A theoretical model of efficient phagocytosis driven by curved membrane proteins and active cytoskeleton forces.

Phagocytosis is the process of engulfment and internalization of comparatively large particles by cells, and plays a central role in the functioning of our immune system. We study the process of phagocytosis by considering a simplified coarse grained model of a three-dimensional vesicle, having a uniform adhesion interaction with a rigid particle, and containing curved membrane-bound protein complexes or curved membrane nano-domains, which in turn recruit active cytoskeletal forces. Complete engulfment is achieved when the bending energy cost of the vesicle is balanced by the gain in the adhesion energy. The presence of curved (convex) proteins reduces the bending energy cost by self-organizing with a higher density at the highly curved leading edge of the engulfing membrane, which forms the circular rim of the phagocytic cup that wraps around the particle. This allows the engulfment to occur at much smaller adhesion strength. When the curved membrane-bound protein complexes locally recruit actin polymerization machinery, which leads to outward forces being exerted on the membrane, we found that engulfment is achieved more quickly and at a lower protein density. We consider spherical and non-spherical particles and found that non-spherical particles are more difficult to engulf in comparison to the spherical particles of the same surface area. For non-spherical particles, the engulfment time crucially depends on the initial orientation of the particles with respect to the vesicle. Our model offers a mechanism for the spontaneous self-organization of the actin cytoskeleton at the phagocytic cup, in good agreement with recent high-resolution experimental observations.

Membrane tension: A challenging but universal physical parameter in cell biology.

The plasma membrane separates the interior of cells from the outside environment. The membrane tension, defined as the force per unit length acting on a cross-section of membrane, regulates many vital biological processes. In this review, we summarize the first historical findings and the latest advances, showing membrane tension as an important physical parameter in cell biology. We also discuss how this parameter must be better integrated and we propose experimental approaches for key unanswered questions.

Plasma membrane tension orchestrates membrane trafficking, cytoskeletal remodeling, and biochemical signaling during phagocytosis.

Phagocytes clear the body of undesirable particles such as infectious agents and debris. To extend pseudopods over the surface of targeted particles during engulfment, cells must change shape through extensive membrane and cytoskeleton remodeling. We observed that pseudopod extension occurred in two phases. In the first phase, pseudopods extended rapidly, with actin polymerization pushing the plasma membrane forward. The second phase occurred once the membrane area from preexisting reservoirs was depleted, leading to increased membrane tension. Increased tension directly altered the small Rho GTPase Rac1, 3'-phosphoinositide, and cytoskeletal organization. Furthermore, it activated exocytosis of vesicles containing GPI-anchored proteins, increasing membrane area and phagocytosis efficiency for large particles. We thus propose that, during phagocytosis, membrane remodeling, cytoskeletal organization, and biochemical signaling are orchestrated by the mechanical signal of membrane tension. These results put a simple mechanical signal at the heart of understanding immunological responses.

Integrin-dependent force transmission to the extracellular matrix by α-actinin triggers adhesion maturation.

Focal adhesions are mechanosensitive elements that enable mechanical communication between cells and the extracellular matrix. Here, we demonstrate a major mechanosensitive pathway in which α-actinin triggers adhesion maturation by linking integrins to actin in nascent adhesions. We show that depletion of the focal adhesion protein α-actinin enhances force generation in initial adhesions on fibronectin, but impairs mechanotransduction in a subsequent step, preventing adhesion maturation. Expression of an α-actinin fragment containing the integrin binding domain, however, dramatically reduces force generation in depleted cells. This behavior can be explained by a competition between talin (which mediates initial adhesion and force generation) and α-actinin for integrin binding. Indeed, we show in an in vitro assay that talin and α-actinin compete for binding to ß3 integrins, but cooperate in binding to ß1 integrins. Consistently, we find opposite effects of α-actinin depletion and expression of mutants on substrates that bind ß3 integrins (fibronectin and vitronectin) versus substrates that only bind ß1 integrins (collagen). We thus suggest that nascent adhesions composed of ß3 integrins are initially linked to the actin cytoskeleton by talin, and then α-actinin competes with talin to bind ß3 integrins. Force transmitted through α-actinin then triggers adhesion maturation. Once adhesions have matured, α-actinin recruitment correlates with force generation, suggesting that α-actinin is the main link transmitting force between integrins and the cytoskeleton in mature adhesions. Such a multistep process enables cells to adjust forces on matrices, unveiling a role of α-actinin that is different from its well-studied function as an actin cross-linker.

