Actin Retrograde Flow Research Articles

3D live imaging and phenotyping of CAR-T cell mediated-cytotoxicity using high-throughput Bessel oblique plane microscopy

Clarification of the cytotoxic function of T cells is crucial for understanding human immune responses and immunotherapy procedures. Here, we report a high-throughput Bessel oblique plane microscopy (HBOPM) platform capable of 3D live imaging and phenotyping of chimeric antigen receptor (CAR)-modified T-cell cytotoxicity against cancer cells. The HBOPM platform has the following characteristics: an isotropic subcellular resolution of 320 nm, large-scale scouting over 400 interacting cell pairs, long-term observation across 5 hours, and quantitative analysis of the Terabyte-scale 3D, multichannel, time-lapse image datasets. Using this advanced microscopy platform, several key subcellular events in CAR-T cells are captured and comprehensively analyzed; these events include the instantaneous formation of immune synapses and the sustained changes in the microtubing morphology. Furthermore, we identify the actin retrograde flow speed, the actin depletion coefficient, the microtubule polarization and the contact area of the CAR-T/target cell conjugates as essential parameters strongly correlated with CAR-T-cell cytotoxic function. Our approach will be useful for establishing criteria for quantifying T-cell function in individual patients for all T-cell-based immunotherapies.

A simple active fluid model unites cytokinesis, cell crawling, and axonal outgrowth.

Axonal outgrowth, cell crawling, and cytokinesis utilize actomyosin, microtubule-based motors, cytoskeletal dynamics, and substrate adhesions to produce traction forces and bulk cellular motion. While it has long been appreciated that growth cones resemble crawling cells and that the mechanisms that drive cytokinesis help power cell crawling, they are typically viewed as unique processes. To better understand the relationship between these modes of motility, here, we developed a unified active fluid model of cytokinesis, amoeboid migration, mesenchymal migration, neuronal migration, and axonal outgrowth in terms of cytoskeletal flow, adhesions, viscosity, and force generation. Using numerical modeling, we fit subcellular velocity profiles of the motions of cytoskeletal structures and docked organelles from previously published studies to infer underlying patterns of force generation and adhesion. Our results indicate that, during cytokinesis, there is a primary converge zone at the cleavage furrow that drives flow towards it; adhesions are symmetric across the cell, and as a result, cells are stationary. In mesenchymal, amoeboid, and neuronal migration, the site of the converge zone shifts, and differences in adhesion between the front and back of the cell drive crawling. During neuronal migration and axonal outgrowth, the primary convergence zone lies within the growth cone, which drives actin retrograde flow in the P-domain and bulk anterograde flow of the axonal shaft. They differ in that during neuronal migration, the cell body is weakly attached to the substrate and thus moves forward at the same velocity as the axon. In contrast, during axonal outgrowth, the cell body strongly adheres to the substrate and remains stationary, resulting in a decrease in flow velocity away from the growth cone. The simplicity with which cytokinesis, cell crawling, and axonal outgrowth can be modeled by varying coefficients in a simple model suggests a deep connection between them.

Influence of Curvature on Cell Motility and Morphology during Cancer Migration in Confined Microchannels.

Microchannels often serve as highways for cancer migration, and their topology largely determines the migration efficiency. Curvature, a topological parameter in biological systems, has recently been reported to be efficient in guiding cell polarization and migration. Curvature varies widely along curved microchannels, while its influence on cell migration remains elusive. Here, we recapitulated the curved microchannels, as observed in clinical tumor tissues with hydrogels, and studied how cancer cells respond to curvature. We found that cells bend more significantly in a larger curvature and exhibit less spreading as well as lower motility. The underlying mechanism is probably based on the hindrance of the movement of cytoskeletal molecules at the curved microchannel walls. Collectively, our results demonstrated that the accelerated actin retrograde flow rate under local curvature has an effective negative regulation on cell motility and morphology, leading to shortened and bent cell morphologies as well as hampered cell migration efficiency.

