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The nucleus measures shape changes for cellular proprioception to control dynamic cell behavior
Abstract
The physical microenvironment regulates cell behavior during tissue development and homeostasis. How single cells decode information about their geometrical shape under mechanical stress and physical space constraints within tissues remains largely unknown. Here, using a zebrafish model, we show that the nucleus, the biggest cellular organelle, functions as an elastic deformation gauge that enables cells to measure cell shape deformations. Inner nuclear membrane unfolding upon nucleus stretching
provides physical information on cellular shape changes and adaptively activates a calcium-dependent mechanotransduction pathway, controlling actomyosin contractility and migration plasticity. Our data support that the nucleus establishes a functional module for cellular proprioception that enables cells to sense shape variations for adapting cellular behavior to their microenvironment.
... The (micro)environment surrounding cells is fundamental to understanding the function of these cells. In recent years, the importance of the physical properties of the environment, which drive different biological processes, including proliferation(1), mobility (2), and differentiation (3)(4)(5)(6)(7)(8) is gaining special relevance. Changes in the physical properties of the tissues have been also related to the effects of aging (9) and disease (9,10). ...
... Most studies try to answer this question through the investigation of the changes in cytoskeleton and/or membrane components (11)(12)(13)(14)(15)(16)(17)(18). Interestingly, although the nucleus has been considered to play a major role in mechanical responses (2,17,(19)(20)(21)(22), the contribution of the chromatin organization to the differential responses to mechanical stress has been rarely addressed. Mostly, the nuclear stiffness has been characterized to be dependent on the laminA concentration (11) and its contribution related to the linker of nucleoskeleton and cytoskeleton (LINC) complex (23). ...
... Mechanoreception also relies on the mechanical properties of the nucleus for cellular proprioception and mechanotransduction. In proprioception, the physical stimuli applied to cells are only sensed upon nuclear deformation (2). In mechanotransduction, the import of key transcription factors to the nucleus is controlled by nuclear stiffness. ...
Cellular differentiation is driven by epigenetic modifiers and readers, including the methyl CpG binding protein 2 (MeCP2), whose level and mutations cause the neurological disorder Rett syndrome. During differentiation, most of the genome gets densely packed into heterochromatin, whose function has been simplistically viewed as gene silencing. However, gene expression changes reported in mutations leading to Rett syndrome have failed to be a predictor of disease severity. Here, we show that MeCP2 increases nuclear stiffness in a concentration dependent manner and dependent on its ability to cluster heterochromatin during differentiation. MeCP2-dependent stiffness increase could not be explained by changes in the expression of mechanobiology-related genes, but we found it is disrupted by Rett syndrome mutations and correlated with disease severity. Our results highlight the impact of chromatin organization in the mechanical properties of the cell as an alternative or complementary mechanism to changes in cytoskeleton components.
Graphical abstract
... Thus, the physical barriers imposed by 25 µm droplet OHGs are more prominent and the proportion of cells in contact with droplets is higher (Fig. 5g). Cell confinement has been found to be sensed via mechanosensitive ion channels and the deformation of the nucleus, causing reduced nuclear envelope wrinkles 6,29,30 . Indeed, OVCAR8 cells in contact with both 70 and 25 µm microdroplets showed reduced nuclear envelope wrinkles compared to cells embedded in collagen (Fig. 5h). ...
... Analysis of spheroids under TRPV4 inhibition revealed that cell invasion into OHGs is not blocked but, as shown previously 31,32 , cell proliferation is reduced (Extended data Fig. 15c-e). Next, we investigated whether the cytosolic phospholipase A2 (cPLA2)-Arachidonic acid-InsP3Rs-myosin II signalling cascade, involved in nuclear sensing of confinement, regulates OVCAR8 invasion into OHGs 29,30 . Inhibition of the signalling cascade did not cause major effects on OVCAR8 invasion in either 70 or 25 µm OHGs (Extended data Fig. 16a). ...
... Cells migrating at the adipocyte/microdroplet-ECM interface are under confinement, especially in small droplet OHGs. Although we observed cellular hallmarks of confinement such as nuclear deformations and nuclear envelop stretching, cells did not present enhanced DNA damage and the inhibition of confinement-related pathways had a minimal effect on invasiveness 29,30 . This suggests that confinement does not reach the threshold to induce these effects, possibly facilitated by the mechanical anisotropy of adipocytes/microdroplets (high compliance radially, but displaying high interfacial shear moduli), leading to the opening of migration tracks. ...
High-grade serous ovarian cancer, the most common and aggressive form of ovarian cancer, generally metastasises to visceral adipose tissues. In these tissues, the extracellular matrix through which ovarian cancer cells adhere and migrate is confined by the presence and preponderance of adipocytes. How cells migrate in this unique environment is not known, yet critical to understanding metastatic progression. To study these processes, we develop biomimetic organo-hydrogels that recreate structural and mechanical properties of human visceral adipose tissues. We show that ovarian cancer cells present invasive tropism towards organo-hydrogels, replicating the behaviour observed in native adipose tissues. This migration is facilitated by the mechanical anisotropy and microstructure of organo-hydrogels and adipose tissues, allowing the formation of cell force-induced migratory tracks, a process regulated by TGFβ in an MMP degradation-independent manner. These results highlight the contribution of adipocytes to tissue biophysical features as a key regulatory factor of ovarian cancer cell migration.
... In addition to the canonical differences between soft and stiff matrices in adhesion assembly and cytoskeletal organization, nuclear morphology is different on soft vs. stiff matrices [11][12][13][14][15] . Because nuclear deformations can induce secondary effects in certain contexts, such as alterations in chromatin conformation [16][17][18][19] , nucleoplasmic shuttling of transcription factors 20,21 , and signaling 22,23 , it is important to understand how mechanical cues from the extracellular matrix (ECM) modulate nuclear morphology. ...
... The pressure is posited to develop through osmotic pressure in the nucleus 37,38 that resists volume compression 39 , and entropic pressure from chromatin 40 . Because the lamina is molecularly coupled with the nuclear envelope, the tension in the lamina may in turn contribute to the regulation of nuclear transport 20 , trigger signaling through a stretch in the nuclear envelope 22,23 , or cause nuclear envelope rupture 41,42 . ...
... The threshold-like mechanical behavior of the nuclear lamina, where tension is supported in the lamina only upon laminar smoothing, is reminiscent of the threshold behavior of the wrinkled nuclear membrane observed in experiments involving compression of cells in microdevices 22,23 . In these experiments, the nuclear envelope became unwrinkled as the nucleus was compressed, resulting ultimately in a membrane strain that opened membrane channels with eventual signaling that increased actomyosin contractility. ...
Extracellular matrix (ECM) stiffness influences cancer cell fate by altering gene expression. Previous studies suggest that stiffness-induced nuclear deformation may regulate gene expression through YAP nuclear localization. We investigated the role of the nuclear lamina in this process. We show that the nuclear lamina exhibits mechanical threshold behavior: once unwrinkled, the nuclear lamina is inextensible. A computational model predicts that the unwrinkled lamina is under tension, which is confirmed using a lamin tension sensor. Laminar unwrinkling is caused by nuclear flattening during cell spreading on stiff ECM. Knockdown of lamin A/C eliminates nuclear surface tension and decreases nuclear YAP localization. These findings show that nuclear deformation in cells conforms to the nuclear drop model and reveal a role for lamin A/C tension in controlling YAP localization in cancer cells.
... Using Lifeact-labelled MSCs, we observed that seconds-scale F-actin dynamics (5-s intervals) and cortical F-actin with dynamic protrusions were significantly enhanced in SG (Fig. 2b-f and only recently gained attention for its function in activating mechanotransduction pathways that modulate cell fates. For example, when stretched under confinement, the nucleus can enhance cortical actomyosin contractility and help cells migrate through restrictive environments 12,13 . ...
... Although the nucleus has been traditionally studied as a storehouse of genetic material, emerging evidence suggests that it is also a mechanosensory element that can sense mechanical forces in cells under confinement [10][11][12][13] . Therefore, we characterized nuclear dynamics by performing live-cell imaging by labelling the nuclei with a live nucleus stain (5-min intervals). ...
... 12,13,37,38 . This process can occur in a rapid seconds-to-minute timescale12 . ...
Cells can deform their local niche in three dimensions via whole-cell movements such as spreading, migration or volume expansion. These behaviours, occurring over hours to days, influence long-term cell fates including differentiation. Here we report a whole-cell movement that occurs in sliding hydrogels at the minutes timescale, termed cell tumbling, characterized by three-dimensional cell dynamics and hydrogel deformation elicited by heightened seconds-to-minutes-scale cytoskeletal and nuclear activity. Studies inhibiting or promoting the cell tumbling of mesenchymal stem cells show that this behaviour enhances differentiation into chondrocytes. Further, it is associated with a decrease in global chromatin accessibility, which is required for enhanced differentiation. Cell tumbling also occurs during differentiation into other lineages and its differentiation-enhancing effects are validated in various hydrogel platforms. Our results establish that cell tumbling is an additional regulator of stem cell differentiation, mediated by rapid niche deformation and nuclear mechanotransduction.
... Whether and how these forces contribute to the T-cells' outstanding sensitivity in antigen discrimination is still under debate 6 . Recent studies suggest, that the nucleus itself can act as a force sensor at the cellular level 7,8 . Leukocytes have been shown to discriminate pore-size using their nucleus as a mechanical gauge 9 , which helps them to determine their migration path of least resistance. ...
... They further provided evidence that microtubule dynamics are the critical cytoskeletal component involved in these processes. Confinement-induced cellular reflexes 7,8 were previously also shown for other cell types like mesenchymal cells 13 , immature mouse dendritic cells 7 or early zebrafish progenitor cells 49 . ...
Mechanical stimuli are an integral part to the natural cellular microenvironment, influencing cell growth, differentiation, and survival, particularly in mechanically challenging environments like tumors. These stimuli are also crucial in...
... Additionally, mechanical forces have also been found to drive chromatin changes by reorganizing the epigenomic landscape through histone modifications [3,8,9], enabling cells to finely tune gene expression in response to external cues to maintain homeostasis [10,11]. The specific molecular mechanisms by which extracellular cues from the cells' periphery are transmitted to the nucleus to influence chromatin organisation, and how these aspects affects cell fate, however, remain poorly understood [3]. ...
... Alterations in nuclear morphology, particularly those involving nuclear lamins, have been reported to affect chromatin organization, leading to modifications in chromatin condensation and accessibility [6,8,10,39,40]. Considering the effects of the SRBW nanomechanostimulation on the cells, we assessed the distribution of H3K27me3 (Fig. 2a), a facultative heterochromatin marker mediated by the Polycomb repressive complex EZH2, and the constitutive heterochromatin marker H3K9me3 (Fig. 2b). The results show an initial slight downregulation of H3K27me3, which precede significant upregulation upon normalization of cytosolic Ca 2+ levels and elevation of cAMP during the sonotransformation phase at 0.5 hrs. ...
Cells effectively balance and integrate numerous pathways to adapt to external signals in an attempt to regain homeostasis, although the complex nuclear mechanotransduction mechanism through which this occurs is not as yet fully understood. Contrary to prevalent thought that the relay of extracellular cues to the nucleus to effect its fate and function predominantly relies on direct transmission through the cytoskeletal structure, we demonstrate, through the use of a unique form of high frequency (10 MHz) nanomechanostimulation, that manipulations to the cells' nuclear chromatin response are primarily influenced by the spatiotemporal dynamics associated with the bidirectional crosstalk between two key second messengers, namely calcium (Ca ²⁺ ) and cyclic adenosine monophosphate (cAMP). In stem cells, we show this conditioning, as an adaptive response to the mechanostimuli, to correlate with a 'mechanopriming' effect that is responsible for their early induction towards an osteogenic lineage in as little as three days with just brief (10 mins) daily bursts of mechanostimulation, without the need for osteogenic factors.