Cell crawling mediates collective cell migration to close undamaged epithelial gaps.

Fundamental biological processes such as morphogenesis and wound healing involve the closure of epithelial gaps. Epithelial gap closure is commonly attributed either to the purse-string contraction of an intercellular actomyosin cable or to active cell migration, but the relative contribution of these two mechanisms remains unknown. Here we present a model experiment to systematically study epithelial closure in the absence of cell injury. We developed a pillar stencil approach to create well-defined gaps in terms of size and shape within an epithelial cell monolayer. Upon pillar removal, cells actively respond to the newly accessible free space by extending lamellipodia and migrating into the gap. The decrease of gap area over time is strikingly linear and shows two different regimes depending on the size of the gap. In large gaps, closure is dominated by lamellipodium-mediated cell migration. By contrast, closure of gaps smaller than 20 µm was affected by cell density and progressed independently of Rac, myosin light chain kinase, and Rho kinase, suggesting a passive physical mechanism. By changing the shape of the gap, we observed that low-curvature areas favored the appearance of lamellipodia, promoting faster closure. Altogether, our results reveal that the closure of epithelial gaps in the absence of cell injury is governed by the collective migration of cells through the activation of lamellipodium protrusion.

Temporary increase in plasma membrane tension coordinates the activation of exocytosis and contraction during cell spreading.

Cell migration and spreading involve the coordination of membrane trafficking, actomyosin contraction, and modifications to plasma membrane tension and area. The biochemical or biophysical basis for this coordination is however unknown. In this study, we show that during cell spreading, lamellipodia protrusion flattens plasma membrane folds and blebs and, once the plasma membrane area is depleted, there is a temporary increase in membrane tension by over twofold that is followed by activation of exocytosis and myosin contraction. Further, an artificial increase in plasma membrane tension stopped lamellipodia protrusion and activated an exocytotic burst. Subsequent decrease in tension restored spreading with activation of contraction. Conversely, blebbistatin inhibition of actomyosin contraction resulted in an even greater increase in plasma membrane tension and exocytosis activation. This spatiotemporal synchronization indicates that membrane tension is the signal that coordinates membrane trafficking, actomyosin contraction, and plasma membrane area change. We suggest that cells use plasma membrane tension as a global physical parameter to control cell motility.

Mechanotransduction through membrane tension: It's all about propagation?

Over the past 25 years, membrane tension has emerged as a primary mechanical factor influencing cell behavior. Although supporting evidences are accumulating, the integration of this parameter in the lifecycle of cells, organs, and tissues is complex. The plasma membrane is envisioned as a bilayer continuum acting as a 2D fluid. However, it possesses almost infinite combinations of proteins, lipids, and glycans that establish interactions with the extracellular or intracellular environments. This results in a tridimensional composite material with non-trivial dynamics and physics, and the task of integrating membrane mechanics and cellular outcome is a daunting chore for biologists. In light of the most recent discoveries, we aim in this review to provide non-specialist readers some tips on how to solve this conundrum.

Mechanically induced topological transition of spectrin regulates its distribution in the mammalian cell cortex.

The cell cortex is a dynamic assembly formed by the plasma membrane and underlying cytoskeleton. As the main determinant of cell shape, the cortex ensures its integrity during passive and active deformations by adapting cytoskeleton topologies through yet poorly understood mechanisms. The spectrin meshwork ensures such adaptation in erythrocytes and neurons by adopting different organizations. Erythrocytes rely on triangular-like lattices of spectrin tetramers, whereas in neurons they are organized in parallel, periodic arrays. Since spectrin is ubiquitously expressed, we exploited Expansion Microscopy to discover that, in fibroblasts, distinct meshwork densities co-exist. Through biophysical measurements and computational modeling, we show that the non-polarized spectrin meshwork, with the intervention of actomyosin, can dynamically transition into polarized clusters fenced by actin stress fibers that resemble periodic arrays as found in neurons. Clusters experience lower mechanical stress and turnover, despite displaying an extension close to the tetramer contour length. Our study sheds light on the adaptive properties of spectrin, which participates in the protection of the cell cortex by varying its densities in response to key mechanical features.