WAVE2 Regulates Actin-Dependent Processes Induced by the B Cell Antigen Receptor and Integrins.

B cell antigen receptor (BCR) signaling induces actin cytoskeleton remodeling by stimulating actin severing, actin polymerization, and the nucleation of branched actin networks via the Arp2/3 complex. This enables B cells to spread on antigen-bearing surfaces in order to increase antigen encounters and to form an immune synapse (IS) when interacting with antigen-presenting cells (APCs). Although the WASp, N-WASp, and WAVE nucleation-promoting factors activate the Arp2/3 complex, the role of WAVE2 in B cells has not been directly assessed. We now show that both WAVE2 and the Arp2/3 complex localize to the peripheral ring of branched F-actin when B cells spread on immobilized anti-Ig antibodies. The siRNA-mediated depletion of WAVE2 reduced and delayed B cell spreading on immobilized anti-Ig, and this was associated with a thinner peripheral F-actin ring and reduced actin retrograde flow compared to control cells. Depleting WAVE2 also impaired integrin-mediated B cell spreading on fibronectin and the LFA-1-induced formation of actomyosin arcs. Actin retrograde flow amplifies BCR signaling at the IS, and we found that depleting WAVE2 reduced microcluster-based BCR signaling and signal amplification at the IS, as well as B cell activation in response to antigen-bearing cells. Hence, WAVE2 contributes to multiple actin-dependent processes in B lymphocytes.

Actin Filaments Couple the Protrusive Tips to the Nucleus through the I-BAR Domain Protein IRSp53 during the Migration of Cells on 1D Fibers.

The cell migration cycle, well-established in2D, proceeds with forming new protrusive structures at the cell membrane andsubsequentredistribution of contractile machinery. Three-dimensional (3D) environments are complexand composed of 1D fibers, and 1D fibers are shown to recapitulateessential features of 3D migration. However, the establishment of protrusiveactivity at the cell membrane and contractilityin1Dfibrous environmentsremains partially understood. Here the role of membrane curvatureregulator IRSp53 is examined as a coupler between actin filaments and plasmamembraneduring cell migration on single, suspended 1D fibers. IRSp53 depletion reducedcell-length spanning actin stress fibers that originate from thecellperiphery, protrusive activity, and contractility, leading touncoupling of the nucleus from cellular movements. A theoreticalmodel capable of predicting the observed transition of IRSp53-depleted cellsfrom rapid stick-slip migration to smooth and slower migration due toreducedactin polymerization at the cell edges is developed, which is verified by directmeasurements of retrograde actin flow using speckle microscopy. Overall, it isfound that IRSp53 mediates actin recruitment at the cellular tips leading tothe establishment of cell-length spanning fibers, thus demonstrating a uniquerole of IRSp53 in controlling cell migration in 3D.

Integrin αIIbβ3 intermediates: From molecular dynamics to adhesion assembly

The platelet integrin αIIbβ3 undergoes long-range conformational transitions associated with its functional conversion from inactive (low-affinity) to active (high-affinity) during hemostasis. Although new conformations that are intermediate between the well-characterized bent and extended states have been identified, their molecular dynamic properties and functions in the assembly of adhesions remain largely unexplored. In this study, we evaluated the properties of intermediate conformations of integrin αIIbβ3 and characterized their effects on the assembly of adhesions by combining all-atom simulations, principal component analysis, and mesoscale modeling. Our results show that in the low-affinity, bent conformation, the integrin ectodomain tends to pivot around the legs; in intermediate conformations, the headpiece becomes partially extended, away from the lower legs. In the fully open, active state, αIIbβ3 is flexible, and the motions between headpiece and lower legs are accompanied by fluctuations of the transmembrane helices. At the mesoscale, bent integrins form only unstable adhesions, but intermediate or open conformations stabilize the adhesions. These studies reveal a mechanism by which small variations in ligand binding affinity and enhancement of the ligand-bound lifetime in the presence of actin retrograde flow stabilize αIIbβ3 integrin adhesions.

