Pierre-François Lenne

Cell surface mechanics and the control of cell shape, tissue patterns and morphogenesis

Embryonic morphogenesis requires the execution of complex mechanisms that regulate the local behaviour of groups of cells. The orchestration of such mechanisms has been mainly deciphered through the identification of conserved families of signalling pathways that spatially and temporally control cell behaviour. However, how this information is processed to control cell shape and cell dynamics is an open area of investigation. The framework that emerges from diverse disciplines such as cell biology, physics and developmental biology points to adhesion and cortical actin networks as regulators of cell surface mechanics. In this context, a range of developmental phenomena can be explained by the regulation of cell surface tension.

Nature and anisotropy of cortical forces orienting Drosophila tissue morphogenesis.

The morphogenesis of developing embryos and organs relies on the ability of cells to remodel their contacts with neighbouring cells. Using quantitative modelling and laser nano-dissection, we probed the mechanics of a morphogenetic process, the elongation of Drosophila melanogaster embryos, which results from polarized cell neighbour exchanges. We show that anisotropy of cortical tension at apical cell junctions is sufficient to drive tissue elongation. We estimated its value through comparisons between in silico and in vivo data using various tissue descriptors. Nano-dissection of the actomyosin network indicates that tension is anisotropically distributed and depends on myosin II accumulation. Junction relaxation after nano-dissection also suggests that cortical elastic forces are dominant in this process. Interestingly, fluctuations in vertex position (points where three or more cells meet) facilitate neighbour exchanges. We delineate the contribution of subcellular tensile activity polarizing junction remodelling, and the permissive role of vertex fluctuations during tissue elongation.

Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork.

It is by now widely recognized that cell membranes show complex patterns of lateral organization. Two mechanisms involving either a lipid‐dependent (microdomain model) or cytoskeleton‐based (meshwork model) process are thought to be responsible for these plasma membrane organizations. In the present study, fluorescence correlation spectroscopy measurements on various spatial scales were performed in order to directly identify and characterize these two processes in live cells with a high temporal resolution, without any loss of spatial information. Putative raft markers were found to be dynamically compartmented within tens of milliseconds into small microdomains (∅<120 nm) that are sensitive to the cholesterol and sphingomyelin levels, whereas actin‐based cytoskeleton barriers are responsible for the confinement of the transferrin receptor protein. A free‐like diffusion was observed when both the lipid‐dependent and cytoskeleton‐based organizations were disrupted, which suggests that these are two main compartmentalizing forces at work in the plasma membrane.

Planar polarized actomyosin contractile flows control epithelial junction remodelling

Force generation by Myosin-II motors on actin filaments drives cell and tissue morphogenesis. In epithelia, contractile forces are resisted at apical junctions by adhesive forces dependent on E-cadherin, which also transmits tension. During Drosophila embryonic germband extension, tissue elongation is driven by cell intercalation, which requires an irreversible and planar polarized remodelling of epithelial cell junctions. We investigate how cell deformations emerge from the interplay between force generation and cortical force transmission during this remodelling in Drosophila melanogaster. The shrinkage of dorsal–ventral-oriented (‘vertical’) junctions during this process is known to require planar polarized junctional contractility by Myosin II (refs 4, 5, 7, 12). Here we show that this shrinkage is not produced by junctional Myosin II itself, but by the polarized flow of medial actomyosin pulses towards ‘vertical’ junctions. This anisotropic flow is oriented by the planar polarized distribution of E-cadherin complexes, in that medial Myosin II flows towards ‘vertical’ junctions, which have relatively less E-cadherin than transverse junctions. Our evidence suggests that the medial flow pattern reflects equilibrium properties of force transmission and coupling to E-cadherin by α-Catenin. Thus, epithelial morphogenesis is not properly reflected by Myosin II steady state distribution but by polarized contractile actomyosin flows that emerge from interactions between E-cadherin and actomyosin networks.

A two-tiered mechanism for stabilization and immobilization of E-cadherin

Epithelial tissues maintain a robust architecture which is important for their barrier function, but they are also remodelled through the reorganization of cell–cell contacts. Tissue stability requires intercellular adhesion mediated by E-cadherin, in particular its trans-association in homophilic complexes supported by actin filaments through β- and α-catenin. How α-catenin dynamic interactions between E-cadherin/β-catenin and cortical actin control both stability and remodelling of adhesion is unclear. Here we focus on Drosophila homophilic E-cadherin complexes rather than total E-cadherin, including diffusing ‘free’ E-cadherin, because these complexes are a better proxy for adhesion. We find that E-cadherin complexes partition in very stable microdomains (that is, bona fide adhesive foci which are more stable than remodelling contacts). Furthermore, we find that stability and mobility of these microdomains depend on two actin populations: small, stable actin patches concentrate at homophilic E-cadherin clusters, whereas a rapidly turning over, contractile network constrains their lateral movement by a tethering mechanism. α-Catenin controls epithelial architecture mainly through regulation of the mobility of homophilic clusters and it is largely dispensable for their stability. Uncoupling stability and mobility of E-cadherin complexes suggests that stable epithelia may remodel through the regulated mobility of very stable adhesive foci.