... The cell membrane plays an important role in conveying messages from the external environment of the cell to its nucleus and vice versa [34,35]. Mechanical pressures that produce changes in the cell shape are sensed by the nucleus to produce relevant alterations in cell behavior [34,35]. ...
... The cell membrane plays an important role in conveying messages from the external environment of the cell to its nucleus and vice versa [34,35]. Mechanical pressures that produce changes in the cell shape are sensed by the nucleus to produce relevant alterations in cell behavior [34,35]. Pressures exerted on the cell are conveyed to its nucleus, which serves as a ruler to tailor cell responses. ...
Renin plays a significant role in the regulation of blood pressure and fluid volume by modulating the renin‒angiotensin‒aldosterone (RAAS) system. Renin suppression reduces serum aldosterone levels and lowers blood pressure in addition to preserving renal function. However, exactly how renin synthesis and action are regulated and how renin suppression preserves renal function are not clear. We propose that arachidonic acid (AA) and its metabolites control renin synthesis, secretion, and action by virtue of its (AA) anti-inflammatory, cytoprotective actions and ability to regulate the secretion of renin. These findings suggest that direct renin suppression results in changes in AA metabolism. This proposal implies that AA and its metabolites may be developed as potential drugs to prevent and manage hypertension and preserve renal function.
... We ignore the other solution for the rest length as it is unstable. Through mechanosensitive mechanisms that are either based on the ion channel Piezo 1 44 or on nuclear shape changes and subsequent calcium release 45,46 , confinement, as in our channels, leads to an increased cortical contractility 17,18 ζ . As a result, the rest length decreases and thus the cell contracts (Fig. 6a, left). ...
Cells move directionally along gradients of substrate stiffness — a process called durotaxis. In the situations studied so far, durotaxis relies on cell-substrate focal adhesions to sense stiffness and transmit forces that drive directed motion. However, whether and how durotaxis can take place in the absence of focal adhesions remains unclear. Here, we show that confined cells can perform durotaxis despite lacking focal adhesions. This durotactic migration depends on an asymmetric myosin distribution and actomyosin retrograde flow. We propose that the mechanism of this focal adhesion-independent durotaxis is that stiffer substrates offer higher friction. We put forward a physical model that predicts that non-adherent cells polarise and migrate towards regions of higher friction — a process that we call frictiotaxis. We demonstrate frictiotaxis in experiments by showing that cells migrate up a friction gradient even when stiffness is uniform. Our results broaden the potential of durotaxis to guide any cell that contacts a substrate, and they reveal a mode of directed migration based on friction. These findings have implications for cell migration during development, immune response and cancer progression, which usually takes place in confined environments that favour adhesion-independent amoeboid migration.
... In another study, a trapped bead was pressed against the ER (in this case from outside of the cell) in order to trigger the release of Ca 2+ through mechanosensitive ion channels in the ER membrane, which was visualised using a genetically encoded ion reporter 75 . In contrast with AFM experiments, which do not allow a direct measurement of nuclear mechanics, indentation of the nucleus by OT has been recently developed in living cells to quantify the local viscoelastic properties of the nucleus in living mammalian cells 76,77 , and in zebrafish cells [78][79][80] . ...
Studying the physical properties of sub-cellular components is increasingly important in understanding cell mechanics. This review focuses on the most advanced techniques available for investigating intracellular mechanics. We distinguish methods that act as force generators and those that act as force sensors. We highlight six state-of-the-art techniques, with increased spatial and temporal resolutions: optogenetics, Brillouin microscopy, bacterial cells and nanorobots, optical tweezers, membrane tension probes, and magnetic particles.
... In practice, the ability to extract the "correct" features has been a bottleneck 16 . Traits commonly used by pathologists (e.g., cell type) are non-trivial to engineer, while histological presentation of novel discriminative features in cancers are often unintuitive due to tissue complexity and tumor variability, making them hard to formulate [17][18][19][20][21][22][23][24][25][26][27][28] . In contrast, deep learning (DL) algorithms, which bypass the task of feature engineering, have excelled on a wide range 9, 29-33 of histopathology-based predictions, matching human performance on traditional classification tasks and enabled predictions of mutation status, gene expression, molecular subtypes and treatment response [34][35][36] . ...
Deep learning (DL) algorithms have demonstrated remarkable proficiency in histopathology classification tasks, presenting an opportunity to discover disease [ndash]related features escaping visual inspection. However, the ″black box″ nature of DL obfuscates the basis of the classification. Here, we develop an algorithm for interpretable Deep Learning (IDL) that sheds light on the links between tissue morphology and cancer biology. We make use of a generative model trained to represent images via a combination of a semantic latent space and a noise vector to capture low level image details. We traversed the latent space so as to induce prototypical image changes associated with the disease state, which we identified via a second DL model. Applied to a dataset of clear cell renal cell carcinoma (ccRCC) tissue images the AI system pinpoints nuclear size and nucleolus density in tumor cells (but not other cell types) as the decisive features of tumor progression from grade 1 to grade 4 – rules that have been used for decades in the clinic and are taught in textbooks. Moreover, the AI system posits a decrease in vasculature with increasing grade. While the association has been illustrated by some, the correlation is not part of currently implemented grading systems. These results indicate the potential of IDL to autonomously formalize the connection between the histopathological presentation of a disease and the underlying tissue architectural drivers.
... Cells constantly sense and respond to mechanical inputs, and the interplay between nuclear deformation, morphology and chromatin organization emerges as a vital process underlying cell mechanoresponse 1,2 . Recent studies show that the nucleus can act as a "ruler" to measure cellular and nuclear shape variations originating from external compression, to interpret and respond to cues important for survival, movement and growth 3,4 . ...
Cells sense external physical cues through complex processes involving signaling pathways, cytoskeletal dynamics, and transcriptional regulation to coordinate a cellular response. A key emerging principle underlying such mechanoresponses is the interplay between nuclear morphology, chromatin organization, and the dynamic behavior of nuclear bodies such as HP1α condensates. Here, applying Airyscan super-resolution live cell imaging, we report a hitherto undescribed level of mechanoresponse triggered by cell confinement below their resting nuclear diameter, which elicits changes in the number, size and dynamics of HP1α nuclear condensates. Utilizing biophysical polymer models, we observe radial redistribution of HP1α condensates within the nucleus, influenced by changes in nuclear geometry. These insights shed new light on the complex relationship between external forces and changes in nuclear shape and chromatin organization in cell mechanoreception.
... 10 This, in turn, influences the nuclear shuttling of transcription factors (e.g., YAP) and ions (e.g., Ca²⁺), ultimately impacting cell functions. 11,12 In our previous study, we demonstrated that altering nuclear morphology using micropillar topography affects nuclear lamin A/C assembly, which, in turn, influences chromatin tethering, packing, and condensation. 13 These changes affect transcriptional accessibility and responsiveness, thereby regulating gene expression and stem cell differentiation. ...
Nuclear morphology, which modulates chromatin architecture, plays a critical role in regulating gene expression and cell functions. While most research has focused on the direct effects of nuclear morphology on cell fate, its impact on the cell secretome and surrounding cells remains largely unexplored, yet is especially crucial for cell-based therapies. In this study, we fabricated implants with a micropillar topography using methacrylated poly(octamethylene citrate)/hydroxyapatite (mPOC/HA) composites to investigate how micropillar-induced nuclear deformation influences cell paracrine signaling for osteogenesis and cranial bone regeneration. In vitro, cells with deformed nuclei showed enhanced secretion of proteins that support extracellular matrix (ECM) organization, which promoted osteogenic differentiation in neighboring human mesenchymal stromal cells (hMSCs). In a mouse model with critical-size cranial defects, nuclear-deformed hMSCs on micropillar mPOC/HA implants elevated Col1a2 expression, contributing to bone matrix formation, and drove cell differentiation toward osteogenic progenitor cells. These findings indicate that micropillars not only enhance the osteogenic differentiation of human mesenchymal stromal cells (hMSCs) but also modulate the secretome, thereby influencing the fate of surrounding cells through paracrine effects.
... This is in agreement with a recent study in NK cells showing MyoII-dependent cell contraction upon cell deformation in 3D microenvironments 49 . Additionally, MyoII activation upon confinement has been evidenced in other cell types [25][26][27]50 . In such systems, MyoII activity increased seconds upon cell confinement in a calcium-dependent manner. ...
As the first responders of the immune system, neutrophils rapidly reach inflamed tissues navigating through the blood circulation. In areas drained by blood capillaries, neutrophils might squeeze and migrate through tubes smaller than their own diameter where they can crawl independently of blood flow. However, the cellular mechanisms they use to migrate in such situations have not been elucidated. In this work, we investigated neutrophil migration in capillary tubes in vivo and in vitro. We found that neutrophil crawling speed at the site of infection was independent of the size of the capillaries they traversed in vivo. Detailed in vitro studies showed that these cells could maintain high speed in tubular microenvironments imposing confinement changes. Mechanistically, neutrophils adapted their actomyosin cytoskeleton to confinement within seconds, a process essential to maintain their high speed in capillaries of small diameter. Actomyosin reorganization was reversible and correlated with the degree of confinement. Importantly, we observed cytoskeleton adaptation to confinement in capillaries in vivo. However, other leukocytes failed to adapt their migration speed to a change in confinement strength. These findings reveal a unique mechanism that enables neutrophils to maintain their speed in blood capillaries, potentially contributing to the efficacy of innate immune reactions.
... to rapidly escape crowded tissue regions 5 . Recent evidence also unveiled polarization of inner membrane surface charge and of lipid composition during cell migration 7 . ...
Glioblastoma (GBM) is a malignant brain tumor with diffuse infiltration. Here, we demonstrate how GBM cells usurp guidance receptor Plexin-B2 for confined migration through restricted space. Using live-cell imaging to track GBM cells negotiating microchannels, we reveal endocytic vesicle accumulation at cell front and filamentous actin assembly at cell rear in a polarized manner. These processes are interconnected and require Plexin-B2 signaling. We further show that Plexin-B2 governs membrane tension and other membrane features such as endocytosis, phospholipid composition, and inner leaflet surface charge, thus providing biophysical mechanisms by which Plexin-B2 promotes GBM invasion. Together, our studies unveil how GBM cells regulate membrane tension and mechano-electrical coupling to adapt to physical constraints and achieve polarized confined migration.
... ; https://doi.org/10.1101/2024.12.31.630838 doi: bioRxiv preprint interaction with the flow of the underlying cortical actin cytoskeleton 37,63 ; or local restriction of free mobility (diffusion and bulk membrane flow) via membrane "corralling" 64 . We began by ruling out the first hypothesis, which is supported by the fact that leader bleb cells under confinement have their secretory and endocytic apparati localized almost exclusively in the cell body and do not extend into the bleb 65,66 . We further validated this assumption by photobleaching EGFR-GFP throughout the entire bleb and analyzing the recovery pattern by time-lapse imaging (Extended Figure 2A). ...
Cells under high confinement form highly polarized hydrostatic pressure–driven, stable leader blebs that enable efficient migration in low adhesion, environments. Here we investigated the basis of the polarized bleb morphology of metastatic melanoma cells migrating in non-adhesive confinement. Using high–resolution time–lapse imaging and specific molecular perturbations, we found that EGF signaling via PI3K stabilizes and maintains a polarized leader bleb. Protein activity biosensors revealed a unique EGFR and PI3K activity gradient decreasing from rear–to–front, promoting PIP3 and Rac1–GTP accumulation at the bleb rear, with its antagonists PIP2 and RhoA–GTP concentrated at the bleb tip, opposite to the front–to–rear organization of these signaling modules in integrin–mediated mesenchymal migration. Optogenetic experiments showed that disrupting this gradient caused bleb retraction, underscoring the role of this signaling gradient in bleb stability. Mathematical modeling and experiments identified a mechanism where, as the bleb initiates, CD44 and ERM proteins restrict EGFR mobility in a membrane–apposed cortical actin meshwork in the bleb rear, establishing a rear–to–front EGFR–PI3K–Rac activity gradient. Thus, our study reveals the biophysical and molecular underpinnings of cell polarity in bleb–based migration of metastatic cells in non-adhesive confinement, and underscores how alternative spatial arrangements of migration signaling modules can mediate different migration modes according to the local microenvironment.