The glycocalyx affects the mechanotransductive perception of the topographical microenvironment

The cell/microenvironment interface is the starting point of integrin-mediated mechanotransduction, but many details of mechanotransductive signal integration remain elusive due to the complexity of the involved (extra)cellular structures, such as the glycocalyx. We used nano-bio-interfaces reproducing the complex nanotopographical features of the extracellular matrix to analyse the glycocalyx impact on PC12 cell mechanosensing at the nanoscale (e.g., by force spectroscopy with functionalised probes). Our data demonstrates that the glycocalyx configuration affects spatio-temporal nanotopography-sensitive mechanotransductive events at the cell/microenvironment interface. Opposing effects of major glycocalyx removal were observed, when comparing flat and specific nanotopographical conditions. The excessive retrograde actin flow speed and force loading are strongly reduced on certain nanotopographies upon strong reduction of the native glycocalyx, while on the flat substrate we observe the opposite trend. Our results highlight the importance of the glycocalyx configuration in a molecular clutch force loading-dependent cellular mechanism for mechanosensing of microenvironmental nanotopographical features.Graphical

Pulling, failing, and adaptive mechanotransduction of macrophage filopodia

Macrophages use filopodia to withdraw particles toward the cell body for phagocytosis. This can require substantial forces, which the cell generates after bio-mechanical stimuli are transmitted to the filopodium. Adaptation mechanisms to mechanical stimuli are essential for cells, but can a cell iteratively improve filopodia pulling? If so, the underlying mechanic adaptation principles organized on the protein level are unclear. Here, we tackle this problem using optically trapped 1 μm beads, which we tracked interferometrically at 1 MHz during connection to the tips of dorsal filopodia of macrophages. We observe repetitive failures while the filopodium tries to pull the bead out of the optical trap. Analyses of mean bead motions and position fluctuations on the nano-meter and microsecond scale indicate mechanical ruptures caused by a force-dependent actin-membrane connection. We found that beads are retracted three times slower under any load between 5 and 40 pN relative to the no-load transport, which has the same speed as the actin retrograde flow obtained from fluorescent speckle tracking. From this duty ratio of pulling velocities, we estimated a continuous on/off binding with τoff = 2⋅τon, with measured off times τoff = 0.1–0.5 s. Remarkably, we see a gradual increase of filopodia pulling forces from 10 to 30 pN over time and after failures, which points toward an unknown adaptation mechanism. Additionally, we see that the attachment strength and friction between the bead and filopodium tip increases under load and over time. All observations are typical for catch-bond proteins such as integrin-talin complexes. We present a mechanistic picture of adaptive mechanotransduction, which formed by the help of mathematical models for repetitive tip ruptures and reconnections. The analytic mathematical model and the stochastic computer simulations, both based on catch-bond lifetimes, confirmed our measurements. Such catch-bond characteristics could also be important for other immune cells taking up counteracting pathogens.

Actin Retrograde Flow Regulated by the Wiskott-Aldrich Syndrome Protein Drives the Natural Killer Cell Response.

