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Stabilization and motility mechanism of blebs in cancer cells
Abstract
Previously associated with apoptosis, blebs have arisen in the past decade as important structures for amoeboid cell migration, particularly for cancer cells. Blebs are formed when the plasma membrane detaches from the actomyosin cortex. They retract exerting friction forces and allowing cells to migrate. In recent years, a few independent studies have reported large and stable blebs in cells under non-adhesive confinement. This universal switch to bleb-based migration has been found in amoeba, choanoflagellates, immortalized cell lines and primary cultures. Unlike previous blebs described, they are able to overcome retraction and stabilize a constant flow. Stable blebs are a new type of cellular structures that amoeboid cells use to migrate, analogous to filopodia or lamellipodia for mesenchymal cells. In a single cell, multiple blebs form and compete against each other, so that eventually a single bleb drives the migration. Thus, it is important to know how single blebs are stabilized to understand how single-bleb amoeboid cells polarize. More generally, stable actomyosin flows constitute the basis of fast migration in numerous cell types, including also immune cells. During my Ph.D. I studied bleb morphogenesis and bleb stabilization in confined cancer cells, using advanced microfluidic techniques to control the confinement of cells. The first part of my project describes the blebs forming as an immediate response of cells to confinement and what differentiates it from a classical retracting bleb. The second part of my project focuses on the mechanism leading to the establishment of a retrograde flow. Based on the results I obtained with my experiments, we propose that bleb stabilization depends on 1) the depletion of actin by myosin contractility and 2) the particular actin filament arrangement at the bleb tip caused by the membrane topology of a confined cell. I completed this work with advanced imaging which allowed observation of single actin filaments and tagged cytoskeleton-associated molecules at the bleb tip, under different perturbations. This unique set of observations allowed to complete a model for the stabilization of motile blebs, with conclusions that can be generally applied to any flowing actomyosin cortex. My results show three cortex regimes in blebs: 1) Assembling loose cortex: localized at the tip, composed of single filaments poorly attached to the membrane. If this region is lost, the bleb retracts. 2) Crosslinked cortex: actin filaments and fibers bind together to form a network which gradually gets denser and reticulated but do not contract (this region is devoid of Myosin II motors). 3) Contractile cortex: towards the base of the bleb. Myosin-II starts to get enriched contracting the dense actin network, driving the entire retrograde actin flow up to the tip of the bleb, generating new actin free regions at the tip and pressurizing the bleb, leading to membrane protrusion at the very front.
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Significance
Bleb-driven cell migration plays important roles in diverse biological processes. Here, we present the mechanism for polarity establishment and maintenance in blebbing cells in vivo. We show that actin polymerization defines the leading edge, the position where blebs form. We show that the cell front can direct the formation of the rear by facilitating retrograde flow of proteins that limit the generation of blebs at the opposite aspect of the cell. Conversely, localization of bleb-inhibiting proteins at one aspect of the cell results in the establishment of the cell front at the opposite side. These antagonistic interactions result in robust polarity that can be initiated in a random direction, or oriented by a chemokine gradient.
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 .
Cell shape is controlled by the submembranous cortex, an actomyosin network mainly generated by two actin nucleators: the Arp2/3 complex and the formin mDia1. Changes in relative nucleator activity may alter cortical organization, mechanics and cell shape. Here we investigate how nucleation-promoting factors mediate interactions between nucleators. In vitro, the nucleation-promoting factor SPIN90 promotes formation of unbranched filaments by Arp2/3, a process thought to provide the initial filament for generation of dendritic networks. Paradoxically, in cells, SPIN90 appears to favour a formin-dominated cortex. Our in vitro experiments reveal that this feature stems mainly from two mechanisms: efficient recruitment of mDia1 to SPIN90–Arp2/3 nucleated filaments and formation of a ternary SPIN90–Arp2/3–mDia1 complex that greatly enhances filament nucleation. Both mechanisms yield rapidly elongating filaments with mDia1 at their barbed ends and SPIN90–Arp2/3 at their pointed ends. Thus, in networks, SPIN90 lowers branching densities and increases the proportion of long filaments elongated by mDia1.
Actin cortex controls cell migration
Cell migration is mainly controlled by local actin polymerization–driven membrane protrusion. However, a second structural mechanism might also regulate membrane protrusions and directed migration: changes in the density of the attachment between the plasma membrane and the underlying F-actin cortex, a parameter related to membrane tension. Many types of attachment and signaling mechanisms are known to alter the density of membrane-proximal cortical actin. Bisaria et al. designed a membrane-proximal F-actin (MPA) reporter that could directly measure local changes in the density of MPA in living cells. Levels of MPA were surprisingly low toward the front of migrating cells despite an opposing high overall concentration of F-actin in the same front region. The researchers propose that MPA density can integrate different signaling processes to direct local membrane protrusions and stabilize cell polarity during cell migration.
Science , this issue p. 1205
Cortical actomyosin flows, among other mechanisms, scale up spontaneous symmetry breaking and thus play pivotal roles in cell differentiation, division, and motility. According to many model systems, myosin motor-induced local contractions of initially isotropic actomyosin cortices are nucleation points for generating cortical flows. However, the positive feedback mechanisms by which spontaneous contractions can be amplified towards large-scale directed flows remain mostly speculative. To investigate such a process on spherical surfaces, we reconstituted and confined initially isotropic minimal actomyosin cortices to the interfaces of emulsion droplets. The presence of ATP leads to myosin-induced local contractions that self-organize and amplify into directed large-scale actomyosin flows. By combining our experiments with theory, we found that the feedback mechanism leading to a coordinated directional motion of actomyosin clusters can be described as asymmetric cluster vibrations, caused by intrinsic non-isotropic ATP consumption with spatial confinement. We identified fingerprints of vibrational states as the basis of directed motions by tracking individual actomyosin clusters. These vibrations may represent a generic key driver of directed actomyosin flows under spatial confinement in vitro and in living systems.
