Matthieu Piel

The extracellular matrix guides the orientation of the cell division axis

The cell division axis determines the future positions of daughter cells and is therefore critical for cell fate. The positioning of the division axis has been mostly studied in systems such as embryos or yeasts, in which cell shape is well defined. In these cases, cell shape anisotropy and cell polarity affect spindle orientation. It remains unclear whether cell geometry or cortical cues are determinants for spindle orientation in mammalian cultured cells. The cell environment is composed of an extracellular matrix (ECM), which is connected to the intracellular actin cytoskeleton via transmembrane proteins. We used micro-contact printing to control the spatial distribution of the ECM on the substrate and demonstrated that it has a role in determining the orientation of the division axis of HeLa cells. On the basis of our analysis of the average distributions of actin-binding proteins in interphase and mitosis, we propose that the ECM controls the location of actin dynamics at the membrane, and thus the segregation of cortical components in interphase. This segregation is further maintained on the cortex of mitotic cells and used for spindle orientation.

Anisotropy of cell adhesive microenvironment governs cell internal organization and orientation of polarity

Control of the establishment of cell polarity is an essential function in tissue morphogenesis and renewal that depends on spatial cues provided by the extracellular environment. The molecular role of cell–cell or cell–extracellular matrix (ECM) contacts on the establishment of cell polarity has been well characterized. It has been hypothesized that the geometry of the cell adhesive microenvironment was directing cell surface polarization and internal organization. To define how the extracellular environment affects cell polarity, we analyzed the organization of individual cells plated on defined micropatterned substrates imposing cells to spread on various combinations of adhesive and nonadhesive areas. The reproducible normalization effect on overall cell compartmentalization enabled quantification of the spatial organization of the actin network and associated proteins, the spatial distribution of microtubules, and the positioning of nucleus, centrosome, and Golgi apparatus. By using specific micropatterns and statistical analysis of cell compartment positions, we demonstrated that ECM geometry determines the orientation of cell polarity axes. The nucleus–centrosome orientations were reproducibly directed toward cell adhesive edges. The anisotropy of the cell cortex in response to the adhesive conditions did not affect the centrosome positioning at the cell centroid. Based on the quantification of microtubule plus end distribution we propose a working model that accounts for that observation. We conclude that, in addition to molecular composition and mechanical properties, ECM geometry plays a key role in developmental processes.

External forces control mitotic spindle positioning

The response of cells to forces is essential for tissue morphogenesis and homeostasis. This response has been extensively investigated in interphase cells, but it remains unclear how forces affect dividing cells. We used a combination of micro-manipulation tools on human dividing cells to address the role of physical parameters of the micro-environment in controlling the cell division axis, a key element of tissue morphogenesis. We found that forces applied on the cell body direct spindle orientation during mitosis. We further show that external constraints induce a polarization of dynamic subcortical actin structures that correlate with spindle movements. We propose that cells divide according to cues provided by their mechanical micro-environment, aligning daughter cells with the external force field.

Regulation of dendritic cell migration by CD74, the MHC class II-associated invariant chain.

Dendritic cells (DCs) sample peripheral tissues of the body in search of antigens to present to T cells. This requires two processes, antigen processing and cell motility, originally thought to occur independently. We found that the major histocompatibility complex II–associated invariant chain (Ii or CD74), a known regulator of antigen processing, negatively regulates DC motility in vivo. By using microfabricated channels to mimic the confined environment of peripheral tissues, we found that wild-type DCs alternate between high and low motility, whereas Ii-deficient cells moved in a faster and more uniform manner. The regulation of cell motility by Ii depended on the actin-based motor protein myosin II. Coupling antigen processing and cell motility may enable DCs to more efficiently detect and process antigens within a defined space.

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.

ESCRT III repairs nuclear envelope ruptures during cell migration to limit DNA damage and cell death.

