffness and geometry, independent of protein or chemical patterning, is described. Endothelial cells in 2 kPa microwells are found to be ≈30 times more likely to migrate to the edge to organize in ring-like patterns than in stiff 35 kPa microwells. This organization is independent of curvature and significantly more pronounced in 2 kPa microwells with aspect ratio (perimeter/depth) < 25. Physical factors of cells and substrates that drive this behavior are systematically investigated and a mathematical model that explains the organization by balancing the dynamic interaction between tangential cytoskeletal tension, cell-cell, and cell-substrate adhesion is presented. These findings demonstrate the importance of combinatorial effects of geometry and stiffness in complex cellular organization that can be leveraged to facilitate the engineering of bionics and integrated model organoid systems with customized nutrient vascular networks.

Kidney epithelial cells are active mechano-biological fluid pumps.

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.

The correlation between cell and nucleus size is explained by an eukaryotic cell growth model.

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 influence the cell growth rate as well as the cell/nucleus volume ratio (C/N 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 cell/nucleus volume ratio, the cell must optimally control groups of kinetic and transport parameters together, which could explain the quantitative roles of canonical growth pathways. Finally, although not explicitly demonstrated in this work, we point out that it is possible to construct a detailed proteome distribution using our model and RNAseq data, provided that a quantitative cell division mechanism is known.

Hydrogen, Bicarbonate, and Their Associated Exchangers in Cell Volume Regulation.

Cells lacking a stiff cell wall, e.g., mammalian cells, must actively regulate their volume to maintain proper cell function. On the time scale that protein production is negligible, water flow in and out of the cell determines the cell volume variation. Water flux follows hydraulic and osmotic gradients; the latter is generated by various ion channels, transporters, and pumps in the cell membrane. Compared to the widely studied roles of sodium, potassium, and chloride in cell volume regulation, the effects of proton and bicarbonate are less understood. In this work, we use mathematical models to analyze how proton and bicarbonate, combined with sodium, potassium, chloride, and buffer species, regulate cell volume upon inhibition of ion channels, transporters, and pumps. The model includes several common, widely expressed ion transporters and focuses on obtaining generic outcomes. Results show that the intracellular osmolarity remains almost constant before and after cell volume change. The steady-state cell volume does not depend on water permeability. In addition, to ensure the stability of cell volume and ion concentrations, cells need to develop redundant mechanisms to maintain homeostasis, i.e., multiple ion channels or transporters are involved in the flux of the same ion species. These results provide insights for molecular mechanisms of cell volume regulation with additional implications for water-driven cell migration.

Hydraulic resistance induces cell phenotypic transition in confinement.

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.

The importance of water and hydraulic pressure in cell dynamics.

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.