Eukaryotic cells migrate by coupling the intracellular force of the actin cytoskeleton to the environment. While force coupling is usually mediated by transmembrane adhesion receptors, especially those of the integrin family, amoeboid cells such as leukocytes can migrate extremely fast despite very low adhesive forces¹. Here we show that leukocytes cannot only migrate under low adhesion but can also transmit forces in the complete absence of transmembrane force coupling. When confined within three-dimensional environments, they use the topographical features of the substrate to propel themselves. Here the retrograde flow of the actin cytoskeleton follows the texture of the substrate, creating retrograde shear forces that are sufficient to drive the cell body forwards. Notably, adhesion-dependent and adhesion-independent migration are not mutually exclusive, but rather are variants of the same principle of coupling retrograde actin flow to the environment and thus can potentially operate interchangeably and simultaneously. As adhesion-free migration is independent of the chemical composition of the environment, it renders cells completely autonomous in their locomotive behaviour.
Centering and decentering of cellular components is essential for internal organization of cells and their ability to perform basic cellular functions such as division and motility. How cells achieve proper localization of their organelles is still not well-understood, especially in large cells such as oocytes. Here, we study actin-based positioning mechanisms in artificial cells with persistently contracting actomyosin networks, generated by encapsulating cytoplasmic Xenopus egg extracts into cell-sized ‘water-in-oil’ droplets. We observe size-dependent localization of the contraction center, with a symmetric configuration in larger cells and a polar one in smaller cells. Centering is achieved via a hydrodynamic mechanism based on Darcy friction between the contracting network and the surrounding cytoplasm. During symmetry breaking, transient attachments to the cell boundary drive the contraction center to a polar location. The centering mechanism is cell-cycle dependent and weakens considerably during interphase. Our findings demonstrate a robust, yet tunable, mechanism for subcellular localization.
Mechano-sensation of cells is an important prerequisite for cellular function, e.g. in the context of cell migration , tissue organisation and morphogenesis. An important mechano-chemical-transducer is the actin cytoskeleton. In fact, previous studies have shown that actin cross-linkers, such as α-actinin-4, exhibit mechanosensitive properties in its binding dynamics to actin polymers. However, to date, a quantitative analysis of tension-dependent binding dynamics in live cells is lacking. Here, we present a new technique that allows to quantitatively characterize the dependence of cross-linking lifetime of actin cross-linkers on mechanical tension in the actin cortex of live cells. We use an approach that combines parallel plate confinement of round cells, fluorescence recovery after photo-bleaching, and a mathematical mean-field model of cross-linker binding. We apply our approach to the actin cross-linker α-actinin-4 and show that the cross-linking time of α-actinin-4 homodimers increases approximately twofold within the cellular range of cortical mechanical tension rendering α-actinin-4 a catch bond in physiological tension ranges.
Recent in vivo studies reveal that several membrane proteins are driven to form nanoclusters by active contractile flows arising from localized dynamic patterning of F-actin and myosin at the cortex. Since myosin-II assemble as minifilaments with tens of myosin heads, one might worry that steric considerations would obstruct the emergence of nanoclustering. Using coarse-grained, agent-based simulations that account for steric constraints, we find that the patterns exhibited by actomyosin in two dimensions, do not resemble the steady-state patterns in our in vitro reconstitution of actomyosin on a supported bilayer. We perform simulations in a thin rectangular slab, separating the layer of actin filaments from myosin-II minifilaments. This recapitulates the observed features of in vitro patterning. Using super resolution microscopy, we find evidence for such stratification in our in vitro system. Our study suggests that molecular stratification may be an important organizing feature of the cortical cytoskeleton in vivo.
The plasma membrane and the underlying cytoskeletal cortex constitute active platforms for a variety of cellular processes. Recent work has shown that the remodeling acto-myosin network modifies local membrane organization, but the molecular details are only partly understood due to difficulties with experimentally accessing the relevant time and length scales. Here, we use interferometric scattering (iSCAT) microscopy to investigate a minimal acto-myosin network linked to a supported lipid bilayer membrane. Using the magnitude of the interferometric contrast, which is proportional to molecular mass, and fast acquisition rates, we detect, and image individual membrane attached actin filaments diffusing within the acto-myosin network and follow individual myosin II filament dynamics. We quantify myosin II filament dwell times and processivity as functions of ATP concentration, providing experimental evidence for the predicted ensemble behavior of myosin head domains. Our results show how decreasing ATP concentrations lead to both increasing dwell times of individual myosin II filaments and a global change from a remodeling to a contractile state of the acto-myosin network.
The assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell movement, shape change, and mechanics, but how different sets of proteins localize to these structures within a shared cytoplasm remains unclear. Here, we show that the actin-binding domains of accessory proteins can be sensitive to filament conformational changes induced by perturbations that include other binding proteins, stabilizing drugs, and physical constraints on the filament. Using a combination of live cell imaging and in vitro single molecule binding measurements, we demonstrate that the affinity of tandem calponin homology domain (CH1-CH2) mutants varies as actin filament twist is altered, and we show differential localization of native and mutant CH1-CH2 domains to actin networks at the front and rear of motile cells. These findings suggest that conformational heterogeneity of actin filaments in cells could influence the biochemical composition of cytoskeletal structures through a biophysical feedback loop.