Simple SummaryImmune cells sense mechanical stimuli from their microenvironment by transducing them into biochemical signals through mechanosensors, in a process called mechanotransduction. The cytoskeleton mediates mechanotransduction in response to local environmental changes. The cytoskeletal component, actin, provides the structural basis for the NK cell immunological synapse (NKIS) and polarization of the signaling complex and secretory apparatus. Actin-mediated mechanotransduction is essential to adjust and to improve the natural killer (NK) cell response to changes induced by the microenvironment. Wiskott–Aldrich Syndrome protein (WASp), another key cytoskeletal component, plays a role in actin filament nucleation, and hence it’s branching toward the membrane. Here, we found that WASp regulates mechanotransduction by controlling F-actin movement thereby modulating NK cell signaling and the equilibrium between activation versus inhibition. WASp regulation of the actin retrograde flow (ARF) provides a novel mechanism to modulate the functional outcome of NK cells in response to their environment.Understanding the crosstalk between natural killer (NK) cells and the tumor microenvironment (TME) has enhanced the potential of exploiting the interplay between activation and inhibition of NK cells for immunotherapy. This interaction is crucial for understanding how tumor cells escape NK cell immune surveillance. NK cell dysfunction is regulated by two molecular mechanisms, downregulated activating receptor ligand expression on the tumor cells, and upregulated inhibitory signals delivered to NK cells. Recent studies demonstrated the role of mechanotransduction in modulating NK cell responses in the TME. The immunological synapse represents a functional interface between the NK cell and its target, regulated by Actin Retrograde Flow (ARF), which drives the adhesion molecules and receptors toward the central zone of the immunological synapse (IS). Here, we further characterize the role of ARF in controlling the immune response of NK cells, using CRISPR/cas9-mediated Wiskott–Aldrich Syndrome protein (WASp) gene silencing of NK cells. We demonstrate that WASp regulates ARF velocity, affecting the conformation and function of the key NK inhibitory regulator, SH2-domain containing protein tyrosine phosphatase-1 (SHP-1), and consequently, the NK cell response. Our results demonstrate the potential of modulating the biophysical and intracellular regulation of NK activation as a promising approach for improving immunotherapy.

Adhesion-independent topography-based leukocyte migration.

Cells need to couple intracellular actin flows with the substrate to generate forward movement. This has traditionally been studied in the context of specific transmembrane receptors, particularly integrin adhesion receptors, which link extracellular adhesive molecules to the actin cytoskeleton. However, leukocytes and other cells can also migrate using integrin-independent strategies both in vivo and in vitro, though the cellular and environmental requirements for this mode are not fully understood. In seminal recent work, Reversat et al.1 develop a range of innovative 2D and 3D engineered microdevices and probe the biophysical mechanisms underlying T lymphocytes and dendritic cells in conditions of limited substrate adhesion. They identify a physical principle of mechano-coupling between retrograde actin flow and irregular extracellular confinement, which allows the cell to generate mechanical resistance and move in the absence of receptor-mediated adhesion. Through the combined use of experiments and theoretical modeling, this work resolves a long-standing question in cell biology and establishes mechanical interaction with an irregular-shaped 3D environment which may be relevant to cell migration in a range of tissue contexts.

On the significance of membrane unfolding in mechanosensitive cell spreading: Its individual and synergistic effects.

Mechanosensitivity of cell spread area to substrate stiffness has been established both through experiments and different types of mathematical models of varying complexity including both the mechanics and biochemical reactions in the cell. What has not been addressed in previous mathematical models is the role of cell membrane dynamics on cell spreading, and an investigation of this issue is the goal of this work. We start with a simple mechanical model of cell spreading on a deformable substrate and progressively layer mechanisms to account for the traction dependent growth of focal adhesions, focal adhesion induced actin polymerization, membrane unfolding/exocytosis and contractility. This layering approach is intended to progressively help in understanding the role each mechanism plays in reproducing experimentally observed cell spread areas. To model membrane unfolding we introduce a novel approach based on defining an active rate of membrane deformation that is dependent on membrane tension. Our modeling approach allows us to show that tension-dependent membrane unfolding plays a critical role in achieving the large cell spread areas experimentally observed on stiff substrates. We also demonstrate that coupling between membrane unfolding and focal adhesion induced polymerization works synergistically to further enhance cell spread area sensitivity to substrate stiffness. This enhancement has to do with the fact that the peripheral velocity of spreading cells is associated with contributions from the different mechanisms by either enhancing the polymerization velocity at the leading edge or slowing down of the retrograde flow of actin within the cell. The temporal evolution of this balance in the model corresponds to the three-phase behavior observed experimentally during spreading. In the initial phase membrane unfolding is found to be particularly important.

