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Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Institut de Science et d'Ingénierie Supramoléculaires (ISIS), CNRS and Université de Strasbourg

Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS and Université de Strasbourg

The structure of mitotic chromosomes is still poorly understood. Here we describe the use of a novel approach based on elasticity measurements of a single chromosome for studying the organization of these objects. The data reveal that mitotic chromosomes exhibit a non-homogenous structure consisting of rigid elastic axes surrounded by a soft chromatin envelope. The chemical continuity of DNA, but not RNA, was required for the maintenance of these axes. The axes show a modular structure, and the structural maintenance of chromosomes (SMC) proteins participate in their organization. Topoisomerase II was not involved in either the organization of the axes or the maintenance of the mitotic chromosomes. A model for the assembly and the structure of the mitotic chromosome is proposed. According this model, the chromosome axes are dynamic structures that assemble at the onset and disassemble the end of mitosis, respectively. The SMC proteins, in addition to maintaining axis elasticity, are essential for the determination of the rod-like chromosome shape. The extreme compaction of mitotic chromosomes is determined mainly by the high amount of bivalent ions bound to DNA at mitosis.

The mitotic chromosome is an assembly of rigid elastic axes organized by structural maintenance of chromosomes (SMC) proteins and surrounded by a soft chromatin envelope.

Adherens junctions (AJ) are adhesive motifs joining neighboring cells. The physical contacts are fingerlike structures which grow perpendicular to the cell-cell interface. While their length may vary with the applied force, their density is independent of length. Here we measure AJ contact lengths while varying the mean contractile force of the cell using nocodazole. Using this assay and a simple mechanical model, we measure an effective spring constant of about 30 pN/microm per finger. This measurement may enable the estimation of cell monolayer force distributions from a simple AJ image.

Force-extension relationship of cell-cell contacts.

Cell-cell contact formation relies on the recruitment of cadherin molecules and their anchoring to actin. However, the precise chronology of events from initial cadherin trans-interactions to adhesion strengthening is unclear, in part due to the lack of access to the distribution of cadherins within adhesion zones. Using N-cadherin expressing cells interacting with N-cadherin coated surfaces, we characterized the formation of cadherin adhesions at the ventral cell surface. TIRF and RIC microscopies revealed streak-like accumulations of cadherin along actin fibers. FRAP analysis indicated that engaged cadherins display a slow turnover at equilibrium, compatible with a continuous addition and removal of cadherin molecules within the adhesive contact. Association of cadherin cytoplasmic tail to actin as well as actin cables and myosin II activity are required for the formation and maintenance of cadherin adhesions. Using time lapse microscopy we deciphered how cadherin adhesions form and grow. As lamellipodia protrude, cadherin foci stochastically formed a few microns away from the cell margin. Neo-formed foci coalesced aligned and coalesced with preformed foci either by rearward sliding or gap filling to form cadherin adhesions. Foci experienced collapse at the rear of cadherin adhesions. Based on these results, we present a model for the nucleation, directional growth and shrinkage of cadherin adhesions.

Nucleation and growth of cadherin adhesions.

Specifically expressed at intercellular adherens junctions of endothelial cells, VE-cadherin is a receptor that exhibits particular self-association properties. Indeed, in vitro studies demonstrated that the extracellular part of VE-cadherin elaborates Ca(++)-dependent hexameric structures. We hypothesized that this assembly could be at the basis of a new cadherin-mediated cell-cell adhesion mechanism. To verify this assumption, we first demonstrated that VE-cadherin can elaborate hexamers at the cell surface of confluent endothelial cells. Second, mutations were introduced within the extracellular part of VE-cadherin to destabilize the hexamer. Following an in vitro screening, three mutants were selected, among which, one is able to elaborate only dimers. The selected mutations were expressed as C-terminal green fluorescent protein fusions in CHO cells. Despite their capacity to elaborate nascent cell-cell contacts, the mutants seem to be rapidly degraded and/or internalized. Altogether, our results suggest that the formation of VE-cadherin hexamers protects this receptor and might allow the elaboration of mature endothelial cell-cell junctions.