The shape of many eukaryotic cells depends on the actin cytoskeleton; and localized changes in actin assembly dynamics underlie many changes in cell shape. Polymerases of the Ena/VASP family modulate cell shape by locally accelerating actin filament assembly and slowing filament capping. When concentrated into discrete foci at the leading edge, VASP promotes formation of filopodia, but the mechanisms that drive VASP clustering are poorly understood. Here we show that, in migrating B16F1 cells, VASP molecules assemble on pre-existing foci of the adaptor protein, lamellipodin, and that dimerization of lamellipodin is essential for cluster formation. VASP/lamellipodin clusters grow by accumulating monomers and by fusing, but their growth is limited by a previously undescribed, size-dependent instability. Our results demonstrate that assembly and disassembly dynamics of filopodia tip complexes are determined, in part, by a network of multivalent interactions between VASP, lamellipodin, and actin.
Contractile actomyosin networks generate intracellular forces essential for the regulation of cell shape, migration, and cell-fate decisions, ultimately leading to the remodeling and patterning of tissues. Although actin filaments aligned in bundles represent the main source of traction-force production in adherent cells, there is increasing evidence that these bundles form interconnected and interconvertible structures with the rest of the intracellular actin network. In this study, we explored how these bundles are connected to the surrounding cortical network and the mechanical impact of these interconnected structures on the production and distribution of traction forces on the extracellular matrix and throughout the cell. By using a combination of hydrogel micropatterning, traction-force microscopy and laser photoablation, we measured the relaxation of the cellular traction field in response to local photoablations at various positions within the cell. Our experimental results and modeling of the mechanical response of the network revealed that bundles were fully embedded along their entire length in a continuous and contractile network of cortical filaments. Moreover, the propagation of the contraction of these bundles throughout the entire cell was dependent on this embedding. In addition, these bundles appeared to originate from the alignment and coalescence of thin and unattached cortical actin filaments from the surrounding mesh.
Significance
Cells migrating within the body perform vital functions in development and for defense and repair of tissues. In this dense environment, cells encounter mechanical forces and constraints not experienced when moving under buffer, and, accordingly, many change how they move. We find that gentle squashing, which mimics mechanical resistance, causes cells to move using blebs—a form of projection driven by fluid pressure—rather than pseudopods. This behavior depends on the Piezo stretch-operated ion channel in the cell membrane and calcium fluxes into the cell. Piezo is highly conserved and is required for light touch sensation; this work extends its functions into migrating cells.
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 their local environment remains largely unknown. Here we show that the nucleus, the biggest cellular organelle, functions as a non-dissipative cellular shape deformation gauge that enables cells to continuously measure shape variations on the time scale of seconds. Inner nuclear membrane unfolding together with the relative spatial intracellular positioning of the nucleus provides physical information on the amplitude and type of cellular shape deformation. This adaptively activates a calcium-dependent mechano-transduction pathway, controlling the level of 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 behaviour to their microenvironment.
One Sentence Summary
The nucleus functions as an active deformation sensor that enables cells to adapt their behavior to the tissue microenvironment.
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.
How local interactions of actin regulators yield large-scale organization of cell shape and movement is not well understood. For example, why does the WAVE complex build lamellipodia, the broad sheet-like protrusions that power cell migration, whereas the homologous actin regulator N-WASP forms spiky finger-like actin networks? N-WASP is known to oligomerize into focal condensates that generate an actin finger. In contrast, the WAVE complex exhibits the linear distribution needed to generate an actin sheet. This linear organization of the WAVE complex could either arise from interactions with the actin cytoskeleton or could represent an ability of the complex to self-organize into a linear template. Using super-resolution microscopy, we find that the WAVE complex forms higher-order linear oligomers that curve into 270 nanometer-wide ring structures in the absence of actin polymer. These rings localize to the necks of membrane invaginations, which display saddle point geometries with positive curvature in one axis and negative curvature in the orthogonal axis. To investigate the molecular mechanism of saddle curvature enrichment, we show that the WAVE complex and IRSp53, a membrane curvature-sensitive protein, collaborate to recognize saddle curvature that IRSp53 cannot sense alone. This saddle preference for the WAVE complex could explain emergent cell behaviors, such as expanding and self-straightening lamellipodia as well as the ability of endothelial cells to recognize and seal transcellular holes. Our work highlights how partnering protein interactions enable complex shape sensing and how feedback between cell shape and actin regulators yields self-organized cell morphogenesis.
Cell migration is hypothesized to involve a cycle of behaviours beginning with leading edge extension. However, recent evidence suggests that the leading edge may be dispensable for migration, raising the question of what actually controls cell directionality. Here, we exploit the embryonic migration of Drosophila macrophages to bridge the different temporal scales of the behaviours controlling motility. This approach reveals that edge fluctuations during random motility are not persistent and are weakly correlated with motion. In contrast, flow of the actin network behind the leading edge is highly persistent. Quantification of actin flow structure during migration reveals a stable organization and asymmetry in the cell-wide flowfield that strongly correlates with cell directionality. This organization is regulated by a gradient of actin network compression and destruction, which is controlled by myosin contraction and cofilin-mediated disassembly. It is this stable actin-flow polarity, which integrates rapid fluctuations of the leading edge, that controls inherent cellular persistence.
The actin cytoskeleton drives many essential biological processes, from cell morphogenesis to motility. Assembly of functional actin networks requires control over the speed at which actin filaments grow. How this can be achieved at the high and variable levels of soluble actin subunits found in cells is unclear. Here we reconstitute assembly of mammalian, non-muscle actin filaments from physiological concentrations of profilin-actin. We discover that under these conditions, filament growth is limited by profilin dissociating from the filament end and the speed of elongation becomes insensitive to the concentration of soluble subunits. Profilin release can be directly promoted by formin actin polymerases even at saturating profilin-actin concentrations. We demonstrate that mammalian cells indeed operate at the limit to actin filament growth imposed by profilin and formins. Our results reveal how synergy between profilin and formins generates robust filament growth rates that are resilient to changes in the soluble subunit concentration.