Modeling cell protrusion predicts how myosin II and actin turnover affect adhesion-based signaling

Orchestration of cell migration is essential for development, tissue regeneration, and the immune response. This dynamic process integrates adhesion, signaling, and cytoskeletal subprocesses across spatial and temporal scales. In mesenchymal cells, adhesion complexes bound to extracellular matrix mediate both biochemical signal transduction and physical interaction with the F-actin cytoskeleton. Here, we present a mathematical model that offers insight into both aspects, considering spatiotemporal dynamics of nascent adhesions, active signaling molecules, mechanical clutching, actin treadmilling, and nonmuscle myosin II contractility. At the core of the model is a positive feedback loop, whereby adhesion-based signaling promotes generation of barbed ends at, and protrusion of, the cell’s leading edge, which in turn promotes formation and stabilization of nascent adhesions. The model predicts a switch-like transition and optimality of membrane protrusion, determined by the balance of actin polymerization and retrograde flow, with respect to extracellular matrix density. The model, together with new experimental measurements, explains how protrusion can be modulated by mechanical effects (nonmuscle myosin II contractility and adhesive bond stiffness) and F-actin turnover.

Cyclical phosphorylation of LRAP35a and CLASP2 by GSK3β and CK1δ regulates EB1-dependent MT dynamics in cell migration.

Mammalian cell cytoskeletal reorganization for efficient directional movement requires tight coordination of actomyosin and microtubule networks. In this study, we show that LRAP35a potentiates microtubule stabilization by promoting CLASP2/EB1 interaction besides its complex formation with MRCK/MYO18A for retrograde actin flow. The alternate regulation of these two networks by LRAP35a is tightly regulated by a series of phosphorylation events that dictated its specificity. Sequential phosphorylation of LRAP35a by Protein Kinase A (PKA) and Glycogen Synthase Kinase-3β (GSK3β) initiates the association of LRAP35a with CLASP2, while subsequent binding and further phosphorylation by Casein Kinase 1δ (CK1δ) induce their dissociation, which facilitates LRAP35a/MRCK association in driving lamellar actomyosin flow. Importantly, microtubule dynamics is directly moderated by CK1δ activity on CLASP2 to regulate GSK3β phosphorylation of the SxIP motifs that blocks EB1 binding, an event countered by LRAP35a interaction and its competition for CK1δ activity. Overall this study reveals an essential role for LRAP35a in coordinating lamellar contractility and microtubule polarization in cell migration.

Mechanosensation and Mechanotransduction in Natural Killer Cells

Natural killer (NK) cells are a main subset of innate lymphocytes that contribute to host immune protection against viruses and tumors by mediating target cell killing and secreting a wide array of cytokines. Their functions are finely regulated by a balance between activating and inhibitory receptors and involve also adhesive interactions. Mechanotransduction is the process in which physical forces sensed by mechanosensors are translated into chemical signaling. Herein, we report findings on the involvement of this mechanism that is mainly mediated by actin cytoskeleton, in the regulation of NK cell adhesion, migration, tissue infiltration and functions. Actin represents the structural basis for NK cell immunological synapse (NKIS) and polarization of secretory apparatus. NK-target cell interaction involves the formation of both uropods and membrane nanotubes that allow target cell interaction over long distances. Actin retrograde flow (ARF) regulates NK cell signaling and controls the equilibrium between activation versus inhibition. Activating NKIS is associated with rapid lamellipodial ARF, whereas lower centripetal actin flow is present during inhibitory NKIS where β actin can associate with the tyrosine phosphatase SHP-1. Overall, a better knowledge of mechanotransduction might represent a future challenge: Realization of nanomaterials tailored for NK cells, would be important to translate in vitro studies in in vivo new immunotherapeutic approaches.

The Actin-Disassembly Protein Glia Maturation Factor γ Enhances Actin Remodeling and B Cell Antigen Receptor Signaling at the Immune Synapse.