... The nucleus is a key sensor of the local microenvironment. When a cell is confined or subjected to mechanical stress, the increase in NE tension triggers a signaling cascade for actomyosin contractility, allowing the cell to migrate out of confinement [162,163]. Additionally, the nucleus can act as a piston, exerting pressure on the front of the cell [164], thus influencing the efficiency of cell migration [165]. Deformation of the nucleus under mechanical stress can reorganize the chromatin state and increase the condensation of proteins in the nucleoplasm, thus influencing cellular function [166]. ...
The nucleus serves as a pivotal regulatory and control hub in the cell, governing numerous aspects of cellular functions, including DNA replication, transcription, and RNA processing. Therefore, any deviations in nuclear morphology, structure, or organization can strongly affect cellular activities. In this review, we provide an updated perspective on the structure and function of nuclear components, focusing on the linker of nucleoskeleton and cytoskeleton complex, the nuclear envelope, the nuclear lamina, and chromatin. Additionally, nuclear size should be considered a fundamental parameter for the cellular state. Its regulation is tightly linked to environmental changes, development, and various diseases, including cancer. Hence, we also provide a concise overview of different mechanisms by which nuclear size is determined, the emerging role of the nucleus as a mechanical sensor, and the implications of altered nuclear morphology on the physiology of diseased cells.
... The tissue shrinkage caused by compressive force can also increase the local concentration of growth factors and cytokines that may promote tumor growth (Tschumperlin et al. 2004). Intriguingly, the compressive stresses due to tissue confinement also deform the nucleus below a resting nuclear size, causing it to stretch its envelope to activate actomyosin signaling and induce cell contractility (Lomakin et al. 2020;Venturini et al. 2020;Kalukula et al. 2022). Additionally, elevated interstitial fluid pressure (IFP) due to fluid buildup from leaky blood vessels and dysfunctional lymphatics can further restrict the vasculature, impede drug delivery, and hinder immune cell infiltration and clearance (Heldin et al. 2004;Jain 2005;Jain et al. 2014;Kalli et al. 2023). ...
Development and disease are regulated by the interplay between genetics and the signaling pathways stimulated by morphogens, growth factors, and cytokines. Experimental data highlight the importance of mechanical force in regulating embryonic development, tissue morphogenesis, and malignancy. Force not only sculpts tissue movements to drive embryogenesis and morphogenesis but also modifies the context of biochemical signaling and gene expression to regulate cell and tissue fate. Not surprisingly, experiments have demonstrated that perturbations in cell tension drive malignancy and metastasis by altering biochemical signaling and gene expression through modifications in cytoskeletal tension, transmembrane receptor structure and function, and organelle phenotype that enhance cell growth and survival, alter metabolism, and foster cell migration and invasion. At the tissue level, tumor-associated forces disrupt cell–cell adhesions to perturb tissue organization, compromise vascular integrity to induce hypoxia, and interfere with antitumor immunity to foster metastasis and treatment resistance. Exciting new approaches now exist with which to clarify the relationship between mechanotransduction, biochemical signaling, and gene expression in development and disease. Indeed, gaining insight into these interactions is essential to unravel molecular mechanisms that regulate development and clarify the molecular basis of cancer.
... However, the nuclear lamina resists extensional deformation only in extreme nuclear shapes where the lamina is smooth 12,15 , such as the flattened nuclei in cell culture. We and others have reported that the nuclear lamina in non-extreme shapes, such as those in 3D culture or in elongated or rounded nuclei, tend to form folds and wrinkles [16][17][18][19][20][21][22][23][24] . These folds, which we have quantified as excess surface area relative to a sphere of the same volume as the nucleus 25 , imply that the nuclear lamina does not resist extensional strain so long as these folds are present. ...
Nuclear atypia is a hallmark of cancer. A recent model posits that excess surface area, visible as folds/wrinkles in the lamina of a rounded nucleus, allows the nucleus to take on diverse shapes with little mechanical resistance. Whether this model is applicable to normal and cancer nuclei in human tissues is unclear. We image nuclear lamins in patient tissues and find: (a) nuclear laminar wrinkles are present in control and cancer tissue but are obscured in hematoxylin and eosin (H&E) images, (b) nuclei rarely have a smooth lamina, and (c) wrinkled nuclei assume diverse shapes. Deep learning reveals the presence of extreme nuclear laminar wrinkling in cancer tissues, which is confirmed by Fourier analysis. These data support a model in which excess surface area in the nuclear lamina enables nuclear shape diversity in vivo. Extreme laminar wrinkling is a marker of cancer, and imaging the lamina may benefit cancer diagnosis.
... Cytoskeletal activation of NHE1 deforms the cell nucleus and modifies the transcriptomic profile Recent studies have revealed the important role of the nucleus in sensing mechanical stimuli. [64][65][66][67][68][69] To examine whether the cell nucleus responds to hypotonic stress, we simultaneously monitored cell volume and nucleus volume dynamics during hypotonic shock using a recently developed technique, N2FXm (nuclear double fluorescence exclusion method, Figure 5A). 70 In HT1080 cells, hypotonic shock increased the nuclear volume, followed by a monotonic RVD similar to the cell volume ( Figure 5B). ...
... Quantification of the fraction of nuclei with complete synapsis was carried out as previously described (45). For curvature analysis, n = 11 (− auxin) or 15 (+ auxin) latepachytene nuclei (pooled from at least four animals in each condition) were analyzed for the deformation of NE contours, which was manually segmented for at least 20 consecutive frames acquired every 5 s using Fiji and subsequently processed in MATLAB (R2023a, Mathworks) (114). Curvature measurement at any given moment for each nucleus over the whole tracked duration were combined for plotting the histogram. ...
Sexual reproduction relies on robust quality control during meiosis. Assembly of the synaptonemal complex between homologous chromosomes (synapsis) regulates meiotic recombination and is crucial for accurate chromosome segregation in most eukaryotes. Synapsis defects can trigger cell cycle delays and, in some cases, apoptosis. We developed and deployed a chemically induced proximity system to identify key elements of this quality control pathway in Caenorhabditis elegans . Persistence of the polo-like kinase PLK-2 at pairing centers—specialized chromosome regions that interact with the nuclear envelope—induced apoptosis of oocytes in response to phosphorylation and destabilization of the nuclear lamina. Unexpectedly, the Piezo1/PEZO-1 channel localized to the nuclear envelope and was required to transduce this signal to promote apoptosis in maturing oocytes.
... As research in the field of cell biology progresses, new discoveries are poised to revolutionize our understanding of the cellular world and its profound implications for all living organisms. The mysteries of the cell are yet to be fully uncovered; however, the ongoing exploration promises to uncover the hidden wonders and secrets that lie within, expanding our knowledge and broadening our perspective on the intricate web of life [25,26,27,28,29,30,31,32,33] . ...
... Forces induced by cell-substrate interactions are transmitted to cytoskeletal forces imposed directly on the NE, which have profound downstream effects on gene expression profiles and cell fate (Carley et al., 2021;Kalukula et al., 2022;Miroshnikova and Wickström, 2022;Nava et al., 2020). Recently, NE tension resulting from hypotonic shock due to cell injury or from physical confinement was shown to promote association of the C2 domain of the enzyme cPLA2 with the INM (Enyedi et al., 2016;Lomakin et al., 2020;Venturini et al., 2020). It has been proposed that cPLA2 recognizes increased packing defects at the INM; however, the extent to which different mechanical inputs affect lipid order at the INM beyond this example remains unexplored. ...
Amphipathic helices (AHs) detect differences in bulk membrane properties, but how AHs detect the nuclear membrane surrounding the genome is not well understood. Here, we computationally screened for candidate AHs in a curated list of characterized and putative human inner nuclear membrane (INM) proteins. Cell biological and in vitro experimental assays combined with computational calculations demonstrate that AHs detect lipid packing defects over electrostatics to bind to the INM, indicating that the INM is loosely packed under basal conditions. Membrane tension resulting from hypotonic shock further promoted AH binding to the INM, whereas cell-substrate stretch did not recruit membrane tension-sensitive AHs. Thus, distinct mechanical inputs enhance lipid loosening at the INM to different degrees, which AHs in INM proteins may harness for downstream biochemical functions. Our resource provides a framework for future studies on the contributions of lipid-protein interactions at the INM and enables exploration of the membrane properties of the INM under different conditions.
Cell migration occurs throughout development, tissue homeostasis and regeneration, as well as in diseases such as cancer. Cells migrate along two-dimensional (2D) surfaces or interfaces, within microtracks, or in confining three-dimensional (3D) extracellular matrices. Although the basic mechanisms of 2D migration are known, recent studies have elucidated unexpected migration behaviors associated with more complex substrates and have provided insights into their underlying molecular mechanisms. Studies using engineered biomaterials for 3D culture and microfabricated channels to replicate cell confinement observed in vivo have revealed distinct modes of migration. Across these contexts, the mechanical features of the surrounding microenvironment have emerged as major regulators of migration. In this Cell Science at a Glance article and the accompanying poster, we describe physiological contexts wherein 2D and 3D cell migration are essential, report how mechanical properties of the microenvironment regulate individual and collective cell migration, and review the mechanisms mediating these diverse modes of cell migration.
From border cell migration during Drosophila embryogenesis to solid stresses inside tumors, cells are often compressed during physiological and pathological processes, triggering major cell responses. Cell compression can be observed in vivo but also controlled in vitro through tools such as micro-channels or planar confinement assays. Such tools have recently become commercially available, allowing a broad research community to tackle the role of cell compression in a variety of contexts. This has led to the discovery of conserved compression-triggered migration modes, cell fate determinants and mechanosensitive pathways, among others. In this Review, we will first address the different ways in which cells can be compressed and their biological contexts. Then, we will discuss the distinct mechanosensing and mechanotransducing pathways that cells activate in response to compression. Finally, we will describe the different in vitro systems that have been engineered to compress cells.
The media, a primary layer of the aortic wall, is rich in smooth muscle cells (SMCs) that regulate the vessel diameter and maintain the mechanical balance of the aortic ring in vivo. Embedded in the medial extracellular matrix, SMCs adapt to their surrounding mechanical environment via cyclic stretch during vascular contraction and relaxation. Thus, the circumferential stress that constantly acts on the hypertensive aorta is expected to further increase with increasing blood pressure (hypertension), resulting in a thickened medial wall. This thickening is considered an active biomechanical response of SMCs to maintain constant circumferential stress, ensuring homeostasis. Therefore, understanding how external forces or mechanical stimuli acting on SMCs are transmitted through intracellular components is crucial. Nuclei may sense mechanical changes through stress fibers (SFs) and focal adhesions (FAs). However, limited quantitative information exists regarding the mechanical contributions of SFs and FAs to whole-cell mechanical events, such as the response to uniaxial stretching. In this study, we developed a finite element model of a cultured vascular SMC with contractile SFs anchored on a silicone substrate via FAs and applied uniaxial stretching to investigate the mechanotransduction pathways in SMCs. We revealed that the initial orientation angle of the cell relative to the stretching direction strongly correlated with the resultant magnitude of the biomechanical forces acting on the nuclei surface exerted by the SFs.