Biomimetic Approach of Brain Vasculature Rapidly Characterizes Inter- and Intra-Patient Migratory Diversity of Glioblastoma.

Glioblastomas exhibit remarkable heterogeneity at various levels, including motility modes and mechanoproperties that contribute to tumor resistance and recurrence. In a recent study using gridded micropatterns mimicking the brain vasculature, glioblastoma cell motility modes, mechanical properties, formin content, and substrate chemistry are linked. Now is presented, SP2G (SPheroid SPreading on Grids), an analytic platform designed to identify the migratory modes of patient-derived glioblastoma cells and rapidly pinpoint the most invasive sub-populations. Tumorspheres are imaged as they spread on gridded micropatterns and analyzed by this semi-automated, open-source, Fiji macro suite that characterizes migration modes accurately. SP2G can reveal intra-patient motility heterogeneity with molecular correlations to specific integrins and EMT markers. This system presents a versatile and potentially pan-cancer workflow to detect diverse invasive tumor sub-populations in patient-derived specimens and offers a valuable tool for therapeutic evaluations at the individual patient level.

Cytoskeletal coherence requires myosin-IIA contractility.

Maintaining a physical connection across cytoplasm is crucial for many biological processes such as matrix force generation, cell motility, cell shape and tissue development. However, in the absence of stress fibers, the coherent structure that transmits force across the cytoplasm is not understood. We find that nonmuscle myosin-II (NMII) contraction of cytoplasmic actin filaments establishes a coherent cytoskeletal network irrespective of the nature of adhesive contacts. When NMII activity is inhibited during cell spreading by Rho kinase inhibition, blebbistatin, caldesmon overexpression or NMIIA RNAi, the symmetric traction forces are lost and cell spreading persists, causing cytoplasm fragmentation by membrane tension that results in 'C' or dendritic shapes. Moreover, local inactivation of NMII by chromophore-assisted laser inactivation causes local loss of coherence. Actin filament polymerization is also required for cytoplasmic coherence, but microtubules and intermediate filaments are dispensable. Loss of cytoplasmic coherence is accompanied by loss of circumferential actin bundles. We suggest that NMIIA creates a coherent actin network through the formation of circumferential actin bundles that mechanically link elements of the peripheral actin cytoskeleton where much of the force is generated during spreading.

Physarum polycephalum percolation as a paradigm for topological phase transitions in transportation networks.

We study the formation of transportation networks of the true slime mold Physarum polycephalum after fragmentation by shear. Small fragments, called microplasmodia, fuse to form macroplasmodia in a percolation transition. At this topological phase transition, one single giant component forms, connecting most of the previously isolated microplasmodia. Employing the configuration model of graph theory for small link degree, we have found analytically an exact solution for the phase transition. It is generally applicable to percolation as seen, e.g., in vascular networks.

Early endosomes associated with dynamic F-actin structures are required for late trafficking of H. pylori VacA toxin.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are endocytosed by a clathrin- independent pathway into vesicles named GPI-AP-enriched early endosomal compartments (GEECs). We recently showed that the vacuolating toxin VacA secreted by Helicobacter pylori is endocytosed into the GEECs (Gauthier, N.C., P. Monzo, V. Kaddai, A. Doye, V. Ricci, and P. Boquet. 2005. Mol. Biol. Cell. 16:4852-4866). Unlike GPI-APs that are mostly recycled back to the plasma membrane, VacA reaches early endosomes (EEs) and then late endosomes (LEs), where vacuolation occurs. In this study, we used VacA to study the trafficking pathway between GEECs and LEs. We found that VacA routing from GEECs to LEs required polymerized actin. During this trafficking, VacA was transferred from GEECs to EEs associated with polymerized actin structures. The CD2-associated protein (CD2AP), a docking protein implicated in intracellular trafficking, bridged the filamentous actin (F-actin) structures with EEs containing VacA. CD2AP regulated those F-actin structures and was required to transfer VacA from GEECs to LEs. These results demonstrate that sorting from GEECs to LEs requires dynamic F-actin structures on EEs.