Migratory cells use distinct motility modes to navigate different microenvironments, but it is unclear whether these modes rely on the same core set of polarity components. To investigate this, we disrupted actin-related protein 2/3 (Arp2/3) and the WASP-family verprolin homologous protein (WAVE) complex, which assemble branched actin networks that are essential for neutrophil polarity and motility in standard adherent conditions. Surprisingly, confinement rescues polarity and movement of neutrophils lacking these components, revealing a processive bleb-based protrusion program that is mechanistically distinct from the branched actin-based protrusion program but shares some of the same core components and underlying molecular logic. We further find that the restriction of protrusion growth to one site does not always respond to membrane tension directly, as previously thought, but may rely on closely linked properties such as local membrane curvature. Our work reveals a hidden circuit for neutrophil polarity and indicates that cells have distinct molecular mechanisms for polarization that dominate in different microenvironments.
Eukaryotic cells migrate by coupling the intracellular force of the actin cytoskeleton to the environment. While force-coupling is usually mediated by transmembrane adhesion receptors, especially these of the integrin family, amoeboid cells like leukocytes can migrate extremely fast despite very low adhesive forces. We show that leukocytes cannot only migrate under low adhesion but indeed can transduce forces in the complete absence of transmembrane force coupling. When confined within three-dimensional environments, they use the topographic features of the substrate to propel themselves. Here, the retrograde flow of the actin cytoskeleton follows the texture of the substrate, creating shear forces sufficient to drive deformations towards the back of the cell. Notably, adhesion-dependent and adhesion-independent migration are not exclusive but rather variants of the same principle of coupling retrograde actin flow to the environment and thus can potentially operate simultaneously. As adhesion free migration is independent of the chemical composition of the environment it renders cells completely autonomous in their locomotive behavior.
A key factor of actin cytoskeleton organization in cells is the interplay between the dynamical properties of actin filaments and cell geometry, which restricts, confines and directs their orientation. Crosslinking interactions among actin filaments, together with geometrical cues and regulatory proteins can give rise to contractile rings in dividing cells and actin rings in neurons. Motivated by recent in vitro experiments, in this work we performed computer simulations to study basic aspects of the interplay between confinement and attractive interactions between actin filaments. We used a spring-bead model and Brownian dynamics to simulate semiflexible actin filaments that polymerize in a confining sphere with a rate proportional to the monomer concentration. We model crosslinking, or attraction through the depletion interaction, implicitly as an attractive short-range potential between filament beads. In confining geometries smaller than the persistence length of actin filaments, we show rings can form by curving of filaments of length comparable to, or longer than the confinement diameter. Rings form for optimal ranges of attractive interactions that exist in between open bundles, irregular loops, aggregated and unbundled morphologies. The probability of ring formation is promoted by attraction to the confining sphere boundary and decreases for large radii and initial monomer concentrations, in agreement with prior experimental data. The model reproduces ring formation along the flat axis of oblate ellipsoids. This article is protected by copyright. All rights reserved.
Cells change direction of migration by sensing rigidity of environment and traction force, yet its underlying mechanism is unclear. Here we show that tip actin barbed ends serve as an active ‘force sensor’ at the leading edge. We established a method to visualize intracellular single‐molecule fluorescent actin through an elastic culture substrate. We found that immediately after cell edge stretch, actin assembly increased specifically at the lamellipodium tip. The rate of actin assembly increased with increasing stretch speed. Furthermore, tip actin polymerization remained elevated at the subsequent hold step, which was accompanied by a decrease in the load on the tip barbed ends. Stretch‐induced tip actin polymerization was still observed without either the WAVE complex or Ena/VASP proteins. The observed relationships between forces and tip actin polymerization are consistent with a force‐velocity relationship as predicted by the Brownian ratchet mechanism. Stretch caused extra membrane protrusion with respect to the stretched substrate and increased local tip polymerization by >5% of total cellular actin in 30 sec. Our data reveal that augmentation of lamellipodium tip actin assembly is directly coupled to the load decrease, which may serve as a force sensor for directed cell protrusion.
Contraction of cortical actomyosin networks driven by myosin activation controls cell shape changes and tissue morphogenesis during animal development. In vitro studies suggest that contractility also depends on the geometrical organization of actin filaments. Here we analyze the function of actomyosin network topology in vivo using optogenetic stimulation of myosin-II in Drosophila embryos. We show that early during cellularization, hexagonally arrayed actomyosin fibers are resilient to myosin-II activation. Actomyosin fibers then acquire a ring-like conformation and become contractile and sensitive to myosin-II. This transition is controlled by Bottleneck, a Drosophila unique protein expressed for only a short time during early cellularization, which we show regulates actin bundling. In addition, it requires two opposing actin cross-linkers, Filamin and Fimbrin. Filamin acts synergistically with Bottleneck to facilitate hexagonal patterning, while Fimbrin controls remodeling of the hexagonal network into contractile rings. Thus, actin cross-linking regulates the spatio-temporal organization of actomyosin contraction in vivo, which is critical for tissue morphogenesis.
