Paolo Maiuri

Confinement and Low Adhesion Induce Fast Amoeboid Migration of Slow Mesenchymal Cells

The mesenchymal-amoeboid transition (MAT) was proposed as a mechanism for cancer cells to adapt their migration mode to their environment. While the molecular pathways involved in this transition are well documented, the role of the microenvironment in the MAT is still poorly understood. Here, we investigated how confinement and adhesion affect this transition. We report that, in the absence of focal adhesions and under conditions of confinement, mesenchymal cells can spontaneously switch to a fast amoeboid migration phenotype. We identified two main types of fast migration--one involving a local protrusion and a second involving a myosin-II-dependent mechanical instability of the cell cortex that leads to a global cortical flow. Interestingly, transformed cells are more prone to adopt this fast migration mode. Finally, we propose a generic model that explains migration transitions and predicts a phase diagram of migration phenotypes based on three main control parameters: confinement, adhesion, and contractility.

ESCRT Machinery Is Required for Plasma Membrane Repair

Introduction Plasma membrane damage can result from numerous threats, including mechanical stress or biochemical agents such as pore-forming toxins. Different mechanisms for plasma membrane repair have been described in a variety of cellular models, including patching with endomembranes, endocytosis, and extracellular budding. We found that the endosomal sorting complex required for transport (ESCRT), which is implicated in numerous membrane fission events (such as during cytokinesis or for the budding of several viruses) was also required for the rapid closure of small wounds made at the plasma membrane. ESCRT recruitment mediates pinching out of wounded plasma membrane. (A) Cells expressing the ESCRT subunit CHMP4B-EGFP and wounded (arrow) in the presence of propidium iodide (PI) were observed by means of fluorescence imaging. (B) Model for ESCRT-mediated detection and shedding of wounded plasma membrane. Methods We used micropipettes, detergents, pore-forming toxins, and laser wounding to damage the plasma membrane of mammalian cells in tissue culture. Ultraviolet or two-photon lasers were used to induce small, localized wounds, and cell reactions were followed with time-lapse imaging. Propidium iodide (PI) entry in wounded cells was used to allow imaging of the plasma membrane opening and to quantify the rate of closure of single wounds. Mathematical fit of PI entry kinetics was used to estimate the diameter and the rate of closure of individual wounds. Characterization of PI fluorescence and diffusion gave us an estimation of wound sizes. Transfection of small interfering RNA or dominant-negative mutants of ESCRT subunits allowed us to assess their importance during plasma membrane repair. Last, using correlative-scanning electron microscopy we examined the ultrastructure of wounded plasma membranes. Results The various wounding methods used here revealed a systematic recruitment of ESCRTs to the plasma membrane. Wounding with a laser beam showed that ESCRTs—and in particular, ESCRT-III proteins—were specifically recruited to wound sites and were accumulated until wound closure. This recruitment depended on calcium, which is known to be a crucial signaling molecule for wound repair. The depletion of important ESCRT subunits such as CHMP4B, CHMP2A, or Vps4 was deleterious for a subpopulation of cells bearing small wounds (less than 100 nm in diameter). Correlative scanning electron microscopy and time-lapse imaging revealed that wounding was followed by ESCRT-positive membrane budding and shedding. Energy depletion did not prevent—and rather increased—ESCRT accumulation but prevented both membrane shedding and repair. Discussion These results show that ESCRT proteins play an important role in the detection and removal through the extracellular shedding of small wounds present at the plasma membrane. We propose that different mechanisms for membrane repair (patching, budding, or endocytosis) can be used by cells depending on the type and size of the wound. These mechanisms are stimulated by common early signaling events, such as calcium, but downstream events are likely to depend on the physiochemical characteristics of the wounds. ESCRT-positive plasma membrane shedding has been observed in a variety of normal and pathological conditions. It remains unclear whether these phenomena are linked to local plasma membrane damage and whether ESCRT-III proteins are involved in these processes. ESCRT Your Wound Away The ESCRT (endosomal sorting complex required for transport) protein complex plays a role in budding into multivesicular bodies, in cytokinesis, and in HIV budding. Now, Jimenez et al. (p. 10.1126/science.1247136, published online 30 January) propose a role for ESCRT proteins in wound repair at the plasma membrane. In vivo imaging, modeling, and electron microscopy were used to reveal how the ESCRTs participate in a rapid energy-independent, calcium-dependent, membrane-shedding process at the plasma membrane that reseals small wounds caused by toxins or laser treatment. ESCRT proteins repair small wounds in the plasma membrane by shearing off damaged portions. Plasma membrane damage can be triggered by numerous phenomena, and efficient repair is essential for cell survival. Endocytosis, membrane patching, or extracellular budding can be used for plasma membrane repair. We found that endosomal sorting complex required for transport (ESCRT), involved previously in membrane budding and fission, plays a critical role in plasma membrane repair. ESCRT proteins were recruited within seconds to plasma membrane wounds. Quantitative analysis of wound closure kinetics coupled to mathematical modeling suggested that ESCRTs are involved in the repair of small wounds. Real-time imaging and correlative scanning electron microscopy (SEM) identified extracellular buds and shedding at the site of ESCRT recruitment. Thus, the repair of certain wounds is ensured by ESCRT-mediated extracellular shedding of wounded portions.