Signaling by the B cell antigen receptor (BCR) initiates actin remodeling. The assembly of branched actin networks that are nucleated by the Arp2/3 complex exert outward force on the plasma membrane, allowing B cells to form membrane protrusions that can scan the surface of antigen-presenting cells (APCs). The resulting Arp2/3 complex-dependent actin retrograde flow promotes the centripetal movement and progressive coalescence of BCR microclusters, which amplifies BCR signaling. Glia maturation factor γ (GMFγ) is an actin disassembly-protein that releases Arp2/3 complex-nucleated actin filaments from actin networks. By doing so, GMFγ could either oppose the actions of the Arp2/3 complex or support Arp2/3 complex-nucleated actin polymerization by contributing to the recycling of actin monomers and Arp2/3 complexes. We now show that reducing the levels of GMFγ in human B cell lines via transfection with a specific siRNA impairs the ability of B cells to spread on antigen-coated surfaces, decreases the velocity of actin retrograde flow, diminishes the coalescence of BCR microclusters into a central cluster at the B cell-APC contact site, and decreases APC-induced BCR signaling. These effects of depleting GMFγ are similar to what occurs when the Arp2/3 complex is inhibited. This suggests that GMFγ cooperates with the Arp2/3 complex to support BCR-induced actin remodeling and amplify BCR signaling at the immune synapse.

Local actin dynamics couple speed and persistence in a cellular Potts model of cell migration

Cell migration is astoundingly diverse. Molecular signatures, cell-cell interactions, and environmental structures each play their part in shaping cell motion, yielding numerous morphologies and migration modes. Nevertheless, in recent years, a simple unifying law was found to describe cell migration across many different cell types and contexts: faster cells turn less frequently. This universal coupling between speed and persistence (UCSP) was explained by retrograde actin flow from front to back, but it remains unclear how this mechanism generalizes to cells with complex shapes and cells migrating in structured environments, which may not have a well-defined front-to-back orientation. Here, we present an in-depth characterization of an existing cellular Potts model, in which cells polarize dynamically from a combination of local actin dynamics (stimulating protrusions) and global membrane tension along the perimeter (inhibiting protrusions). We first show that the UCSP emerges spontaneously in this model through a cross talk of intracellular mechanisms, cell shape, and environmental constraints, resembling the dynamic nature of cell migration in vivo. Importantly, we find that local protrusion dynamics suffice to reproduce the UCSP—even in cases in which no clear global, front-to-back polarity exists. We then harness the spatial nature of the cellular Potts model to show how cell shape dynamics limit both the speed and persistence a cell can reach and how a rigid environment such as the skin can restrict cell motility even further. Our results broaden the range of potential mechanisms underlying the speed-persistence coupling that has emerged as a fundamental property of migrating cells.

Ecm29-Dependent Proteasome Localization Regulates Cytoskeleton Remodeling at the Immune Synapse.

The formation of an immune synapse (IS) enables B cells to capture membrane-tethered antigens, where cortical actin cytoskeleton remodeling regulates cell spreading and depletion of F-actin at the centrosome promotes the recruitment of lysosomes to facilitate antigen extraction. How B cells regulate both pools of actin, remains poorly understood. We report here that decreased F-actin at the centrosome and IS relies on the distribution of the proteasome, regulated by Ecm29. Silencing Ecm29 decreases the proteasome pool associated to the centrosome of B cells and shifts its accumulation to the cell cortex and IS. Accordingly, Ecm29-silenced B cells display increased F-actin at the centrosome, impaired centrosome and lysosome repositioning to the IS and defective antigen extraction and presentation. Ecm29-silenced B cells, which accumulate higher levels of proteasome at the cell cortex, display decreased actin retrograde flow in lamellipodia and enhanced spreading responses. Our findings support a model where B the asymmetric distribution of the proteasome, mediated by Ecm29, coordinates actin dynamics at the centrosome and the IS, promoting lysosome recruitment and cell spreading.