Cancer cells migrate by using different membrane protrusions including plasma membrane blebs. Blebs are formed when the plasma membrane delaminates from the underlying cortex due to actomyosin contractility-induced increase in cytoplasmic pressure, and this causes influx of water and cytosolic fluid through aquaporins into the detached membrane resulting in its enlargement. In this study, we used a combination of molecular biology and microscopy techniques to examine the involvement of aquaporin 1 (AQP1) in bleb formation, and report for the first time that, siRNA-mediated knockdown of AQP1 inhibited bleb formation in both HT1080 and ACHN cell lines embedded in 3D matrigel matrix. In HT1080 cells, blebs resurfaced 48 h post-siRNA transfection, and this paralleled AQP1 protein expression, as shown by Western blotting. By using confocal and phase-contrast time-lapse microscopy to track bleb dynamics, we demonstrate that the re-emergent blebs had longer lifespan due to longer time of expansion, stabilization and retraction when compared with wildtype blebs. Furthermore, overexpression of GFP-tagged AQP1 significantly increased bleb size, and also reduced bleb lifespan by conferring on the cells a shorter and faster bleb retraction time. To unravel the mechanism underpinning AQP1-facilitated bleb retraction, our data showed a requirement for the sodium-hydrogen exchanger (Na + /H +) activity, as inhibition of the pump's activity attenuated bleb retraction. Similarly, and importantly, we demonstrate that GFP-AQP1 overexpression sufficiently induced blebbing phenotype in non-blebbing MDA-MB-231 cell line. Thus, AQP1 is a potential therapeutic target in combating cancer cell migration.
Cellular protrusions are typically considered as distinct structures associated with specific regulators. However, we found that these regulators coordinately localize as propagating cortical waves, suggesting a common underlying mechanism. These molecular events fell into two excitable networks, the signal transduction network STEN and the cytoskeletal network CEN with different wave substructures. Computational studies using a coupled-network model reproduced these features and showed that the morphology and kinetics of the waves depended on strengths of feedback loops. Chemically induced dimerization at multiple nodes produced distinct, coordinated alterations in patterns of other network components. Taken together, these studies indicate: STEN positive feedback is mediated by mutual inhibition between Ras/Rap and PIP2, while negative feedback depends on delayed PKB activation; PKBs link STEN to CEN; CEN includes positive feedback between Rac and F-actin, and exerts fast positive and slow negative feedbacks to STEN The alterations produced protrusions resembling filopodia, ruffles, pseudopodia, or lamellipodia, suggesting that these structures arise from a common regulatory mechanism and that the overall state of the STEN-CEN system determines cellular morphology. © 2019 The Authors. Published under the terms of the CC BY 4.0 license.
Blebs are cellular protrusions observed in migrating cells and in cells undergoing spreading, cytokinesis, and apoptosis. Here we investigate the flow of cytoplasm during bleb formation and the concurrent changes in cell volume using zebrafish primordial germ cells (PGCs) as an in vivo model. We show that bleb inflation occurs concomitantly with cytoplasmic inflow into it and that during this process the total cell volume does not change. We thus show that bleb formation in primordial germ cells results primarily from redistribution of material within the cell rather than being driven by flow of water from an external source.
Persistent directional movement of neutrophils in shallow chemotactic gradients raises the possibility that cells can increase their sensitivity to the chemotactic signal at the front, relative to the back. Redistribution of chemoattractant receptors to the anterior pole of a polarized neutrophil could impose asymmetric sensitivity by increasing the relative strength of detected signals at the cell's leading edge. Previous experiments have produced contradictory observations with respect to receptor location in moving neutro-phils. To visualize a chemoattractant receptor directly during chemotaxis, we expressed a green fluorescent protein (GFP)-tagged receptor for a complement component, C5a, in a leukemia cell line, PLB-985. Differentiated PLB-985 cells, like neutrophils, adhere, spread, and polarize in response to a uniform concentration of chemoattractant, and orient and crawl toward a micropipette containing chemoattractant. Recorded in living cells, fluorescence of the tagged receptor, C5aR-GFP, shows no apparent increase anywhere on the plasma membrane of polarized and moving cells, even at the leading edge. During chemotaxis, however, some cells do exhibit increased amounts of highly folded plasma membrane at the leading edge, as detected by a fluorescent probe for membrane lipids; this is accompanied by an apparent increase of C5aR-GFP fluorescence, which is directly proportional to the accumulation of plasma membrane. Thus neutrophils do not actively concentrate chemoattractant receptors at the leading edge during chemotaxis, although asymmetrical distribution of membrane may enrich receptor number, relative to adjacent cytoplasmic volume, at the anterior pole of some polarized cells. This enrichment could help to maintain persistent migration in a shallow gradient of che-moattractant.
Rodent CNS neuroblasts show parallel and perpendicular contact guidance behaviors on aligned neurite bundles in microexplant cultures (Nakatsuji, N. and Nagata, I. (1989) Development, 106, 441–447; N. I. and N. N. (1991) ibid., 112, 581–590). To test the hypothesis that the physical surface structure of the neurite bundle causes the perpendicular contact guidance, we cultured dissociated neuroblasts on quartz plates on which grating-like microstructures were fabricated by lithographic techniques. Various types of CNS neuroblasts, but not PNS neurons, oriented their processes and migrated both perpendicular and parallel to the axis of the microstructure. Perpendicular orientation was frequently observed when the microstructured grooves had depths between 0.3 micron and 0.8 micron and a width of 1 micron, which roughly mimics a tightly aligned neurite bundle. Thus, CNS neuroblasts have the ability to extend their processes and migrate perpendicular to aligned surface microstructures.
The cell cortex is a contractile actin meshwork, which determines cell shape and is essential for cell mechanics, migration and division. Because the cortical thickness is below optical resolution, it has been generally considered as a thin uniform two-dimensional layer. Using two mutually attracted magnetic beads, one inside the cell and the other in the extracellular medium, we pinch the cortex of dendritic cells and provide an accurate and time resolved measure of its thickness. Our observations draw a new picture of the cell cortex as a highly dynamic layer, harboring large fluctuations in its third dimension due to actomyosin contractility. We propose that the cortex dynamics might be responsible for the fast shape changing capacity of highly contractile cells that use amoeboid-like migration.