Actin Flows Mediate a Universal Coupling between Cell Speed and Cell Persistence

Cell movement has essential functions in development, immunity, and cancer. Various cell migration patterns have been reported, but no general rule has emerged so far. Here, we show on the basis of experimental data in vitro and in vivo that cell persistence, which quantifies the straightness of trajectories, is robustly coupled to cell migration speed. We suggest that this universal coupling constitutes a generic law of cell migration, which originates in the advection of polarity cues by an actin cytoskeleton undergoing flows at the cellular scale. Our analysis relies on a theoretical model that we validate by measuring the persistence of cells upon modulation of actin flow speeds and upon optogenetic manipulation of the binding of an actin regulator to actin filaments. Beyond the quantitative prediction of the coupling, the model yields a generic phase diagram of cellular trajectories, which recapitulates the full range of observed migration patterns.

ESCRT-III Assembly and Cytokinetic Abscission Are Induced by Tension Release in the Intercellular Bridge

Making the Final Cut Abscission, the final separation of two daughter cells, was long thought to be an unimportant step in cytokinesis, triggered merely by the cells pulling strongly enough on the bridge to rupture it. Research over the past 10 years, however, has challenged this notion. Defects in cutting the cytokinetic bridge can lead to the formation of large networks of connected cells or to binucleate cells. Lafaurie-Janvore et al. (p. 1625) now show that the forces postmitotic cells exert on the cytokinetic bridge play an important role in abscission: Surprisingly, increasing the tension in the bridge inhibits abscission, while reducing tension induces abscission. This could provide a sensing mechanism to ensure that daughter cells establish sound connections with their surrounding cells and matrix before detaching from one another. When a daughter cell lets go, the mother cell cuts it loose. The last step of cell division, cytokinesis, produces two daughter cells that remain connected by an intercellular bridge. This state often represents the longest stage of the division process. Severing the bridge (abscission) requires a well-described series of molecular events, but the trigger for abscission remains unknown. We found that pulling forces exerted by daughter cells on the intercellular bridge appear to regulate abscission. Counterintuitively, these forces prolonged connection, whereas a release of tension induced abscission. Tension release triggered the assembly of ESCRT-III (endosomal sorting complex required for transport–III), which was followed by membrane fission. This mechanism may allow daughter cells to remain connected until they have settled in their final locations, a process potentially important for tissue organization and morphogenesis.

The first World Cell Race

Summary Motility is a common property of animal cells. Cell motility is required for embryogenesis [1], tissue morphogenesis [2] and the immune response [3] but is also involved in disease processes, such as metastasis of cancer cells [4]. Analysis of cell migration in native tissue in vivo has yet to be fully explored, but motility can be relatively easily studied in vitro in isolated cells. Recent evidence suggests that cells plated in vitro on thin lines of adhesive proteins printed onto culture dishes can recapitulate many features of in vivo migration on collagen fibers [5,6]. However, even with controlled in vitro measurements, the characteristics of motility are diverse and are dependent on the cell type, origin and external cues. One objective of the first World Cell Race was to perform a large-scale comparison of motility across many different adherent cell types under standardized conditions. To achieve a diverse selection, we enlisted the help of many international laboratories, who submitted cells for analysis. The large-scale analysis, made feasible by this competition-oriented collaboration, demonstrated that higher cell speed correlates with the persistence of movement in the same direction irrespective of cell origin.