The central goal of mechanobiology is to understand how the mechanical forces and material properties of organelles, cells, and tissues influence biological processes and functions. Since the first description of biomolecular condensates, it was hypothesized that they obtain material properties that are tuned to their functions inside cells. Thus, they represent an intriguing playground for mechanobiology. The idea that biomolecular condensates exhibit diverse and adaptive material properties highlights the need to understand how different material states respond to external forces and whether these responses are linked to their physiological roles within the cell. For example, liquids buffer and dissipate, while solids store and transmit mechanical stress, and the relaxation time of a viscoelastic material can act as a mechanical frequency filter. Hence, a liquid–solid transition of a condensate in the force transmission pathway can determine how mechanical signals are transduced within and in-between cells, affecting differentiation, neuronal network dynamics, and behavior to external stimuli. Here, we first review our current understanding of the molecular drivers and how rigidity phase transitions are set forth in the complex cellular environment. We will then summarize the technical advancements that were necessary to obtain insights into the rich and fascinating mechanobiology of condensates, and finally, we will highlight recent examples of physiological liquid–solid transitions and their connection to specific cellular functions. Our goal is to provide a comprehensive summary of the field on how cells harness and regulate condensate mechanics to achieve specific functions.
The ability of cells to sense and respond to mechanical signals is essential for many biological processes that form the basis of cell identity, tissue development and maintenance. This process, known as mechanotransduction, involves crucial feedback between mechanical force and biochemical signals, including epigenomic modifications that establish transcriptional programs. These programs, in turn, reinforce the mechanical properties of the cell and its ability to withstand mechanical perturbation. The nucleus has long been hypothesized to play a key role in mechanotransduction due to its direct exposure to forces transmitted through the cytoskeleton, its role in receiving cytoplasmic signals and its central function in gene regulation. However, parsing out the specific contributions of the nucleus from those of the cell surface and cytoplasm in mechanotransduction remains a substantial challenge. In this Review, we examine the latest evidence on how the nucleus regulates mechanotransduction, both via the nuclear envelope (NE) and through epigenetic and transcriptional machinery elements within the nuclear interior. We also explore the role of nuclear mechanotransduction in establishing a mechanical memory, characterized by a mechanical, epigenetic and transcriptomic cell state that persists after mechanical stimuli cease. Finally, we discuss current challenges in the field of nuclear mechanotransduction and present technological advances that are poised to overcome them.
Muscle stem cells (MuSCs) play a crucial role in skeletal muscle regeneration, residing in a niche that undergoes dimensional and mechanical changes throughout the regeneration process. This study investigates how 3D confinement and stiffness encountered by MuSCs during the later stages of regeneration regulate their function, including stemness, activation, proliferation, and differentiation. An asymmetric 3D hydrogel bilayer platform is engineered with tunable physical constraints to mimic the regenerating MuSC niche. These results demonstrate that increased 3D confinement maintains Pax7 expression, reduces MuSC activation and proliferation, inhibits differentiation, and is associated with smaller nuclear size and decreased H4K16ac levels, suggesting that mechanical confinement modulates both nuclear architecture and epigenetic regulation. MuSCs in unconfined 2D environments exhibit larger nuclei and higher H4K16ac expression compared to those in more confined 3D conditions, leading to progressive activation, expansion, and myogenic commitment. This study highlights the importance of 3D mechanical cues in MuSC fate regulation, with 3D confinement acting as a mechanical brake on myogenic commitment, offering novel insights into the mechano‐epigenetic mechanisms that govern MuSC behavior during muscle regeneration.
Cells are continuously subjected to physical and chemical cues from the extracellular environment, and sense and respond to mechanical cues via mechanosensation and mechanotransduction. Although the role of the cytoskeleton in these processes is well known, the contribution of intracellular membranes has been long neglected. Recently, it has become evident that various organelles play active roles in both mechanosensing and mechanotransduction. In this Review, we focus on mechanosensitive roles of the endoplasmic reticulum (ER), the functions of which are crucial for maintaining cell homeostasis. We discuss the effects of mechanical stimuli on interactions between the ER, the cytoskeleton and other organelles; the role of the ER in intracellular Ca2+ signalling via mechanosensitive channels; and how the unfolded protein response and lipid homeostasis contribute to mechanosensing. The expansive structure of the ER positions it as a key intracellular communication hub, and we additionally explore how this may be leveraged to transduce mechanical signals around the cell. By synthesising current knowledge, we aim to shed light on the emerging roles of the ER in cellular mechanosensing and mechanotransduction.
Enhanced drug testing efficiency has driven the prominence of high‐content and high‐throughput screening (HCHTS) in drug discovery and development. However, traditional HCHTS in well‐plates often lack complexity of in vivo conditions. 3D cell cultures, like cellular spheroids/organoids, offer a promising alternative by replicating in vivo conditions and improving the reliability of drug responses. Integrating spheroids/organoids into HCHTS requires strategies to ensure uniform formation, systemic function, and compatibility with analysis techniques. This study introduces an easy‐to‐fabricate, low‐cost, safe, and scalable approach to create a bioinert hydrogel‐based inverted colloidal crystal (BhiCC) framework for uniform and high‐yield spheroid cultivation. Highly uniform alginate microgels are fabricated and assembled into a colloidal crystal template with controllable contact area, creating engineered void spaces and interconnecting channels within agarose‐based BhiCC through the template degradation by alginate lyase and buffer. This results in a multi‐layered iCC domain, enabling the generation of in‐vitro 3D culture models with over 1000 spheroids per well in a 96‐well plate. The unique hexagonal‐close‐packed geometry of iCC structure enables HCHTS through conventional plate reader analysis and fluorescent microscopy assisted by house‐developed automated data processing algorithm. This advancement offers promising applications in tissue engineering, disease modeling, and drug development in biomedical research.
Cells navigating in complex 3D microenvironments frequently encounter narrow spaces that physically challenge migration. While in vitro studies identified nuclear stiffness as a key rate-limiting factor governing the movement of many cell types through artificial constraints, how cells migrating in vivo respond dynamically to confinement imposed by local tissue architecture, and whether these encounters trigger molecular adaptations, is unclear. Here, we establish an innovative in vivo model for mechanistic analysis of nuclear plasticity as Drosophila immune cells transition into increasingly confined microenvironments. Integrating live in vivo imaging with molecular genetic analyses, we demonstrate how rapid molecular adaptation upon environmental confinement (including fine-tuning of the nuclear lamina) primes leukocytes for enhanced nuclear deformation while curbing damage (including rupture and micronucleation), ultimately accelerating movement through complex tissues. We find nuclear dynamics in vivo are further impacted by large organelles (phagosomes) and the plasticity of neighbouring cells, which themselves deform during leukocyte passage. The biomechanics of cell migration in vivo are thus shaped both by factors intrinsic to individual immune cells and the malleability of the surrounding microenvironment.
Historically considered downstream effects of tumorigenesis—arising from changes in DNA content or chromatin organization—nuclear alterations have long been seen as mere prognostic markers within a genome‐centric model of cancer. However, recent findings have placed the nuclear envelope (NE) at the forefront of tumor progression, highlighting its active role in mediating cellular responses to mechanical forces. Despite significant progress, the precise interplay between NE components and cancer progression remains under debate. In this review, we provide a comprehensive and up‐to‐date overview of how changes in NE composition affect nuclear mechanics and facilitate malignant transformation, grounded in the latest molecular and functional studies. We also review recent research that uses advanced technologies, including artificial intelligence, to predict malignancy risk and treatment outcomes by analyzing nuclear morphology. Finally, we discuss how progress in understanding nuclear mechanics has paved the way for mechanotherapy—a promising cancer treatment approach that exploits the mechanical differences between cancerous and healthy cells. Shifting the perspective on NE alterations from mere diagnostic markers to potential therapeutic targets, this review calls for further investigation into the evolving role of the NE in cancer, highlighting the potential for innovative strategies to transform conventional cancer therapies.
Errors during cell division lead to aneuploidy, which is associated with genomic instability and cell transformation. In response to aneuploidy, cells activate the tumour suppressor p53 to elicit a surveillance mechanism that halts proliferation and promotes senescence. The molecular sensors that trigger this checkpoint are unclear. Here, using a tunable system of chromosome mis-segregation, we show that mitotic errors trigger nuclear deformation, nuclear softening, and lamin and heterochromatin alterations, leading to rapid p53/p21 activation upon mitotic exit in response to changes in nuclear mechanics. We identify mTORC2 and ATR as nuclear deformation sensors upstream of p53/p21 activation. While triggering mitotic arrest, the chromosome mis-segregation-induced alterations of nuclear envelope mechanics provide a fitness advantage for aneuploid cells by promoting nuclear deformation resilience and enhancing pro-invasive capabilities. Collectively, this work identifies a nuclear mechanical checkpoint triggered by altered chromatin organization that probably plays a critical role in cellular transformation and cancer progression.
Quantifying the mechanical response of the biological milieu (such as the cell’s interior) and complex fluids (such as biomolecular condensates) would enable a better understanding of cellular differentiation and aging and accelerate drug discovery. Here we present time-shared optical tweezer microrheology to determine the frequency- and age-dependent viscoelastic properties of biological materials. Our approach involves splitting a single laser beam into two near-instantaneous time-shared optical traps to carry out simultaneous force and displacement measurements and quantify the mechanical properties ranging from millipascals to kilopascals across five decades of frequency. To create a practical and robust nanorheometer, we leverage both numerical and analytical models to analyse typical deviations from the ideal behaviour and offer solutions to account for these discrepancies. We demonstrate the versatility of the technique by measuring the liquid–solid phase transitions of MEC-2 stomatin and CPEB4 biomolecular condensates, and quantify the complex viscoelastic properties of intracellular compartments of zebrafish progenitor cells. In Caenorhabditis elegans, we uncover how mutations in the nuclear envelope proteins LMN-1 lamin A, EMR-1 emerin and LEM-2 LEMD2, which cause premature aging disorders in humans, soften the cytosol of intestinal cells during organismal age. We demonstrate that time-shared optical tweezer microrheology offers the rapid phenotyping of material properties inside cells and protein blends, which can be used for biomedical and drug-screening applications.
Liver metastasis is the main cause of cancer‐related mortality. During the metastasis process, circulating carcinoma cells hardly pass through narrow capillaries, leading to nuclear deformation. However, the effects of nuclear deformation and its underlying mechanisms on metastasis need further study. Here, it is shown that mechanical force‐induced nuclear deformation exacerbates liver metastasis by activating the cGAS‐STING pathway, which promotes splenocyte infiltration in the liver. Mechanical force results in nuclear deformation and rupture of the nuclear envelope with inevitable DNA leakage. Cytoplasmic DNA triggers the activation of cGAS‐STING pathway, enhancing the production of IL6, TNFα, and CCL2. Additionally, splenocyte recruitment by the proinflammatory cytokines support carcinoma cell survival and colonization in the liver. Importantly, both intervening activity of cGAS and blocking of splenocyte migration to the liver efficiently ameliorate liver metastasis. Overall, these findings reveal a mechanism by which mechanical force‐induced nuclear deformation exacerbates liver metastasis by regulating splenocyte infiltration into the liver and support targeting cGAS and blocking splenocyte recruitment as candidate therapeutic approaches for liver metastasis.
Osteoarthritis (OA) is a prevalen degenerative joint disease with no FDA‐approved therapies that can halt or reverse its progression. Current treatments address symptoms like pain and inflammation, but not underlying disease mechanisms. OA progression is marked by increased inflammation and extracellular matrix (ECM) degradation of the joint cartilage. While the role of biochemical cues has been widely studied for OA, how matrix mechanical cues influence OA phenotype remains poorly understood. Using sliding hydrogels (SGs) as a tool, we examine how local matrix compliance in 3D modulates OA chondrocyte phenotype and associated mechanosensing. We demonstrate that local matrix compliance reduces the inflammatory phenotype of OA chondrocytes, as indicated by decreased gene expression of catabolic markers and proinflammatory cytokine secretion. This is achieved via significantly reduced nuclear NF‐κB expression and signaling in OA chondrocytes. Live cell imaging shows enhanced cellular and nuclear dynamics with increased matrix deformation in the compliant SG. Blocking cellular dynamics negates SG compliance‐induced benefits in reducing OA inflammatory phenotype. Further, SG alters nuclear mechanosensing in OA as indicated by increased nuclear lamin reinforcement and chromatin condensation. Finally, we demonstrate that a drug inhibiting histone lysine demethylase to modulate chromatin accessibility reduces OA inflammation in 3D hydrogels. These findings advance our understanding of how ECM mechanics regulate OA mechanobiology and progression and highlight potential disease‐modifying treatments via epigenetic and mechanosensing‐based therapies.