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.
Networks of branched actin filaments formed by Arp2/3 complex generate and experience mechanical forces during essential cellular functions, including cell motility and endocytosis. External forces regulate the assembly and architecture of branched actin networks both in vitro and in cells. Considerably less is known about how mechanical forces influence the disassembly of actin filament networks, specifically, the dissociation of branches. We used microfluidics to apply force to branches formed from purified muscle actin and fission yeast Arp2/3 complex and observed debranching events in real time with total internal reflection fluorescence microscopy. Low forces in the range of 0 pN to 2 pN on branches accelerated their dissociation from mother filaments more than two orders of magnitude, from hours to <1 min. Neither force on the mother filament nor thermal fluctuations in mother filament shape influenced debranching. Arp2/3 complex at branch junctions adopts two distinct mechanical states with different sensitivities to force, which we name "young/strong" and "old/weak." The "young/strong" state 1 has adenosine 5'-diphosphate (ADP)-P
i
bound to Arp2/3 complex. Phosphate release converts Arp2/3 complex into the "old/weak" state 2 with bound ADP, which is 20 times more sensitive to force than state 1. Branches with ADP-Arp2/3 complex are more sensitive to debranching by fission yeast GMF (glia maturation factor) than branches with ADP-P
i
-Arp2/3 complex. These findings suggest that aging of branch junctions by phosphate release from Arp2/3 complex and mechanical forces contribute to disassembling "old" actin filament branches in cells.
Motile cells have developed a variety of migration modes relying on diverse traction-force-generation mechanisms. Before the behavior of intracellular components could be easily imaged, cell movements were mostly classified by different types of cellular shape dynamics. Indeed, even though some types of cells move without any significant change in shape, most cell propulsion mechanisms rely on global or local deformations of the cell surface. In this review, focusing mostly on metazoan cells, we discuss how different types of local and global shape changes underlie distinct migration modes. We then discuss mechanical differences between force-generation mechanisms and finish by speculating on how they may have evolved.
Self-assembly and force generation are two central processes in biological systems that usually are considered in separation. However, the signals that activate nonmuscle myosin II molecular motors simultaneously lead to self-assembly into myosin II minifilaments as well as progression of the motor heads through the cross-bridge cycle. Here we investigate theoretically the possible effects of coupling these two processes. Our assembly model, which builds on a consensus architecture of the minifilament, predicts a critical aggregation concentration at which the assembly kinetics slows down dramatically. The combined model predicts that increasing actin filament concentration and force both lead to a decrease in the critical aggregation concentration. We suggest that due to these effects, myosin II minifilaments in a filamentous context might be in a critical state that reacts faster to varying conditions than in solution. We finally compare our model to experiments by simulating fluorescence recovery after photobleaching.
Mimicking the cellular microenvironment is important for organoids and organ on-a-chip studies. One of the current issues is to introduce vessel-like structures into the culture system to improve the cellular and tissue functions, which deserves particular efforts in design and systematic consideration. Based on a standard device configuration, we fabricated a vessel-like component which can be easily integrated for cell co-culture. This component consists of an embedded monolayer of gelatin nanofibers on the top of an open channel. It can then be enclosed with an upper plastic plate with molded chamber, channels and standard Luer connectors. Human umbilical vein endothelial cells (HUVECs) were firstly introduced into the vessel-like channel and cultivated three-dimensionally with the help of a rotational device. Then, a flow was applied for cytoskeleton remolding, resulted in a dense and aligned HUVECs layer. Afterward, human glioblastoma cells (U87) were introduced in the upside of the fiber layer and a flow was also applied for the upper cell layer culture. Our results show adjunct formation of HUVEC and U87 cell layers on both sides of the monolayer of gelatin nanofibers, thereby providing a reliable support for a variety of co-culture assays.
The field of biomechanics significantly progressed in the last two decades. The importance of the feedback between biochemical signaling and physical properties was revealed in many studies. Cells within tissues constantly generate and experience mechanical forces. Biochemical perturbations inside the cells as well as alterations in the mechanical environment can shift the tiny balance of normal physiological state and lead to pathologies, e.g. cancer. Although the mechanical properties of individual cells can alter when they are within the tissues, the understanding of single cell mechanics is still important. Differentiation, immune cell migration, and cancer invasion strongly depend on the mechanical properties of individual cells. Mechanical deformations can lead to a change in cell surface area and volume. We are particularly interested in single mammalian cell volume regulation in the context of deformations of different timescales. For the moment, volume regulation in this context was out from the research interest, probably due to the difficulties of accurate measurements, and cell volume often considered as a constant parameter. We developed a method for cell volume measurements based on a fluorescent exclusion that allowed us to perform precise volume measurements of individual live cells. In the present study, we mainly focused on cell volume regulation while dynamic spreading on a substrate (timescale – minutes). We demonstrated that there are different regimes for volume regulation while spreading: cells decrease, increase or do not change volume, and a type of the regime depends on the state of the actomyosin cortex and spreading speed. We obtained that faster-spreading cells tend to lose more volume. Our hypothesis is that during fast Arp2/3-driven lamellipodia extension actin pull on the membrane that generates tension and activation of ion transport and regulatory volume loss. Inhibition of actin polymerization or Arp2/3-dependent actin branching decreases spreading speed and volume loss. Next, we showed that inhibition of contractility increases spreading speed and volume loss. However, inhibition of Arp2/3 complex in cells with low contractility leads to fast spreading without volume loss. Our explanation is that inhibition of Arp2/3 induces cell blebbing and even fast deformation does not lead to volume loss as a cell can relax tension by membrane unfolding. We also showed that volume regulation in response to fast mechanical compression (timescale – milliseconds) independent of adhesion also depends on the actomyosin cortex state. Control cells lose up to 30% of volume under confinement, as the cell membrane is attached to the cortex and cannot be unfolded in response to the tension increase. Disruption of actin cortex leads to membrane detachment and prevents volume loss under confinement. Additionally, we showed that cell volume response to the osmotic shock (timescale – seconds) is more complex than it used to be known in the literature. For instance, our data indicate that at the level of individual cells initial volume response to the change of external osmolarity is not a uniform passive process. Using osmotic shock technique, we also confirmed that cells have a large excess of membrane folded in reservoirs. Taken together, our data show that cell volume and surface area are coupled through surface tension homeostasis and as deformations induce surface tension increase, they lead to change volume and surface area.