The actin-disassembly protein glia maturation factor γ enhances actin remodeling and B cell antigen receptor signaling at the immune synapse

Abstract Signaling by the B cell antigen receptor (BCR) initiates actin remodeling. The assembly of branched actin networks that are nucleated by the Arp2/3 complex exert outward force on the plasma membrane, allowing B cells to form membrane protrusions that can scan the surface of antigen-presenting cells (APCs). The resulting Arp2/3 complex-dependent actin retrograde flow promotes the centripetal movement and progressive coalescence of BCR microclusters, which amplifies BCR signaling. Glia maturation factor γ (GMFγ) is an actin disassembly-protein that releases Arp2/3 complex-nucleated actin filaments from actin networks. By doing so, GMFγ could either oppose the actions of the Arp2/3 complex or support Arp2/3 complex-nucleated actin polymerization by contributing to the recycling of actin monomers and Arp2/3 complexes. We now show that depleting GMFγ in human B cell lines impairs the ability of B cells to spread on antigen-coated surfaces, decreases the coalescence of BCR microclusters into a central cluster at the B cell-APC contact site, and reduces APC-induced BCR signaling. These effects of depleting GMFγ are similar to what occurs when the Arp2/3 complex is inhibited. This suggests that GMFγ cooperates with the Arp2/3 complex to support BCR-induced actin remodeling and amplify BCR signaling at the immune synapse.

Structural and functional analysis of LIM domain-dependent recruitment of paxillin to \u03b1v\u03b23 integrin-positive focal adhesions

The LIM domain-dependent localization of the adapter protein paxillin to β3 integrin-positive focal adhesions (FAs) is not mechanistically understood. Here, by combining molecular biology, photoactivation and FA-isolation experiments, we demonstrate specific contributions of each LIM domain of paxillin and reveal multiple paxillin interactions in adhesion-complexes. Mutation of β3 integrin at a putative paxillin binding site (β3VE/YA) leads to rapidly inward-sliding FAs, correlating with actin retrograde flow and enhanced paxillin dissociation kinetics. Induced mechanical coupling of paxillin to β3VE/YA integrin arrests the FA-sliding, thereby disclosing an essential structural function of paxillin for the maturation of β3 integrin/talin clusters. Moreover, bimolecular fluorescence complementation unveils the spatial orientation of the paxillin LIM-array, juxtaposing the positive LIM4 to the plasma membrane and the β3 integrin-tail, while in vitro binding assays point to LIM1 and/or LIM2 interaction with talin-head domain. These data provide structural insights into the molecular organization of β3 integrin-FAs.

Chemokine-biased robust self-organizing polarization of migrating cells in vivo

To study the mechanisms controlling front-rear polarity in migrating cells, we used zebrafish primordial germ cells (PGCs) as an in vivo model. We find that polarity of bleb-driven migrating cells can be initiated at the cell front, as manifested by actin accumulation at the future leading edge and myosin-dependent retrograde actin flow toward the other side of the cell. In such cases, the definition of the cell front, from which bleb-inhibiting proteins such as Ezrin are depleted, precedes the establishment of the cell rear, where those proteins accumulate. Conversely, following cell division, the accumulation of Ezrin at the cleavage plane is the first sign for cell polarity and this aspect of the cell becomes the cell back. Together, the antagonistic interactions between the cell front and back lead to a robust polarization of the cell. Furthermore, we show that chemokine signaling can bias the establishment of the front-rear axis of the cell, thereby guiding the migrating cells toward sites of higher levels of the attractant. We compare these results to a theoretical model according to which a critical value of actin treadmilling flow can initiate a positive feedback loop that leads to the generation of the front-rear axis and to stable cell polarization. Together, our in vivo findings and the mathematical model, provide an explanation for the observed nonoriented migration of primordial germ cells in the absence of the guidance cue, as well as for the directed migration toward the region where the gonad develops.