Innate control of actin nucleation determines two distinct migration behaviours in dendritic cells

Dendritic cell (DC) migration in peripheral tissues serves two main functions: antigen sampling by immature DCs, and chemokine-guided migration towards lymphatic vessels (LVs) on maturation. These migratory events determine the efficiency of the adaptive immune response. Their regulation by the core cell locomotion machinery has not been determined. Here, we show that the migration of immature DCs depends on two main actin pools: a RhoA–mDia1-dependent actin pool located at their rear, which facilitates forward locomotion; and a Cdc42–Arp2/3-dependent actin pool present at their front, which limits migration but promotes antigen capture. Following TLR4–MyD88-induced maturation, Arp2/3-dependent actin enrichment at the cell front is markedly reduced. Consequently, mature DCs switch to a faster and more persistent mDia1-dependent locomotion mode that facilitates chemotactic migration to LVs and lymph nodes. Thus, the differential use of actin-nucleating machineries optimizes the migration of immature and mature DCs according to their specific function.

Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells

The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space.

Triggering Cell Adhesion, Migration or Shape Change with a Dynamic Surface Coating

Theres an APP for that: cell-repellent APP (azido-[polylysine-g-PEG]) is used to create substrates for spatially controlled dynamic cell adhesion. The simple addition of a functional peptide to the culture medium rapidly triggers cell adhesion. This highly accessible yet powerful technique allows diverse applications, demonstrated through tissue motility assays, patterned coculturing and triggered cell shape change.

Geometric Friction Directs Cell Migration

In the absence of environmental cues, a migrating cell performs an isotropic random motion. Recently, the breaking of this isotropy has been observed when cells move in the presence of asymmetric adhesive patterns. However, up to now the mechanisms at work to direct cell migration in such environments remain unknown. Here, we show that a nonadhesive surface with asymmetric microgeometry consisting of dense arrays of tilted micropillars can direct cell motion. Our analysis reveals that most features of cell trajectories, including the bias, can be reproduced by a simple model of active Brownian particle in a ratchet potential, which we suggest originates from a generic elastic interaction of the cell body with the environment. The observed guiding effect, independent of adhesion, is therefore robust and could be used to direct cell migration both in vitro and in vivo.

ZASP Interacts with the Mechanosensing Protein Ankrd2 and p53 in the Signalling Network of Striated Muscle

ZASP is a cytoskeletal PDZ-LIM protein predominantly expressed in striated muscle. It forms multiprotein complexes and plays a pivotal role in the structural integrity of sarcomeres. Mutations in the ZASP protein are associated with myofibrillar myopathy, left ventricular non-compaction and dilated cardiomyopathy. The ablation of its murine homologue Cypher results in neonatal lethality. ZASP has several alternatively spliced isoforms, in this paper we clarify the nomenclature of its human isoforms as well as their dynamics and expression pattern in striated muscle. Interaction is demonstrated between ZASP and two new binding partners both of which have roles in signalling, regulation of gene expression and muscle differentiation; the mechanosensing protein Ankrd2 and the tumour suppressor protein p53. These proteins and ZASP form a triple complex that appears to facilitate poly-SUMOylation of p53. We also show the importance of two of its functional domains, the ZM-motif and the PDZ domain. The PDZ domain can bind directly to both Ankrd2 and p53 indicating that there is no competition between it and p53 for the same binding site on Ankrd2. However there is competition for this binding site between p53 and a region of the ZASP protein lacking the PDZ domain, but containing the ZM-motif. ZASP is negative regulator of p53 in transactivation experiments with the p53-responsive promoters, MDM2 and BAX. Mutations in the ZASP ZM-motif induce modification in protein turnover. In fact, two mutants, A165V and A171T, were not able to bind Ankrd2 and bound only poorly to alpha-actinin2. This is important since the A165V mutation is responsible for zaspopathy, a well characterized autosomal dominant distal myopathy. Although the mechanism by which this mutant causes disease is still unknown, this is the first indication of how a ZASP disease associated mutant protein differs from that of the wild type ZASP protein.