Establishment of cell-cell junctions depends on the oligomeric states of VE-cadherin.

To form adherens junctions (AJ), cells first establish contact by sending out lamellipodia onto neighboring cells. We investigated the role of contacting cells in AJ assembly by studying an asymmetric AJ motif: finger-like AJ extending across the cell-cell interface. Using a cytoskeleton replica and immunofluorescence, we observed that actin bundles embedded in the lamellipodia are co-localized with stress fibers in the neighboring cell at the AJ. This suggests that donor lamellipodia present actin fingers, which are stabilized by acceptor lamellae via acto-myosin contractility. Indeed, we show that changes in actin network geometry promoted by Rac overexpression lead to corresponding changes in AJ morphology. Moreover, contractility inhibition and enhancement (via drugs or local traction) lead respectively to the disappearance and further growth of AJ fingers. Thus, we propose that receiving lamellae exert a local pull on AJ, promoting further polymerization of the donor actin bundles. In spite of different compositions, AJ and focal contacts both act as cellular mechanosensors.

The asymmetric self-assembly mechanism of adherens junctions: a cellular push-pull unit.

Yeast is a powerful genetic model system, but its rigid cell wall has prohibited microinjection. Using microfabricated channels to constrain the fission yeast Schizosaccharomyces pombe, we sheared local regions of individual cells with a piezoelectric unit. The cells remained viable, we detected actin patches in the cell after introduction of fluorescent phalloidin into the medium, and the cytokinetic ring was disrupted after injection of the myosin II inhibitor blebbistatin.

'Injecting' yeast.

Lengths and shapes are approached in different ways in different fields: they serve as a read-out for classifying genes or proteins in cell biology whereas they result from scaling arguments in condensed matter physics. Here, we propose a combined approach with examples illustrated for the fission yeast Schizosaccharomyces pombe.

Explaining lengths and shapes of yeast by scaling arguments.

Major transformations of cells during embryonic development are traditionally associated with the activation or inhibition of genes and with protein modifications. The contributions of mechanical properties intrinsic to the matter an organism is made of, however, are often overlooked. The emerging field "physics of living matter" is addressing active material properties of the cytoskeleton and tissues like the spontaneous generation of stress, which may lead to shape changes and tissue flows, and their implications for embryonic development. Here, we discuss spontaneous mechanical oscillations to present some basic elements for understanding this physics, and we illustrate its application to developing embryos. We highlight the role of state diagrams to quantitatively probe the significance of the corresponding physical concepts for understanding development.

Spontaneous mechanical oscillations: implications for developing organisms.

Adhesion between cells and the extracellular matrix is mediated by different types of transmembraneous proteins. Their associations to specific partners lead to the assembly of contacts such as focal adhesions and hemidesmosomes. The spatial overlap between both contacts within cells has however limited the study of each type of contact. Here we show that with "stampcils" focal contacts and hemidesmosomes can be spatially separated: cells are plated within the cavities of a stencil and the grids of the stencil serve as stamps for grafting an extracellular matrix protein-fibronectin. Cells engage new contacts on stamped zones leading to the segregation of adhesions and their associated cytoskeletons, i.e., actin and intermediate filaments of keratins. This new method should provide new insights into cell contacts compositions and dynamics.

Separation of distinct adhesion complexes and associated cytoskeleton by a micro-stencil-printing method.

Cellular protrusions involved in motile processes are driven by site-directed assembly of actin filaments in response to Rho-GTPase signalling. So far, only chemical compounds depolymerizing actin or stabilizing filaments, inhibiting N-WASP, Arp2/3 or formins, have been used to eliminate the formation of protrusions, while Rho-GTPase-dominant positive strategies have been designed to stimulate protrusions. Here we describe the design of four polyamines (macrocyclic and branched acyclic), and show that they enter the cell and induce specific growth of actin-enriched lamellipodia within minutes. The largest increase in cell area is obtained with micromolar amounts of a branched polyamine harbouring an 8-carbon chain. These polyamines specifically target actin both in vitro and in vivo. Analysis of their effects on filament assembly dynamics and its regulation indicates that the polyamines act by slowing down filament dynamics and by enhancing actin nucleation. These compounds provide new opportunities to study the actin cytoskeleton in motile and morphogenetic processes.