Clustering of alpha(5)beta(1) integrins determines adhesion strength whereas alpha(v)beta(3) and talin enable mechanotransduction.

A key molecular link between cells and the extracellular matrix is the binding between fibronectin and integrins alpha(5)beta(1) and alpha(v)beta(3). However, the roles of these different integrins in establishing adhesion remain unclear. We tested the adhesion strength of fibronectin-integrin-cytoskeleton linkages by applying physiological nanonewton forces to fibronectin-coated magnetic beads bound to cells. We report that the clustering of fibronectin domains within 40 nm led to integrin alpha(5)beta(1) recruitment, and increased the ability to sustain force by over six-fold. This force was supported by alpha(5)beta(1) integrin clusters. Importantly, we did not detect a role of either integrin alpha(v)beta(3) or talin 1 or 2 in maintaining adhesion strength. Instead, these molecules enabled the connection to the cytoskeleton and reinforcement in response to an applied force. Thus, high matrix forces are primarily supported by clustered alpha(5)beta(1) integrins, while less stable links to alpha(v)beta(3) integrins initiate mechanotransduction, resulting in reinforcement of integrin-cytoskeleton linkages through talin-dependent bonds.

Building the phagocytic cup on an actin scaffold.

Cells ingest large particles, such as bacteria, viruses, or apoptotic cells, via the process of phagocytosis, which involves formation of an actin-rich structure known as the phagocytic cup. Phagocytic cup assembly and closure results from a concerted action of phagocytic receptors, regulators of actin polymerization, and myosin motors. Recent studies using advanced imaging approaches and biophysical techniques have revealed new information regarding phagocytic cup architecture, regulation of actin assembly, and the distribution, direction, and magnitude of the forces produced by the cytoskeletal elements that form the cup. These findings provide insights into the mechanisms leading to the assembly, expansion, and closure of phagocytic cups. The new data show that engulfment and internalization of phagocytic targets rely on several distinct yet complementary mechanisms that support the robust uptake of foreign objects and may be precisely tailored to the demands of specific phagocytic pathways.

Protocol to assess human glioma propagating cell migration on linear micropatterns mimicking brain invasion tracks.

Glioblastoma (GBM) cells invade the brain by following linear structures like blood vessel walls and white matter tracts by using specific motility modes. In this protocol, we describe two micropatterning techniques allowing recapitulation of these linear tracks in vitro: micro-contact printing and deep UV photolithography. We also detail how to maintain, transfect, and prepare human glioma propagating cells (hGPCs) for migration assays on linear tracks, followed by image acquisition and analysis, to measure key parameters of their motility. For complete details on the use and execution of this protocol, please refer to Monzo et al. (2016) and Monzo et al. (2021a).

F-actin organization and target constriction during primary macrophage phagocytosis is balanced by competing activity of myosin-I and myosin-II.

Phagocytosis requires rapid remodeling of the actin cytoskeleton for extension of membrane protrusions and force generation to ultimately drive the engulfment of targets. The detailed mechanisms of phagocytosis have almost exclusively been studied in immortalized cell lines. Here, we make use of high-resolution imaging and novel biophysical approaches to determine the structural and mechanical features of phagocytosis by primary bone marrow-derived macrophages. We find that the signature behavior of these primary cells is distinct from macrophage-like cell lines; specifically, it is gentle, with only weak target constriction and modest polarization of the F-actin distribution inside the phagocytic cup. We show that long-tailed myosins 1e/f are critical for this organization. Deficiency of myo1e/f causes dramatic shifts in F-actin localization, reducing F-actin at the phagocytic cup base and enhancing F-actin-mediated constriction at the cup rim. Surprisingly, these changes can be almost fully reverted upon inhibition of another myosin motor protein, myosin-II. Hence, we show that the biomechanics and large-scale organization of phagocytic cups is tightly regulated through competing contributions from myosin-Ie/f and myosin-II.