Significance
Dendritic cells are immune cells that migrate within the human body in search of pathogens. This search is performed by a random walk, which combines persistent and diffuse movements. The mechanism underlying this random walk is currently not known. We analyzed very long trajectories of dendritic cells ex vivo, and we describe here their characteristic persistent and diffusive patterns. Together with theoretical analysis, we provide evidence that the random walk of dendritic cells could be a consequence of the intrinsic actin dynamics without need for molecular noise or external polarization cues. Our results hint at the possibility that these cells can adapt their random search strategies by changing the spontaneous dynamics of their actin cytoskeleton.
Oscillations occur in a wide variety of essential cellular processes, such as cell cycle progression, circadian clocks and calcium signaling in response to stimuli. It remains unclear how intrinsic stochasticity can influence these oscillatory systems. Here, we focus on oscillations of Cdc42 GTPase in fission yeast. We extend our previous deterministic model by Xu and Jilkine to construct a stochastic model, focusing on the fast diffusion case. We use SSA (Gillespie's algorithm) to numerically explore the low copy number regime in this model, and use analytical techniques to study the long-time behavior of the stochastic model and compare it to the equilibria of its deterministic counterpart. Numerical solutions suggest noisy limit cycles exist in the parameter regime in which the deterministic system converges to a stable limit cycle, and quasi-cycles exist in the parameter regime where the deterministic model has a damped oscillation. Near an infinite period bifurcation point, the deterministic model has a sustained oscillation, while stochastic trajectories start with an oscillatory mode and tend to approach deterministic steady states. In the low copy number regime, metastable transitions from oscillatory to steady behavior occur in the stochastic model. Our work contributes to the understanding of how stochastic chemical kinetics can affect a finite-dimensional dynamical system, and destabilize a deterministic steady state leading to oscillations.
Lamellipodial locomotion of fish keratocytes is one of the simplest examples of actin-based motility. In the last four decades, fruitful collaborations between experimentalists and theorists have resulted in a detailed mechanistic understanding of the self-organized lamellipodial engine powering keratocyte motility. Here we review the mechanical mechanisms underlying keratocyte migration, highlighting the interplay between modeling and experiments that led to insights regarding the dynamics of actin network organization, cell shape, and self-polarization. We discuss how to apply lessons learnt from keratocytes to understand cell migration in more complex, physiological contexts.
Cell migration is essential for physiological processes as diverse as development, immune defence and wound healing. It is also a hallmark of cancer malignancy. Thousands of publications have elucidated detailed molecular and biophysical mechanisms of cultured cells migrating on flat, 2D substrates of glass and plastic. However, much less is known about how cells successfully navigate the complex 3D environments of living tissues. In these more complex, native environments, cells use multiple modes of migration, including mesenchymal, amoeboid, lobopodial and collective, and these are governed by the local extracellular microenvironment, specific modalities of Rho GTPase signalling and non-muscle myosin contractility. Migration through 3D environments is challenging because it requires the cell to squeeze through complex or dense extracellular structures. Doing so requires specific cellular adaptations to mechanical features of the extracellular matrix (ECM) or its remodelling. In addition, besides navigating through diverse ECM environments and overcoming extracellular barriers, cells often interact with neighbouring cells and tissues through physical and signalling interactions. Accordingly, cells need to call on an impressively wide diversity of mechanisms to meet these challenges. This Review examines how cells use both classical and novel mechanisms of locomotion as they traverse challenging 3D matrices and cellular environments. It focuses on principles rather than details of migratory mechanisms and draws comparisons between 1D, 2D and 3D migration.
We present ilastik, an easy-to-use interactive tool that brings machine-learning-based (bio)image analysis to end users without substantial computational expertise. It contains pre-defined workflows for image segmentation, object classification, counting and tracking. Users adapt the workflows to the problem at hand by interactively providing sparse training annotations for a nonlinear classifier. ilastik can process data in up to five dimensions (3D, time and number of channels). Its computational back end runs operations on-demand wherever possible, allowing for interactive prediction on data larger than RAM. Once the classifiers are trained, ilastik workflows can be applied to new data from the command line without further user interaction. We describe all ilastik workflows in detail, including three case studies and a discussion on the expected performance. ilastik is an user-friendly interactive tool for machine-learning-based image segmentation, object classification, counting and tracking.
Recent development of innovative tools for live imaging of actin filaments (F-actin) enabled the detection of surprising nuclear structures responding to various stimuli, challenging previous models that actin is substantially monomeric in the nucleus. We review these discoveries, focusing on double-strand break (DSB) repair responses. These studies revealed a remarkable network of nuclear filaments and regulatory mechanisms coordinating chromatin dynamics with repair progression and led to a paradigm shift by uncovering the directed movement of repair sites. In this Review, Caridi et al. discuss actin filaments in the nucleus and the functions of nuclear F-actin in response to DNA double-strand break repair.