Repairing tears in the nuclear envelope The nuclear envelope segregates genomic DNA from the cytoplasm and regulates protein trafficking between the cytosol and the nucleus. Maintaining nuclear envelope integrity during interphase is considered crucial. However, Raab et al. and Denais et al. show that migrating immune and cancer cells experience frequent and transitory nuclear envelope ruptures when they move through tight spaces (see the Perspective by Burke). The nuclear envelope reseals rapidly during interphase, assisted by components of the ESCRT III membrane-remodeling machinery. Science, this issue pp. 359 and 353; see also p. 295 When cells migrate, they stress their nuclear envelopes and need to guard against leakage of the nuclear contents. [Also see Perspective by Burke] In eukaryotic cells, the nuclear envelope separates the genomic DNA from the cytoplasmic space and regulates protein trafficking between the two compartments. This barrier is only transiently dissolved during mitosis. Here, we found that it also opened at high frequency in migrating mammalian cells during interphase, which allowed nuclear proteins to leak out and cytoplasmic proteins to leak in. This transient opening was caused by nuclear deformation and was rapidly repaired in an ESCRT (endosomal sorting complexes required for transport)–dependent manner. DNA double-strand breaks coincided with nuclear envelope opening events. As a consequence, survival of cells migrating through confining environments depended on efficient nuclear envelope and DNA repair machineries. Nuclear envelope opening in migrating leukocytes could have potentially important consequences for normal and pathological immune responses.

Contractility of the cell rear drives invasion of breast tumor cells in 3D Matrigel

Cancer cells use different modes of migration, including integrin-dependent mesenchymal migration of elongated cells along elements of the 3D matrix as opposed to low-adhesion-, contraction-based amoeboid motility of rounded cells. We report that MDA-MB-231 human breast adenocarcinoma cells invade 3D Matrigel with a characteristic rounded morphology and with F-actin and myosin-IIa accumulating at the cell rear in a uropod-like structure. MDA-MB-231 cells display neither lamellipodia nor bleb extensions at the leading edge and do not require Arp2/3 complex activity for 3D invasion in Matrigel. Accumulation of phospho-MLC and blebbing activity were restricted to the uropod as reporters of actomyosin contractility, and velocimetric analysis of fluorescent beads embedded within the 3D matrix showed that pulling forces exerted to the matrix are restricted to the side and rear of cells. Inhibition of actomyosin contractility or β1 integrin function interferes with uropod formation, matrix deformation, and invasion through Matrigel. These findings support a model whereby actomyosin-based uropod contractility generates traction forces on the β1 integrin adhesion system to drive cell propulsion within the 3D matrix, with no contribution of lamellipodia extension or blebbing to movement.

PARP-3 localizes preferentially to the daughter centriole and interferes with the G1/S cell cycle progression

A novel member of the poly(ADP-ribose) polymerase (PARP) family, hPARP-3, is identified here as a core component of the centrosome. hPARP-3 is preferentially localized to the daughter centriole throughout the cell cycle. The N-terminal domain (54 amino acids) of hPARP-3 is responsible for its centrosomal localization. Full-length hPAPR-3 (540 amino acids, with an apparent mass of 67 kDa) synthesizes ADP-ribose polymers during its automodification. Overexpression of hPARP-3 or its N-terminal domain does not influence centrosomal duplication or amplification but interferes with the G1/S cell cycle progression. PARP-1 also resides for part of the cell cycle in the centrosome and interacts with hPARP-3. The presence of both PARP-1 and PARP-3 at the centrosome may link the DNA damage surveillance network to the mitotic fidelity checkpoint.

Comparative study and improvement of current cell micro-patterning techniques

The original micropatterning technique on gold, although very efficient, is not accessible to most biology labs and is not compatible with their techniques for image acquisition. Other solutions have been developed on silanized glass coverslips. These methods are still hardly accessible to biology labs and do not provide sufficient reproducibility to become incorporated in routine biological protocols. Here, we analyzed cell behavior on micro-patterns produced by various alternative techniques. Distinct cell types displayed different behavior on micropatterns, while some were easily constrained by the patterns others escaped or ripped off the patterned adhesion molecules. We report methods to overcome some of these limitations on glass coverslips and on plastic dishes which are compatible with our experimental biological applications. Finally, we present a new method based on UV crosslinking of adhesion proteins with benzophenone to easily and rapidly produce highly reproducible micropatterns without the use of a microfabricated elastomeric stamp.