Phagocytic 'teeth' and myosin-II 'jaw' power target constriction during phagocytosis.

Phagocytosis requires rapid actin reorganization and spatially controlled force generation to ingest targets ranging from pathogens to apoptotic cells. How actomyosin activity directs membrane extensions to engulf such diverse targets remains unclear. Here, we combine lattice light-sheet microscopy (LLSM) with microparticle traction force microscopy (MP-TFM) to quantify actin dynamics and subcellular forces during macrophage phagocytosis. We show that spatially localized forces leading to target constriction are prominent during phagocytosis of antibody-opsonized targets. This constriction is largely driven by Arp2/3-mediated assembly of discrete actin protrusions containing myosin 1e and 1f ('teeth') that appear to be interconnected in a ring-like organization. Contractile myosin-II activity contributes to late-stage phagocytic force generation and progression, supporting a specific role in phagocytic cup closure. Observations of partial target eating attempts and sudden target release via a popping mechanism suggest that constriction may be critical for resolving complex in vivo target encounters. Overall, our findings present a phagocytic cup shaping mechanism that is distinct from cytoskeletal remodeling in 2D cell motility and may contribute to mechanosensing and phagocytic plasticity.

Adaptive mechanoproperties mediated by the formin FMN1 characterize glioblastoma fitness for invasion.

Glioblastoma are heterogeneous tumors composed of highly invasive and highly proliferative clones. Heterogeneity in invasiveness could emerge from discrete biophysical properties linked to specific molecular expression. We identified clones of patient-derived glioma propagating cells that were either highly proliferative or highly invasive and compared their cellular architecture, migratory, and biophysical properties. We discovered that invasiveness was linked to cellular fitness. The most invasive cells were stiffer, developed higher mechanical forces on the substrate, and moved stochastically. The mechano-chemical-induced expression of the formin FMN1 conferred invasive strength that was confirmed in patient samples. Moreover, FMN1 expression was also linked to motility in other cancer and normal cell lines, and its ectopic expression increased fitness parameters. Mechanistically, FMN1 acts from the microtubule lattice and promotes a robust mechanical cohesion, leading to highly invasive motility.

Squeezing in a Meal: Myosin Functions in Phagocytosis.

Phagocytosis is a receptor-mediated, actin-dependent process of internalization of large extracellular particles, such as pathogens or apoptotic cells. Engulfment of phagocytic targets requires the activity of myosins, actin-dependent molecular motors, which perform a variety of functions at distinct steps during phagocytosis. By applying force to actin filaments, the plasma membrane, and intracellular proteins and organelles, myosins can generate contractility, directly regulate actin assembly to ensure proper phagocytic internalization, and translocate phagosomes or other cargo to appropriate cellular locations. Recent studies using engineered microenvironments and phagocytic targets have demonstrated how altering the actomyosin cytoskeleton affects phagocytic behavior. Here, we discuss how studies using genetic and biochemical manipulation of myosins, force measurement techniques, and live-cell imaging have advanced our understanding of how specific myosins function at individual steps of phagocytosis.

Complementary mesoscale dynamics of spectrin and acto-myosin shape membrane territories during mechanoresponse.

The spectrin-based membrane skeleton is a major component of the cell cortex. While expressed by all metazoans, its dynamic interactions with the other cortex components, including the plasma membrane or the acto-myosin cytoskeleton, are poorly understood. Here, we investigate how spectrin re-organizes spatially and dynamically under the membrane during changes in cell mechanics. We find spectrin and acto-myosin to be spatially distinct but cooperating during mechanical challenges, such as cell adhesion and contraction, or compression, stretch and osmolarity fluctuations, creating a cohesive cortex supporting the plasma membrane. Actin territories control protrusions and contractile structures while spectrin territories concentrate in retractile zones and low-actin density/inter-contractile regions, acting as a fence that organize membrane trafficking events. We unveil here the existence of a dynamic interplay between acto-myosin and spectrin necessary to support a mesoscale organization of the lipid bilayer into spatially-confined cortical territories during cell mechanoresponse.