Synthetic polyamines promote rapid lamellipodial growth by regulating actin dynamics.

At scales below micrometers, Brownian motion dictates most of the behaviors. The simple observation of a colloid is striking: a permanent and random motion is seen, whereas inertial forces play a negligible role. This Physics, where velocity is proportional to force, has opened new horizons in biology. The random feature is challenged in living systems where some proteins--molecular motors--have a directed motion whereas their passive behaviors of colloid should lead to a Brownian motion. Individual proteins, polymers of living matter such as DNA, RNA, actin or microtubules, molecular motors, all these objects can be viewed as chains of colloids. They are submitted to shocks from molecules of the solvent. Shapes taken by these biopolymers or dynamics imposed by motors can be measured and modeled from single molecules to their collective effects. Thanks to the development of experimental methods such as optical tweezers, Atomic Force Microscope (AFM), micropipettes, and quantitative fluorescence (such as Förster Resonance Energy Transfer, FRET), it is possible to manipulate these individual biomolecules in an unprecedented manner: experiments allow to probe the validity of models; and a new Physics has thereby emerged with original biological insights. Theories based on statistical mechanics are needed to explain behaviors of these systems. When force-extension curves of these molecules are extracted, the curves need to be fitted with models that predict the deformation of free objects or submitted to a force. When velocity of motors is altered, a quantitative analysis is required to explain the motions of individual molecules under external forces. This lecture will give some elements of introduction to the lectures of the session 'Nanophysics for Molecular Biology'.

'Single molecule': theory and experiments, an introduction.

Many physiological phenomena involve directional cell migration. It is usually attributed to chemical gradients in vivo. Recently, other cues have been shown to guide cells in vitro, including stiffness/adhesion gradients or micropatterned adhesive motifs. However, the cellular mechanism leading to these biased migrations remains unknown, and, often, even the direction of motion is unpredictable. In this study, we show the key role of fluctuating protrusions on ratchet-like structures in driving NIH3T3 cell migration. We identified the concept of efficient protrusion and an associated direction index. Our analysis of the protrusion statistics facilitated the quantitative prediction of cell trajectories in all investigated conditions. We varied the external cues by changing the adhesive patterns. We also modified the internal cues using drug treatments, which modified the protrusion activity. Stochasticity affects the short- and long-term steps. We developed a theoretical model showing that an asymmetry in the protrusion fluctuations is sufficient for predicting all measures associated with the long-term motion, which can be described as a biased persistent random walk.

Protrusion fluctuations direct cell motion.

Cell migration is a crucial event during development and in disease. Mechanical constraints and chemical gradients can contribute to the establishment of cell direction, but their respective roles remain poorly understood. Using a microfabricated topographical ratchet, we show that the nucleus dictates the direction of cell movement through mechanical guidance by its environment. We demonstrate that this direction can be tuned by combining the topographical ratchet with a biochemical gradient of fibronectin adhesion. We report competition and cooperation between the two external cues. We also quantitatively compare the measurements associated with the trajectory of a model that treats cells as fluctuating particles trapped in a periodic asymmetric potential. We show that the cell nucleus contributes to the strength of the trap, whereas cell protrusions guided by the adhesive gradients add a constant tunable bias to the direction of cell motion.

Cells as active particles in asymmetric potentials: motility under external gradients.

Actin is a major actor in the determination of cell shape. On the one hand, site-directed assembly/disassembly cycles of actin filaments drive protrusive force leading to lamellipodia and filopodia dynamics. Force produced by actin similarly contributes in membrane scission in endocytosis or Golgi remodeling. On the other hand, cellular processes like adhesion, immune synapse, cortex dynamics or cytokinesis are achieved by combining acto-myosin contractility and actin assembly in a complex and not fully understood manner. New chemical compounds are therefore needed to disentangle acto-myosin and actin dynamics. We have found that synthetic, cell permeant, short polyamines are promising new actin regulators in this context. They generate growth and stabilization of lamellipodia within minutes by slowing down the actin assembly/disassembly cycle and facilitating nucleation. We now report that these polyamines also slow down cytokinetic ring closure in fission yeast. This shows that these synthetic compounds are active also in yeasts, and these experiments specifically highlight that actin depolymerization is involved in the ring closure. Thus, synthetic polyamines appear to be potentially powerful agents in a quantitative approach to the role of actin in complex processes in cell biology, developmental biology and potentially cancer research.