Force Generation, Transmission, and Integration during Cell and Tissue Morphogenesis

Cell shape changes underlie a large set of biological processes ranging from cell division to cell motility. Stereotyped patterns of cell shape changes also determine tissue remodeling events such as extension or invagination. In vitro and cell culture systems have been essential to understanding the fundamental physical principles of subcellular mechanics. These are now complemented by studies in developing organisms that emphasize how cell and tissue morphogenesis emerge from the interplay between force-generating machines, such as actomyosin networks, and adhesive clusters that transmit tensile forces at the cell cortex and stabilize cell-cell and cell-substrate interfaces. Both force production and transmission are self-organizing phenomena whose adaptive features are essential during tissue morphogenesis. A new era is opening that emphasizes the similarities of and allows comparisons between distant dynamic biological phenomena because they rely on core machineries that control universal features of cytomechanics.

Raft nanodomains contribute to Akt/PKB plasma membrane recruitment and activation

Membrane rafts are thought to be sphingolipid- and cholesterol-dependent lateral assemblies involved in diverse cellular functions. Their biological roles and even their existence, however, remain controversial. Using an original fluorescence correlation spectroscopy strategy that recently enabled us to identify nanoscale membrane organizations in live cells, we report here that highly dynamic nanodomains exist in both the outer and inner leaflets of the plasma membrane. Through specific inhibition of biosynthesis, we show that sphingolipids and cholesterol are essential and act in concert for formation of nanodomains, thus corroborating their raft nature. Moreover, we find that nanodomains play a crucial role in triggering the phosphatidylinositol-3 kinase/Akt signaling pathway, by facilitating Akt recruitment and activation upon phosphatidylinositol-3,4,5-triphosphate accumulation in the plasma membrane. Thus, through direct monitoring and controlled alterations of rafts in living cells, we demonstrate that rafts are critically involved in the activation of a signaling axis that is essential for cell physiology.

Dynamics in the plasma membrane: how to combine fluidity and order

Cell membranes are fascinating supramolecular aggregates that not only form a barrier between compartments but also harbor many chemical reactions essential to the existence and functioning of a cell. Here, it is proposed to review the molecular dynamics and mosaic organization of the plasma membrane, which are thought to have important functional implications. We will first summarize the basic concepts of Brownian diffusion and lipid domain formation in model membranes and then track the development of ideas and tools in this field, outlining key results obtained on the dynamic processes at work in membrane structure and assembly. We will focus in particular on findings made using fluorescent labeling and imaging procedures to record these dynamic processes. We will also discuss a few examples showing the impact of lateral diffusion on cell signal transduction, and outline some future methodological challenges which must be met before we can answer some of the questions arising in this field of research.

States and transitions during forced unfolding of a single spectrin repeat.

Spectrin is a vital and abundant protein of the cytoskeleton. It has an elongated structure that is made by a chain of so‐called spectrin repeats. Each repeat contains three antiparallel α‐helices that form a coiled‐coil structure. Spectrin forms an oligomeric structure that is able to cross‐link actin filaments. In red cells, the spectrin/actin meshwork underlying cell membrane is thought to be responsible for special elastic properties of the cell. In order to determine mechanical unfolding properties of the spectrin repeat, we have used single molecule force spectroscopy to study the states of unfolding of an engineered polymeric protein consisting of identical spectrin domains. We demonstrate that the unfolding of spectrin domains can occur in a stepwise fashion during stretching. The force–extension patterns exhibit features that are compatible with the existence of at least one intermediate between the folded and the completely unfolded conformation. Only those polypeptides that still contain multiple intact repeats display intermediates, indicating a stabilisation effect. Precise force spectroscopy measurements on single molecules using engineered protein constructs reveal states and transitions during the mechanical unfolding of spectrin. Single molecule force spectroscopy appears to open a new window for the analysis of transition probabilities between different conformational states.

Principles of E-Cadherin Supramolecular Organization In Vivo

BACKGROUND E-cadherin plays a pivotal role in tissue morphogenesis by forming clusters that support intercellular adhesion and transmit tension. What controls E-cadherin mesoscopic organization in clusters is unclear. RESULTS We use 3D superresolution quantitative microscopy in Drosophila embryos to characterize the size distribution of E-cadherin nanometric clusters. The cluster size follows power-law distributions over three orders of magnitude with exponential decay at large cluster sizes. By exploring the predictions of a general theoretical framework including cluster fusion and fission events and recycling of E-cadherin, we identify two distinct active mechanisms setting the cluster-size distribution. Dynamin-dependent endocytosis targets large clusters only, thereby imposing a cutoff size. Moreover, interactions between E-cadherin clusters and actin filaments control the fission in a size-dependent manner. CONCLUSIONS E-cadherin clustering depends on key cortical regulators, which provide tunable and local control over E-cadherin organization. Our data provide the foundation for a quantitative understanding of how E-cadherin distribution affects adhesion and might regulate force transmission in vivo.