Intervertebral disc degeneration, inflammation, and bioactive lipids Intervertebral disc degeneration Intervertebral disc (IVD) degeneration is a common condition that is associated with significant morbidity. There is no specific treatment available for IVD except for surgical intervention. IVD is an inflammatory condition. I propose that IVD can be prevented and managed by local administration of lipoxin A4 (LXA4), a potent anti-inflammatory, cytoprotective and anti-osteoporotic metabolite formed from arachidonic acid (AA). Keywords: intervertebral disc, degeneration, lipoxin A4, arachidonic acid, inflammation.
Leukocytes detect distant wounds within seconds to minutes, which is essential for effective pathogen defense, tissue healing, and regeneration. Blood vessels must detect distant wounds just as rapidly to initiate local leukocyte extravasation, but the mechanism behind this immediate vascular response remains unclear. Using high-speed imaging of live zebrafish larvae, we investigated how blood vessels achieve rapid wound detection. We monitored two hallmark vascular responses: vessel dilation and serum exudation. Our experiments—including genetic, pharmacologic, and osmotic perturbations, along with chemogenetic leukocyte depletion—revealed that the cPla 2 nuclear shape sensing pathway in perivascular macrophages converts a fast (∼50 μm/s) osmotic wound signal into a vessel-permeabilizing, 5-lipoxygenase (Alox5a) derived lipid within seconds of injury.
These findings demonstrate that perivascular macrophages act as physicochemical relays, bridging osmotic wound signals and vascular responses. By uncovering this novel type of communication, we provide new insights into the coordination of immune and vascular responses to injury.
Nuclear deformation by osmotic shock or necrosis activates the cytosolic phospholipase A2 (cPla2) nuclear shape sensing pathway, a key regulator of tissue inflammation and repair. Ca²⁺ and inner nuclear membrane (INM) tension (TINM) are believed to mediate nucleoplasmic cPla2 activation. The concept implies that TINM persists long enough to stimulate cPla2-INM adsorption. However, TINM may instead be rapidly dissipated by the contiguous endoplasmic reticulum (ER), with cPla2-INM adsorption reporting rather on changes in Ca²⁺ than TINM. The impact of TINM and ER contiguity on nuclear shape sensing and mechanotransduction remains unknown.
To address this gap, we developed the Ca²⁺ insensitive, TINM-only biosensor ALPIN (Amphipathic Lipid Packing sensor domain Inside the Nucleus). By quantitative ALPIN imaging, we found that stress-induced ER fragmentation increases TINM and nuclear membrane mechanotransduction in osmotically shocked or ferroptotic cells, permeabilized cell corpses, and at zebrafish wounds in vivo. Our findings reveal critical roles for the ER and TINM in nuclear shape sensing and introduce ALPIN as promising tool for studying organelle membrane mechanotransduction in health and disease.
The plant cytoskeleton is an intricate network composed of actin filaments and microtubules. The cytoskeleton undergoes continuous dynamic changes that provide the basis for rapidly responding to intrinsic and extrinsic stimuli, including mechanical stress. Microtubules can respond to alterations of mechanical stress and reorient along the direction of maximal tensile stress in plant cells. The cytoskeleton can also generate driving force for cytoplasmic streaming, organelle movement, and vesicle transportation. In this review, we discuss the progress of how the plant cytoskeleton responds to mechanical stress. We also summarize the roles of the cytoskeleton in generating force that drive organelles and nuclear transportation in plant cells. Finally, some hypotheses concerning the link between the roles of the cytoskeleton in force response and organelle movement, as well as several key questions that remain to be addressed in the field, are highlighted.
The nucleus makes the rules
Single cells continuously experience and react to mechanical challenges in three-dimensional tissues. Spatial constraints in dense tissues, physical activity, and injury all impose changes in cell shape. How cells can measure shape deformations to ensure correct tissue development and homeostasis remains largely unknown (see the Perspective by Shen and Niethammer). Working independently, Venturini et al. and Lomakin et al. now show that the nucleus can act as an intracellular ruler to measure cellular shape variations. The nuclear envelope provides a gauge of cell deformation and activates a mechanotransduction pathway that controls actomyosin contractility and migration plasticity. The cell nucleus thereby allows cells to adapt their behavior to the local tissue microenvironment.
Science , this issue p. eaba2644 , p. eaba2894 ; see also p. 295
The evolution of different cell types was a key process of early animal evolution ¹⁻³ . Two fundamental cell types, epithelial cells and amoeboid cells, are broadly distributed across the animal tree of life 4,5 but their origin and early evolution are unclear. Epithelial cells are polarized, have a fixed shape and often bear an apical cilium and microvilli. These features are shared with choanoflagellates - the closest living relatives of animals - and are thought to have been inherited from their last common ancestor with animals 1,6,7 . The deformable amoeboid cells of animals, on the other hand, seem strikingly different from choanoflagellates and instead evoke more distantly related eukaryotes, such as diverse amoebae - but it has been unclear whether that similarity reflects common ancestry or convergence ⁸ . Here, we show that choanoflagellates subjected to spatial confinement differentiate into an amoeboid phenotype by retracting their flagella and microvilli, generating blebs, and activating myosin-based motility. Choanoflagellate cell crawling is polarized by geometrical features of the substrate and allows escape from confined microenvironments. The confinement-induced amoeboid switch is conserved across diverse choanoflagellate species and greatly expands the known phenotypic repertoire of choanoflagellates. The broad phylogenetic distribution of the amoeboid cell phenotype across animals ⁹⁻¹⁴ and choanoflagellates, as well as the conserved role of myosin, suggests that myosin-mediated amoeboid motility was present in the life history of their last common ancestor. Thus, the duality between animal epithelial and crawling cells might have evolved from a temporal phenotypic switch between flagellate and amoeboid forms in their single-celled ancestors 3,15,16 .
Tissue homeostasis requires maintenance of functional integrity under stress. A central source of stress is mechanical force that acts on cells, their nuclei, and chromatin, but how the genome is protected against mechanical stress is unclear. We show that mechanical stretch deforms the nucleus, which cells initially counteract via a calcium-dependent nuclear softening driven by loss of H3K9me3-marked heterochromatin. The resulting changes in chromatin rheology and architecture are required to insulate genetic material from mechanical force. Failure to mount this nuclear mechanoresponse results in DNA damage. Persistent, high-amplitude stretch induces supracellular alignment of tissue to redistribute mechanical energy before it reaches the nucleus. This tissue-scale mechanoadaptation functions through a separate pathway mediated by cell-cell contacts and allows cells/tissues to switch off nuclear mechanotransduction to restore initial chromatin state. Our work identifies an unconventional role of chromatin in altering its own mechanical state to maintain genome integrity in response to deformation.
The microscopic environment inside a metazoan organism is highly crowded. Whether individual cells can tailor their behavior to the limited space remains unclear. Here, we found that cells measure the degree of spatial confinement using their largest and stiffest organelle, the nucleus. Cell confinement below a resting nucleus size deforms the nucleus, which expands and stretches its envelope. This activates signaling to the actomyosin cortex via nuclear envelope stretch-sensitive proteins, upregulating cell contractility. We established that the tailored contractile response constitutes a nuclear ruler-based signaling pathway involved in migratory cell behaviors. Cells rely on the nuclear ruler to modulate the motive force enabling their passage through restrictive pores in complex three-dimensional (3D) environments, a process relevant to cancer cell invasion, immune responses and embryonic development.
One Sentence Summary
Nuclear envelope expansion above a threshold triggers a contractile cell response and thus acts as a ruler for the degree of cell deformation.
STIM1 (a Ca2+ sensor in the endoplasmic reticulum (ER) membrane) and Orai1 (a pore-forming subunit of the Ca2+-release-activated calcium channel in the plasma membrane) diffuse in the ER membrane and plasma membrane, respectively. Upon depletion of Ca2+ stores in the ER, STIM1 translocates to the ER-plasma membrane junction and binds Orai1 to trigger store-operated Ca2+ entry. However, the motion of STIM1 and Orai1 during this process and its roles to Ca2+ entry is poorly understood. Here, we report real-time tracking of single STIM1 and Orai1 particles in the ER membrane and plasma membrane in living cells before and after Ca2+ store depletion. We found that the motion of single STIM1 and Orai1 particles exhibits anomalous diffusion both before and after store depletion, and their mobility-measured by the radius of gyration of the trajectories, mean-square displacement, and generalized diffusion coefficient-decreases drastically after store depletion. We also found that the measured displacement distribution is non-Gaussian, and the non-Gaussian parameter drastically increases after store depletion. Detailed analyses and simulations revealed that single STIM1 and Orai1 particles are confined in the compartmentalized membrane both before and after store depletion, and the changes in the motion after store depletion are explained by increased confinement and polydispersity of STIM1-Orai1 complexes formed at the ER-plasma membrane junctions. Further simulations showed that this increase in the confinement and polydispersity after store depletion localizes a rapid increase of Ca2+ influx, which can facilitate the rapid activation of local Ca2+ signaling pathways and the efficient replenishing of Ca2+ store in the ER in store-operated Ca2+ entry.
How cells sense hydraulic pressure and make directional choices in confinement remains elusive. Using trifurcating Ψ-like microchannels of different hydraulic resistances and cross-sectional areas, we discovered that the TRPM7 ion channel is the critical mechanosensor, which directs decision-making of blebbing cells toward channels of lower hydraulic resistance irrespective of their cross-sectional areas. Hydraulic pressure–mediated TRPM7 activation triggers calcium influx and supports a thicker cortical actin meshwork containing an elevated density of myosin-IIA. Cortical actomyosin shields cells against external forces and preferentially directs cell entrance in low resistance channels. Inhibition of TRPM7 function or actomyosin contractility renders cells unable to sense different resistances and alters the decision-making pattern to cross-sectional area–based partition. Cell distribution in microchannels is captured by a mathematical model based on the maximum entropy principle using cortical actin as a key variable. This study demonstrates the unique role of TRPM7 in controlling decision-making and navigating migration in complex microenvironments.
The shape of the cell nucleus can vary considerably during developmental and pathological processes, however the impact of nuclear morphology on cell behaviour is not known. Here we observed that the nuclear envelope flattens as cells transit from G1 to S phase and inhibition of myosin II prevents nuclear flattening and impedes progression to S phase. Strikingly, we show that applying compressive force on the nucleus in the absence of myosin II-mediated tension is sufficient to restore G1 to S transition. Using a combination of tools to manipulate nuclear morphology, we observed that nuclear flattening activates a subset of transcription factors, including TEAD and AP1, leading to transcriptional induction of target genes that promotes G1 to S transition. In addition, we found that nuclear flattening mediates TEAD and AP1 activation in response to Rock-generated contractility or cell spreading. Our results reveal that the nuclear envelope can operate as a mechanical sensor whose deformation controls cell growth in response to tension.