Synthetic polyamines: new compounds specific to actin dynamics for mammalian cell and fission yeast.

Many physiological and pathological processes involve directed cell motion. In general, migrating cells are represented with a polarized morphology with extending and retracting protrusions at the leading edge. However, cell motion is a more complex phenomenon. Cells show heterogeneous morphologies and high protrusive dynamics is not always related to cell shape. This prevents the quantitative prediction of cell motion and the identification of cellular mechanisms setting directionality. Here we discuss the importance of protrusion fluctuations in directed cell motion. We show how their spatiotemporal distribution and dynamics determine the fluctuations and directions of cell motion for NIH3T3 fibroblasts plated on micro-patterned adhesive ratchets. (1) We introduce efficient protrusions and direction index which capture short-term cell motility over hours: these new read-outs allow the prediction of parameters characteristic for the long-term motion of cells over days. The results may have important implications for the study of biological phenomena where directed cell migration is involved, in morphogenesis and in cancer.

The cell ratchet: interplay between efficient protrusions and adhesion determines cell motion.

We use the theory of active gels to study theoretically the merging and separation of two actin dense layers akin to cortical layers of animal cells. The layers bind at a distance equal to twice the thickness of a free layer, thus forming a single dense layer, similar in this sense to a lamellipodium. When that unique layer is stretched apart, it is resilient to break apart up to a critical length larger than twice the thickness of a free layer. We show that this behavior can result from the high contractile properties of the actomyosin gel due to the activity of myosin molecular motors. Furthermore, we establish that the stability of the stretched single layer is highly dependent on the properties of the gel. Indeed, the nematic order of the actin filaments along the polymerizing membranes is a destabilizing factor.

Theoretical study of actin layers attachment and separation.

Directed cell migration is usually thought to depend on the presence of long-range gradients of either chemoattractants or physical properties such as stiffness or adhesion. However, in vivo, chemical or mechanical gradients have not systematically been observed. Here we review recent in vitro experiments, which show that other types of spatial guidance cues can bias cell motility. Introducing local geometrical or mechanical anisotropy in the cell environment, such as adhesive/topographical microratchets or tilted micropillars, show that local and periodic external cues can direct cell motion. Together with modeling, these experiments suggest that cell motility can be viewed as a stochastic phenomenon, which can be biased by various types of local cues, leading to directional migration.

Ratchetaxis: Long-Range Directed Cell Migration by Local Cues.

The cytokinetic ring is essential for separating daughter cells during division. It consists of actin filaments and myosin motors that are generally assumed to organize as sarcomeres similar to skeletal muscles. However, direct evidence is lacking. Here we show that the internal organization and dynamics of rings are different from sarcomeres and distinct in different cell types. Using micro-cavities to orient rings in single focal planes, we find in mammalian cells a transition from a homogeneous distribution to a periodic pattern of myosin clusters at the onset of constriction. In contrast, in fission yeast, myosin clusters rotate prior to and during constriction. Theoretical analysis indicates that both patterns result from acto-myosin self-organization and reveals differences in the respective stresses. These findings suggest distinct functional roles for rings: contraction in mammalian cells and transport in fission yeast. Thus self-organization under different conditions may be a generic feature for regulating morphogenesis in vivo.

Daniel Riveline

Laboratory of Cell Physics

Institut de Science et d'Ingénierie Supramoléculaires (ISIS), CNRS and Université de Strasbourg

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Daniel Riveline

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory of Cell Physics

Institut de Science et d'Ingénierie Supramoléculaires (ISIS), CNRS and Université de Strasbourg

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Laboratory of Cell Physics