The size of a cell is determined by a combination of synthesis, self-assembly, incoming matter and the balance of mechanical forces. Such processes operate at the single-cell level, but they are deeply interconnected with cell-cycle progression, resulting in a stable average cell size at the population level. Here, we examine this phenomenon by reviewing the physics of growth processes that operate at vastly different timescales, but result in the controlled production of daughter cells that are close copies of their mothers. We first review the regulatory mechanisms of size at short timescales, focusing on the contribution of fundamental physical forces. We then discuss the multiple relevant regulation processes operating on the timescale of the cell cycle. Finally, we look at how these processes interact: one of the most important challenges to date involves bridging the gap between timescales, connecting the physics of cell growth and the biology of cell-cycle progression.
The spatiotemporal coordination of actin regulators in the lamellipodium determines the dynamics and architecture of branched F-actin networks during cell migration. The WAVE regulatory complex (WRC), an effector of Rac1 during cell protrusion, is concentrated at the lamellipodium tip. Thus, activated Rac1 should operate at this location to activate WRC and trigger membrane protrusion. Yet correlation of Rho GTPase activation with cycles of membrane protrusion previously revealed complex spatiotemporal patterns of Rac1 and RhoA activation in the lamellipodium. Combining single protein tracking (SPT) and super-resolution imaging with loss- or gain-of-function mutants of Rho GTPases, we show that Rac1 immobilizations at the lamellipodium tip correlate with its activation, in contrast to RhoA. Using Rac1 effector loop mutants and wild-type versus mutant variants of WRC, we show that selective immobilizations of activated Rac1 at the lamellipodium tip depend on effector binding, including WRC. In contrast, wild-type Rac1 only displays slower diffusion at the lamellipodium tip, suggesting transient activations. Local optogenetic activation of Rac1, triggered by membrane recruitment of Tiam1, shows that Rac1 activation must occur close to the lamellipodium tip and not behind the lamellipodium to trigger efficient membrane protrusion. However, coupling tracking with optogenetic activation of Rac1 demonstrates that diffusive properties of wild-type Rac1 are unchanged despite enhanced lamellipodium protrusion. Taken together, our results support a model whereby transient activations of Rac1 occurring close to the lamellipodium tip trigger WRC binding. This short-lived activation ensures a local and rapid control of Rac1 actions on its effectors to trigger actin-based protrusion.
Bleb-type cellular protrusions play key roles in a range of biological processes. It was recently found that bleb growth is facilitated by a local supply of membrane from tubular invaginations, but the interplay between the expanding bleb and the membrane tubes remains poorly understood. On the one hand, the membrane area stored in tubes may serve as a reservoir for bleb expansion. On the other hand, the sequestering of excess membrane in stabilized invaginations may effectively increase the cell membrane tension, which suppresses spontaneous protrusions. Here, we investigate this duality through physical modeling and in vivo experiments. In agreement with observations, our model describes the transition into a tube-flattening mode of bleb expansion while also predicting that the blebbing rate is impaired by elevating the concentration of the curved membrane proteins that form the tubes. We show both theoretically and experimentally that the stabilizing effect of tubes could be counterbalanced by the cortical myosin contractility. Our results largely suggest that proteins able to induce membrane tubulation, such as those containing N-BAR domains, can buffer the effective membrane tension-a master regulator of all cell deformations.
Actin networks in the bulk cytoplasm, rather than cortical dynamics, drive ooplasm segregation in zebrafish oocytes. A contracting actin network drags the ooplasm toward the animal pole, while ‘comet tails’ push the yolk granules in the opposite direction.
Cell morphogenesis employs a diversity of membrane protrusions. They are discriminated by differences in force generation. Actin polymerization is the best studied mechanism of force generation, but growing interest in how variable molecular conditions and microenvironments alter morphogenesis has revealed other mechanisms, including intracellular pressure. Here, we show that local depletion of membrane cortex links is an essential step in the initiation of both pressure-based and actin-based protrusions. This observation challenges the quarter-century old Brownian ratchet model of actin-driven membrane protrusion, which requires an optimal balance of actin filament growth and membrane tethering. An updated model confirms membrane-filament detachment is necessary to activate the ratchet mechanism. These findings unify the regulation of different protrusion types, explaining how cells generate robust yet flexible strategies of morphogenesis.
The results of molecular dynamics simulations of the dynamical evolution of assemblies of linear rigid rods of variable aspect ratio, a, and number density, ρ, in the isotropic phase are reported. The rods consist of m equally spaced sites interacting with the Weeks-Chandler-Andersen repulsive pair potential, where 2 < m < 16. With increasing m, features specific to long rods, such as anisotropic self-diffusion, become apparent. There is also an increasing separation between the characteristic relaxation times of the torque, angular velocity, and reorientational time correlation functions with increasing density. The latter is exponential at high densities even for dimers. The isotropic translational diffusion coefficient, Di, and rotational diffusion coefficient, Dr, are reported as a function of m and ρ or volume fraction, ξ. The mDi data scale with ξ throughout much of the simulated range, while the rotational diffusion coefficients scale approximately as m³Dr against ρ at low densities but as ∼m⁶Dr at high ρ, consistent with theories of colloidal and noncolloidal rod-containing liquids. The crossover density between the two regimes is parameterized in analytic form. The probability distribution functions for displacements and angular jumps in a given time show evidence of non-Gaussian behavior with increasing density. The shear viscosity and Di scale approximately as m and m⁻¹, respectively, in the semidilute regime, which is consistent with a Stokes-Einstein-like relationship. At high concentrations, a frustrated or glassy structure formed in which the rods were randomly oriented.