During metazoan development, immune surveillance and cancer dissemination, cells migrate in complex three-dimensional microenvironments1–3. These spaces are crowded by cells and extracellular matrix, generating mazes with differently sized gaps that are typically smaller than the diameter of the migrating cell4,5. Most mesenchymal and epithelial cells and some—but not all—cancer cells actively generate their migratory path using pericellular tissue proteolysis⁶. By contrast, amoeboid cells such as leukocytes use non-destructive strategies of locomotion⁷, raising the question how these extremely fast cells navigate through dense tissues. Here we reveal that leukocytes sample their immediate vicinity for large pore sizes, and are thereby able to choose the path of least resistance. This allows them to circumnavigate local obstacles while effectively following global directional cues such as chemotactic gradients. Pore-size discrimination is facilitated by frontward positioning of the nucleus, which enables the cells to use their bulkiest compartment as a mechanical gauge. Once the nucleus and the closely associated microtubule organizing centre pass the largest pore, cytoplasmic protrusions still lingering in smaller pores are retracted. These retractions are coordinated by dynamic microtubules; when microtubules are disrupted, migrating cells lose coherence and frequently fragment into migratory cytoplasmic pieces. As nuclear positioning in front of the microtubule organizing centre is a typical feature of amoeboid migration, our findings link the fundamental organization of cellular polarity to the strategy of locomotion.
Cancer cell invasion through physical barriers in the extracellular matrix (ECM) requires a complex synergy of traction force against the ECM, mechanosensitive feedback, and subsequent cytoskeletal rearrangement. PDMS microchannels were used to investigate the transition from mesenchymal to amoeboid invasion in cancer cells. Migration was faster in narrow 3 μm-wide channels than in wider 10 μm channels, even in the absence of cell-binding ECM proteins. Cells permeating narrow channels exhibited blebbing and had smooth leading edge profiles, suggesting an ECM-induced transition from mesenchymal invasion to amoeboid invasion. Live cell labeling revealed a mechanosensing period in which the cell attempts mesenchymal-based migration, reorganizes its cytoskeleton, and proceeds using an amoeboid phenotype. Rho/ROCK (amoeboid) and Rac (mesenchymal) pathway inhibition revealed that amoeboid invasion through confined environments relies on both pathways in a time- and ECM-dependent manner. This demonstrates that cancer cells can dynamically modify their invasion programming to navigate physically-confining matrix conditions.
As tissues develop, they are subjected to a variety of mechanical forces. Some of these forces are instrumental in the development of tissues, while others can result in tissue damage. Despite our extensive understanding of force-guided morphogenesis, we have only a limited understanding of how tissues prevent further morphogenesis once the shape is determined after development. Here, through the development of a tissue-stretching device, we uncover a mechanosensitive pathway that regulates tissue responses to mechanical stress through the polarization of actomyosin across the tissue. We show that stretch induces the formation of linear multicellular actomyosin cables, which depend on Diaphanous for their nucleation. These stiffen the epithelium, limiting further changes in shape, and prevent fractures from propagating across the tissue. Overall, this mechanism of force-induced changes in tissue mechanical properties provides a general model of force buffering that serves to preserve the shape of tissues under conditions of mechanical stress.
Cells are the building units of living organisms and consequently adapt to their environment by modulating their intracellular architecture, in particular the position of their nucleus. Important efforts have been made to decipher the molecular mechanisms involved in nuclear positioning. The LINC complex at the nuclear envelope is a very important part of the molecular connectivity between the cell outside and the intranuclear compartment, and thus emerged as a central player in nuclear mechanotransduction. More recent concepts in nuclear mechanotransduction came from studies involving nuclear confined migration, compression or swelling. Also, the effect of nuclear mechanosensitive properties in driving cell differentiation raises the question of nuclear mechanotransduction and gene expression and recent efforts have been done to tackle it, even though it remains difficult to address in a direct manner. Eventually, an original mechanism of nucleus positioning, mechanotransduction and regulation of gene expression in the non-adherent, non-polarized mouse oocyte, highlights the fact that nuclear positioning is an important developmental issue.
ROCK-Myosin II drives fast rounded-amoeboid migration in cancer cells during metastatic dissemination. Analysis of human melanoma biopsies revealed that amoeboid melanoma cells with high Myosin II activity are predominant in the invasive fronts of primary tumors in proximity to CD206⁺CD163⁺ tumor-associated macrophages and vessels. Proteomic analysis shows that ROCK-Myosin II activity in amoeboid cancer cells controls an immunomodulatory secretome, enabling the recruitment of monocytes and their differentiation into tumor-promoting macrophages. Both amoeboid cancer cells and their associated macrophages support an abnormal vasculature, which ultimately facilitates tumor progression. Mechanistically, amoeboid cancer cells perpetuate their behavior via ROCK-Myosin II-driven IL-1α secretion and NF-κB activation. Using an array of tumor models, we show that high Myosin II activity in tumor cells reprograms the innate immune microenvironment to support tumor growth. We describe an unexpected role for Myosin II dynamics in cancer cells controlling myeloid function via secreted factors.
Tissue morphogenesis is driven by mechanical forces that elicit changes in cell size, shape and motion. The extent by which forces deform tissues critically depends on the rheological properties of the recipient tissue. Yet, whether and how dynamic changes in tissue rheology affect tissue morphogenesis and how they are regulated within the developing organism remain unclear. Here, we show that blastoderm spreading at the onset of zebrafish morphogenesis relies on a rapid, pronounced and spatially patterned tissue fluidization. Blastoderm fluidization is temporally controlled by mitotic cell rounding-dependent cell–cell contact disassembly during the last rounds of cell cleavages. Moreover, fluidization is spatially restricted to the central blastoderm by local activation of non-canonical Wnt signalling within the blastoderm margin, increasing cell cohesion and thereby counteracting the effect of mitotic rounding on contact disassembly. Overall, our results identify a fluidity transition mediated by loss of cell cohesion as a critical regulator of embryo morphogenesis. © 2018, The Author(s), under exclusive licence to Springer Nature Limited.
During epithelial contraction, cells generate forces to constrict their surface and, concurrently, fine-tune the length of their adherens junctions to ensure force transmission. While many studies have focused on understanding force generation, little is known on how junctional length is controlled. Here, we show that, during amnioserosa contraction in Drosophila dorsal closure, adherens junctions reduce their length in coordination with the shrinkage of apical cell area, maintaining a nearly constant junctional straightness. We reveal that junctional straightness and integrity depend on the endocytic machinery and on the mechanosensitive activity of the actomyosin cytoskeleton. On one hand, upon junctional stretch and decrease in E-cadherin density, actomyosin relocalizes from the medial area to the junctions, thus maintaining junctional integrity. On the other hand, when junctions have excess material and ruffles, junction removal is enhanced, and high junctional straightness and tension are restored. These two mechanisms control junctional length and integrity during morphogenesis.
Fibroblasts exhibit heterogeneous cell geometries in tissues and integrate both mechanical and biochemical signals in their local microenvironment to regulate genomic programs via chromatin remodelling. While in connective tissues fibroblasts experience tensile and compressive forces (CFs), the role of compressive forces in regulating cell behaviour and in particular, the impact of cell geometry in modulating transcriptional response to such extrinsic mechanical forces is unclear. Here, we show that CF on geometrically well-defined mouse fibroblast cells reduces actomyosin contractility and shuttles Histone Deacetylase 3 (HDAC3), into the nucleus. HDAC3 then triggers an increase in the heterochromatin content by initiating removal of acetylation marks on the histone tails. This suggests that, in response to CF, fibroblasts condense their chromatin and enter into a transcriptionally less active and quiescent states as also revealed by transcriptome analysis. Upon removal of CF, the alteration in chromatin condensation was reversed. We also present a quantitative model linking CF dependent changes in actomyosin contractility leading to chromatin condensation. Further, transcriptome analysis also revealed that the transcriptional response of cells to CF was geometry dependent. Collectively, our results suggest that CFs induce chromatin condensation and geometry dependent differential transcriptional response in fibroblasts that allows maintenance of tissue homeostasis. [Media: see text] [Media: see text] [Media: see text].
The ability of cells to respond to mechanical forces is critical for numerous biological processes. Emerging evidence indicates that external mechanical forces trigger changes in nuclear envelope structure and composition, chromatin organization and gene expression. However, it remains unclear if these processes originate in the nucleus or are downstream of cytoplasmic signals. Here we discuss recent findings that support a direct role of the nucleus in cellular mechanosensing and highlight novel tools to study nuclear mechanotransduction.
To form functional neural circuits, neurons migrate to their final destination and extend axons towards their targets. Whether and how these two processes are coordinated in vivo remains elusive. We use the zebrafish olfactory placode as a system to address the underlying mechanisms. Quantitative live imaging uncovers a choreography of directed cell movements that shapes the placode neuronal cluster: convergence of cells towards the centre of the placodal domain and lateral cell movements away from the brain. Axon formation is concomitant with lateral movements and occurs through an unexpected, retrograde mode of extension, where cell bodies move away from axon tips attached to the brain surface. Convergence movements are active, whereas cell body lateral displacements are of mainly passive nature, likely triggered by compression forces from converging neighbouring cells. These findings unravel a previously unknown mechanism of neuronal circuit formation, whereby extrinsic mechanical forces drive the retrograde extension of axons.
Migrating cells can extend their leading edge by forming myosin-driven blebs and F-actin-driven pseudopods. When coerced to migrate in resistive environments, Dictyostelium cells switch from using predominately pseudopods to blebs. Bleb formation has been shown to be chemotactic and can be influenced by the direction of the chemotactic gradient. In this study, we determine the blebbing responses of developed cells of Dictyostelium discoideum to cAMP gradients of varying steepness produced in microfluidic channels with different confining heights, ranging between 1.7 μm and 3.8 μm. We show that microfluidic confinement height, gradient steepness, buffer osmolarity and Myosin II activity are important factors in determining whether cells migrate with blebs or with pseudopods. Dictyostelium cells were observed migrating within the confines of microfluidic gradient channels. When the cAMP gradient steepness is increased from 0.7 nM/μm to 20 nM/μm, cells switch from moving with a mixture of blebs and pseudopods to moving only using blebs when chemotaxing in channels with confinement heights less than 2.4 μm. Furthermore, the size of the blebs increases with gradient steepness and correlates with increases in myosin-II localization at the cell cortex. Reduction of intracellular pressure by high osmolarity buffer or inhibition of myosin-II by blebbistatin leads to a decrease in bleb formation and bleb size. Together, our data reveal that the protrusion type formed by migrating cells can be influenced by the channel height and the steepness of the cAMP gradient, and suggests that a combination of confinement-induced myosin-II localization and cAMP-regulated cortical contraction leads to increased intracellular fluid pressure and bleb formation.
Inside eukaryotic cells, membrane contact sites (MCSs), regions where two membrane-bound organelles are apposed at less than 30 nm, generate regions of important lipid and calcium exchange. This review principally focuses on the structure and the function of MCSs between the endoplasmic reticulum (ER) and the plasma membrane (PM). Here we describe how tethering structures form and maintain these junctions and, in some instances, participate in their function. We then discuss recent insights into the mechanisms by which specific classes of proteins mediate nonvesicular lipid exchange between the ER and PM and how such phenomena, already known to be crucial for maintaining organelle identity, are also emerging as regulators of cell growth and development Expected final online publication date for the Annual Review of Cell and Developmental Biology Volume 32 is October 06, 2016. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Cells adopt distinct signaling pathways to optimize cell locomotion in different physical microenvironments. However, the underlying mechanism that enables cells to sense and respond to physical confinement is unknown. Using microfabricated devices and substrate-printing methods along with FRET-based biosensors, we report that, as cells transition from unconfined to confined spaces, intracellular Ca2+ level is increased, leading to phosphodiesterase 1 (PDE1)-dependent suppression of PKA activity. This Ca2+ elevation requires Piezo1, a stretch-activated cation channel. Moreover, differential regulation of PKA and cell stiffness in unconfined versus confined cells is abrogated by dual, but not individual, inhibition of Piezo1 and myosin II, indicating that these proteins can independently mediate confinement sensing. Signals activated by Piezo1 and myosin II in response to confinement both feed into a signaling circuit that optimizes cell motility. This study provides a mechanism by which confinement-induced signaling enables cells to sense and adapt to different physical microenvironments.
From embryonic development to cancer metastasis, cell migration plays a central role in health and disease. It is increasingly becoming apparent that cells migrating in three-dimensional (3-D) environments exhibit some striking differences compared with their well-established 2-D counterparts. One key finding is the significant role the nucleus plays during 3-D migration: when cells move in confined spaces, the cell body and nucleus must deform to squeeze through available spaces, and the deformability of the large and relatively rigid nucleus can become rate-limiting. In this review, we highlight recent findings regarding the role of nuclear mechanics in 3-D migration, including factors that govern nuclear deformability, and emerging mechanisms by which cells apply cytoskeletal forces to the nucleus to facilitate nuclear translocation. Intriguingly, the 'physical barrier' imposed by the nucleus also impacts cytoplasmic dynamics that affect cell migration and signaling, and changes in nuclear structure resulting from the mechanical forces acting on the nucleus during 3-D migration could further alter cellular function. These findings have broad relevance to the migration of both normal and cancerous cells inside living tissues, and motivate further research into the molecular details by which cells move their nuclei, as well as the consequences of the mechanical stress on the nucleus.
The Drosophila blastoderm and the vertebrate neural tube are archetypal examples of morphogen-patterned tissues that create precise spatial patterns of different cell types. In both tissues, pattern formation is dependent on molecular gradients that emanate from opposite poles. Despite distinct evolutionary origins and differences in time scales, cell biology and molecular players, both tissues exhibit striking similarities in the regulatory systems that establish gene expression patterns that foreshadow the arrangement of cell types. First, signaling gradients establish initial conditions that polarize the tissue, but there is no strict correspondence between specific morphogen thresholds and boundary positions. Second, gradients initiate transcriptional networks that integrate broadly distributed activators and localized repressors to generate patterns of gene expression. Third, the correct positioning of boundaries depends on the temporal and spatial dynamics of the transcriptional networks. These similarities reveal design principles that are likely to be broadly applicable to morphogen-patterned tissues.
Significance
In animal tissue, most adherent cells round up against confinement to conduct mitosis. Impaired cell rounding is thought to perturb tissue development and homeostasis and contribute to progression of cancer. Due to the lack of suitable experimental tools, however, insight into the mechanical robustness of mitosis in animal cells remains limited. Here we introduce force-feedback–controlled ion beam-sculpted cantilevers to confine single cells and characterize their progression through mitosis. Our approach reveals critical yield forces that trigger cell cortex herniation, loss of F-actin homogeneity, dissipation of intracellular pressure, critical cell-height decrease, mitotic spindle defects, and resultant perturbation of mitotic progression.
Actin and myosin assemble into a thin layer of a highly dynamic network
underneath the membrane of eukaryotic cells. This network generates the forces
that drive cell and tissue-scale morphogenetic processes. The effective
material properties of this active network determine large-scale deformations
and other morphogenetic events. For example,the characteristic time of stress
relaxation (the Maxwell time)in the actomyosin sets the time scale of
large-scale deformation of the cortex. Similarly, the characteristic length of
stress propagation (the hydrodynamic length) sets the length scale of slow
deformations, and a large hydrodynamic length is a prerequisite for long-ranged
cortical flows. Here we introduce a method to determine physical parameters of
the actomyosin cortical layer (in vivo). For this we investigate the relaxation
dynamics of the cortex in response to laser ablation in the one-cell-stage {\it
C. elegans} embryo and in the gastrulating zebrafish embryo. These responses
can be interpreted using a coarse grained physical description of the cortex in
terms of a two dimensional thin film of an active viscoelastic gel. To
determine the Maxwell time, the hydrodynamic length and the ratio of active
stress and per-area friction, we evaluated the response to laser ablation in
two different ways: by quantifying flow and density fields as a function of
space and time, and by determining the time evolution of the shape of the
ablated region. Importantly, both methods provide best fit physical parameters
that are in close agreement with each other and that are similar to previous
estimates in the two systems. We provide an accurate and robust means for
measuring physical parameters of the actomyosin cortical layer.It can be useful
for investigations of actomyosin mechanics at the cellular-scale, but also for
providing insights in the active mechanics processes that govern tissue-scale
morphogenesis.
μManager is an open-source, cross-platform desktop application, to control a wide variety of motorized microscopes, scientific cameras, stages, illuminators, and other microscope accessories. Since its inception in 2005, μManager has grown to support a wide range of microscopy hardware and is now used by thousands of researchers around the world. The application provides a mature graphical user interface and offers open programming interfaces to facilitate plugins and scripts. Here, we present a guide to using some of the recently added advanced μManager features, including hardware synchronization, simultaneous use of multiple cameras, projection of patterned light onto a specimen, live slide mapping, imaging with multi-well plates, particle localization and tracking, and high-speed imaging.
3D amoeboid cell migration is central to many developmental and disease-related processes such as cancer metastasis. Here, we identify a unique prototypic amoeboid cell migration mode in early zebrafish embryos, termed stable-bleb migration. Stable-bleb cells display an invariant polarized balloon-like shape with exceptional migration speed and persistence. Progenitor cells can be reversibly transformed into stable-bleb cells irrespective of their primary fate and motile characteristics by increasing myosin II activity through biochemical or mechanical stimuli. Using a combination of theory and experiments, we show that, in stable-bleb cells, cortical contractility fluctuations trigger a stochastic switch into amoeboid motility, and a positive feedback between cortical flows and gradients in contractility maintains stable-bleb cell polarization. We further show that rearward cortical flows drive stable-bleb cell migration in various adhesive and non-adhesive environments, unraveling a highly versatile amoeboid migration phenotype.
Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
The mesenchymal-amoeboid transition (MAT) was proposed as a mechanism for cancer cells to adapt their migration mode to their environment. While the molecular pathways involved in this transition are well documented, the role of the microenvironment in the MAT is still poorly understood. Here, we investigated how confinement and adhesion affect this transition. We report that, in the absence of focal adhesions and under conditions of confinement, mesenchymal cells can spontaneously switch to a fast amoeboid migration phenotype. We identified two main types of fast migration-one involving a local protrusion and a second involving a myosin-II-dependent mechanical instability of the cell cortex that leads to a global cortical flow. Interestingly, transformed cells are more prone to adopt this fast migration mode. Finally, we propose a generic model that explains migration transitions and predicts a phase diagram of migration phenotypes based on three main control parameters: confinement, adhesion, and contractility.
Copyright © 2015 Elsevier Inc. All rights reserved.
Membrane fusion is an energy-consuming process that requires tight juxtaposition of two lipid bilayers. Little is known about how cells overcome energy barriers to bring their membranes together for fusion. Previously, we have shown that cell-cell fusion is an asymmetric process in which an "attacking" cell drills finger-like protrusions into the "receiving" cell to promote cell fusion. Here, we show that the receiving cell mounts a Myosin II (MyoII)-mediated mechanosensory response to its invasive fusion partner. MyoII acts as a mechanosensor, which directs its force-induced recruitment to the fusion site, and the mechanosensory response of MyoII is amplified by chemical signaling initiated by cell adhesion molecules. The accumulated MyoII, in turn, increases cortical tension and promotes fusion pore formation. We propose that the protrusive and resisting forces from fusion partners put the fusogenic synapse under high mechanical tension, which helps to overcome energy barriers for membrane apposition and drives cell membrane fusion.
Copyright © 2015 Elsevier Inc. All rights reserved.
ATR controls chromosome integrity and chromatin dynamics. We have previously shown that yeast Mec1/ATR promotes chromatin detachment from the nuclear envelope to counteract aberrant topological transitions during DNA replication. Here, we provide evidence that ATR activity at the nuclear envelope responds to mechanical stress. Human ATR associates with the nuclear envelope during S phase and prophase, and both osmotic stress and mechanical stretching relocalize ATR to nuclear membranes throughout the cell cycle. The ATR-mediated mechanical response occurs within the range of physiological forces, is reversible, and is independent of DNA damage signaling. ATR-defective cells exhibit aberrant chromatin condensation and nuclear envelope breakdown. We propose that mechanical forces derived from chromosome dynamics and torsional stress on nuclear membranes activate ATR to modulate nuclear envelope plasticity and chromatin association to the nuclear envelope, thus enabling cells to cope with the mechanical strain imposed by these molecular processes.
Background:
Bone metastases in prostate cancer (CaP) result in CaP-related morbidity/mortality. The omega-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA) and lipophilic statins affect metastasis-like behaviour in CaP cells, regulating the critical metastatic step of CaP migration to the bone marrow stroma.
Methods:
Microscopic analysis and measurement of adhesion and invasion of CaP cells through bone marrow endothelial cells (BMEC) was undertaken with AA stimulation and/or simvastatin (SIM) treatment. Amoeboid characteristics of PC-3, PC3-GFP and DU-145 were analysed by western blotting and Rho assays.
Results:
The CaP cell lines PC-3, PC3-GFP and DU-145 share the ability to migrate across a BMEC layer. Specific amoeboid inhibition decreased transendothelial migration (TEM). AA stimulates amoeboid characteristics, driven by Rho signalling. Selective knock-down of components of the Rho pathway (RhoA, RhoC, Rho-associated protein kinase 1 (ROCK1) and ROCK2) showed that Rho signalling is crucial to TEM. Functions of these components were analysed, regarding adhesion to BMEC, migration in 2D and the induction of the amoeboid phenotype by AA. TEM was reduced by SIM treatment of PC3-GFP and DU-145, which inhibited Rho pathway signalling.
Conclusions:
AA-induced TEM is mediated by the induction of a Rho-driven amoeboid phenotype. Inhibition of this cell migratory process may be an important therapeutic target in high-risk CaP.
Mechanical forces are known to influence cellular processes with consequences at the cellular and physiological level. The cell nucleus is the largest and stiffest organelle, and it is connected to the cytoskeleton for proper cellular function. The connection between the nucleus and the cytoskeleton is in most cases mediated by the linker of nucleoskeleton and cytoskeleton (LINC) complex. Not surprisingly, the nucleus and the associated cytoskeleton are implicated in multiple mechanotransduction pathways important for cellular activities. Herein, we review recent advances describing how the LINC complex, the nuclear lamina, and nuclear pore complexes are involved in nuclear mechanotransduction. We will also discuss how the perinuclear actin cytoskeleton is important for the regulation of nuclear mechanotransduction. Additionally, we discuss the relevance of nuclear mechanotransduction for cell migration, development, and how nuclear mechanotransduction impairment leads to multiple disorders.
The sensation of mechanical force underlies many of our daily activities. As the sense of touch determines the quality of life, the subconscious sense of proprioception and visceral mechanosensation is indispensible for survival. Many internal organs change shape, either as an active part of their physiology or passively due to body movements. Importantly, these shape changes need to be sensed and balanced properly to prevent organ failure and dysfunction. Consequently, a failure to properly sense volume changes of internal organs has a huge clinical relevance, manifested by a plethora of congenital and age-related diseases. Here we review novel data on mammalian stretch reception as well as classical studies from insect and nematode proprioceptors with the aim to highlight the missing link between organ-level deformation and mechanosensing on the molecular level.
There is increasing evidence that both mechanical and biochemical signals play important roles in development and disease. The development of complex organisms, in particular, has been proposed to rely on the feedback between mechanical and biochemical patterning events. This feedback occurs at the molecular level via mechanosensation but can also arise as an emergent property of the system at the cellular and tissue level. In recent years, dynamic changes in tissue geometry, flow, rheology, and cell fate specification have emerged as key platforms of mechanochemical feedback loops in multiple processes. Here, we review recent experimental and theoretical advances in understanding how these feedbacks function in development and disease.
Mechanical signals affect many aspects of biological processes. Physical forces from the extracellular microenvironment are ultimately transmitted to the nucleus and elicit a response that result in the deformation and remodeling of the nucleus. Recent studies have shown that nuclear deformation has several consequences such as reorganization of chromatin, changes in gene expression, and nuclear envelope rupture. It is widely believed that a direct coupling between the cytoskeleton and nucleoskeleton is required for nuclear deformation; however, some studies have proposed alternative mechanisms for nuclear deformation and the transmission of mechanical signals and stresses from the cytoskeleton to the nucleus. Herein, we review the processes, in which the cell nucleus experiences stresses and discuss the evidence of involvement of a direct link between the cytoskeleton and nucleoskeleton in nuclear deformation.
Migration is a vital, intricate, and multi-faceted process that involves the entire cell, entails the integration of multiple external cues and, at times, necessitates high-level coordination among fields of cells that can be physically attached or not, depending on the physiological setting. Recent advances have highlighted the essential role of cellular components that have not been traditionally considered when studying cell migration. This review details how much we recently learned by studying the role of intermediate filaments, the nucleus, extracellular vesicles, and mitochondria during cell migration.
The function of most immune cells depends on their ability to migrate through complex microenvironments, either randomly to patrol for the presence of antigens or directionally to reach their next site of action. The actin cytoskeleton and its partners are key conductors of immune cell migration as they control the intrinsic migratory properties of leukocytes as well as their capacity to respond to cues present in their environment. In this review we focus on the latest discoveries regarding the role of the actomyosin cytoskeleton in optimizing immune cell migration in complex environments, with a special focus on recent insights provided by physical modeling.
Cell migration is an adaptive process that depends on and responds to physical and molecular triggers. Moving cells sense and respond to tissue mechanics and induce transient or permanent tissue modifications, including extracellular matrix stiffening, compression and deformation, protein unfolding, proteolytic remodelling and jamming transitions. Here we discuss how the bi-directional relationship of cell-tissue interactions (mechanoreciprocity) allows cells to change position and contributes to single-cell and collective movement, structural and molecular tissue organization, and cell fate decisions.
YAP is a mechanosensitive transcriptional activator with a critical role in cancer, regeneration, and organ size control. Here, we show that force applied to the nucleus directly drives YAP nuclear translocation by decreasing the mechanical restriction of nuclear pores to molecular transport. Exposure to a stiff environment leads cells to establish a mechanical connection between the nucleus and the cytoskeleton, allowing forces exerted through focal adhesions to reach the nucleus. Force transmission then leads to nuclear flattening, which stretches nuclear pores, reduces their mechanical resistance to molecular transport, and increases YAP nuclear import. The restriction to transport is further regulated by the mechanical stability of the transported protein, which determines both active nuclear transport of YAP and passive transport of small proteins. Our results unveil a mechanosensing mechanism mediated directly by nuclear pores, demonstrated for YAP but with potential general applicability in transcriptional regulation. Force-dependent changes in nuclear pores control protein access to the nucleus.
Despite acting as a barrier for the organs they encase, epithelial cells turn over at some of the fastest rates in the body. However, epithelial cell division must be tightly linked to cell death to preserve barrier function and prevent tumour formation. How does the number of dying cells match those dividing to maintain constant numbers? When epithelial cells become too crowded, they activate the stretch-activated channel Piezo1 to trigger extrusion of cells that later die. However, it is unclear how epithelial cell division is controlled to balance cell death at the steady state. Here we show that mammalian epithelial cell division occurs in regions of low cell density where cells are stretched. By experimentally stretching epithelia, we find that mechanical stretch itself rapidly stimulates cell division through activation of the Piezo1 channel. To stimulate cell division, stretch triggers cells that are paused in early G2 phase to activate calcium-dependent phosphorylation of ERK1/2, thereby activating the cyclin B transcription that is necessary to drive cells into mitosis. Although both epithelial cell division and cell extrusion require Piezo1 at the steady state, the type of mechanical force controls the outcome: stretch induces cell division, whereas crowding induces extrusion. How Piezo1-dependent calcium transients activate two opposing processes may depend on where and how Piezo1 is activated, as it accumulates in different subcellular sites with increasing cell density. In sparse epithelial regions in which cells divide, Piezo1 localizes to the plasma membrane and cytoplasm, whereas in dense regions in which cells extrude, it forms large cytoplasmic aggregates. Because Piezo1 senses both mechanical crowding and stretch, it may act as a homeostatic sensor to control epithelial cell numbers, triggering extrusion and apoptosis in crowded regions and cell division in sparse regions.
Cell migration results from stepwise mechanical and chemical interactions between cells and their extracellular environment. Mechanistic principles that determine single-cell and collective migration modes and their interconversions depend upon the polarization, adhesion, deformability, contractility, and proteolytic ability of cells. Cellular determinants of cell migration respond to extracellular cues, including tissue composition, topography, alignment, and tissue-associated growth factors and cytokines. Both cellular determinants and tissue determinants are interdependent; undergo reciprocal adjustment; and jointly impact cell decision making, navigation, and migration outcome in complex environments. We here review the variability, decision making, and adaptation of cell migration approached by live-cell, in vivo, and in silico strategies, with a focus on cell movements in morphogenesis, repair, immune surveillance, and cancer metastasis. Expected final online publication date for the Annual Review of Cell and Developmental Biology Volume 32 is October 06, 2016. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Tissue damage activates cytosolic phospholipase A2 (cPLA2), releasing arachidonic acid (AA), which is oxidized to proinflammatory eicosanoids by 5-lipoxygenase (5-LOX) on the nuclear envelope. How tissue damage is sensed to activate cPLA2 is unknown. We investigated this by live imaging in wounded zebrafish larvae, where damage of the fin tissue causes osmotic cell swelling at the wound margin and the generation of a chemotactic eicosanoid signal. Osmotic swelling of cells and their nuclei activates cPla2 by translocating it from the nucleoplasm to the nuclear envelope. Elevated cytosolic Ca(2+) was necessary but not sufficient for cPla2 translocation, and nuclear swelling was required in parallel. cPla2 translocation upon nuclear swelling was reconstituted in isolated nuclei and appears to be a simple physical process mediated by tension in the nuclear envelope. Our data suggest that the nucleus plays a mechanosensory role in inflammation by transducing cell swelling and lysis into proinflammatory eicosanoid signaling.
To remove dying or unwanted cells from an epithelium while preserving the barrier function of the layer, epithelia use a unique process called cell extrusion. To extrude, the cell fated to die emits the lipid Sphingosine 1 Phosphate (S1P), which binds the G-protein-coupled receptor Sphingosine 1 Phosphate receptor 2 (S1P2) in the neighboring cells that activates Rho-mediated contraction of an actomyosin ring circumferentially and basally. This contraction acts to squeeze the cell out apically while drawing together neighboring cells and preventing any gaps to the epithelial barrier. Epithelia can extrude out cells targeted to die by apoptotic stimuli to repair the barrier in the face of death or extrude live cells to promote cell death when epithelial cells become too crowded. Indeed, because epithelial cells naturally turn over by cell death and division at some of the highest rates in the body, epithelia depend on crowding-induced live cell extrusion to preserve constant cell numbers. If extrusion is defective, epithelial cells rapidly lose contact inhibition and form masses. Additionally, because epithelia act as the first line of defense in innate immunity, preservation of this barrier is critical for preventing pathogens from invading the body. Given its role in controlling constant cell numbers and maintaining barrier function, a number of different pathologies can result when extrusion is disrupted. Here, we review mechanisms and signaling pathways that control epithelial extrusion and discuss how defects in these mechanisms can lead to multiple diseases. We also discuss tactics pathogens have devised to hijack the extrusion process to infect and colonize epithelia.
Repairing tears in the nuclear envelope
The nuclear envelope segregates genomic DNA from the cytoplasm and regulates protein trafficking between the cytosol and the nucleus. Maintaining nuclear envelope integrity during interphase is considered crucial. However, Raab et al. and Denais et al. show that migrating immune and cancer cells experience frequent and transitory nuclear envelope ruptures when they move through tight spaces (see the Perspective by Burke). The nuclear envelope reseals rapidly during interphase, assisted by components of the ESCRT III membrane-remodeling machinery.
Science , this issue pp. 359 and 353 ; see also p. 295
Repairing tears in the nuclear envelope
The nuclear envelope segregates genomic DNA from the cytoplasm and regulates protein trafficking between the cytosol and the nucleus. Maintaining nuclear envelope integrity during interphase is considered crucial. However, Raab et al. and Denais et al. show that migrating immune and cancer cells experience frequent and transitory nuclear envelope ruptures when they move through tight spaces (see the Perspective by Burke). The nuclear envelope reseals rapidly during interphase, assisted by components of the ESCRT III membrane-remodeling machinery.
Science , this issue pp. 359 and 353 ; see also p. 295
Eukaryotic cell movement is characterized by very diverse migration modes. Recent studies show that cells can adapt to environmental cues, such as adhesion and geometric confinement, thereby readily switching their mode of migration. Among this diversity of motile behavior, actin flows have emerged as a highly conserved feature of both mesenchymal and amoeboid migration, and have also been identified as key regulators of cell polarity. This suggests that the various observed migration modes are continuous variations of elementary locomotion mechanisms, based on a very robust physical property of the actin/myosin system - its ability to sustain flows at the cell scale. This central role of actin/myosin flows is shown to affect the large scale properties of cell trajectories.
When cells move using integrin-based focal adhesions, they pull in the direction of motion with large, ∼100 Pa, stresses that contract the substrate. Integrin-mediated adhesions, however, are not required for in vivo confined migration. During focal adhesion-free migration, the transmission of propelling forces, and their magnitude and orientation, are not understood. Here, we combine theory and experiments to investigate the forces involved in adhesion-free migration. Using a non-adherent blebbing cell line as a model, we show that actin cortex flows drive cell movement through nonspecific substrate friction. Strikingly, the forces propelling the cell forward are several orders of magnitude lower than during focal-adhesion-based motility. Moreover, the force distribution in adhesion-free migration is inverted: it acts to expand, rather than contract, the substrate in the direction of motion. This fundamentally different mode of force transmission may have implications for cell-cell and cell-substrate interactions during migration in vivo.
Kupffer's vesicle (KV) is the zebrafish organ of laterality, patterning the embryo along its left-right (LR) axis. Regional differences in cell shape within the lumen-lining KV epithelium are essential for its LR patterning function. However, the processes by which KV cells acquire their characteristic shapes are largely unknown. Here, we show that the notochord induces regional differences in cell shape within KV by triggering extracellular matrix (ECM) accumulation adjacent to anterior-dorsal (AD) regions of KV. This localized ECM deposition restricts apical expansion of lumen-lining epithelial cells in AD regions of KV during lumen growth. Our study provides mechanistic insight into the processes by which KV translates global embryonic patterning into regional cell shape differences required for its LR symmetry-breaking function.
Copyright © 2014 Elsevier Inc. All rights reserved.
The way in which a cell migrates is influenced by the physical properties of its surroundings, in particular the properties of the extracellular matrix. How the physical aspects of the cell's environment affect cell migration poses a considerable challenge when trying to understand migration in complex tissue environments and hinders the extrapolation of in vitro analyses to in vivo situations. A comprehensive understanding of these problems requires an integrated biochemical and biophysical approach. In this Review, we outline the findings that have emerged from approaches that span these disciplines, with a focus on actin-based cell migration in environments with different stiffness, dimensionality and geometry.
Cells use actomyosin contractility to move through three-dimensional (3D) extracellular matrices. Contractility affects the
type of protrusions cells use to migrate in 3D, but the mechanisms are unclear. In this work, we found that contractility
generated high-pressure lobopodial protrusions in human cells migrating in a 3D matrix. In these cells, the nucleus physically
divided the cytoplasm into forward and rear compartments. Actomyosin contractility with the nucleoskeleton-intermediate filament
linker protein nesprin-3 pulled the nucleus forward and pressurized the front of the cell. Reducing expression of nesprin-3
decreased and equalized the intracellular pressure. Thus, the nucleus can act as a piston that physically compartmentalizes
the cytoplasm and increases the hydrostatic pressure between the nucleus and the leading edge of the cell to drive lamellipodia-independent
3D cell migration.