DS0401 - Etude des systèmes biologiques, de leur dynamique, des interactions et inter-conversions au niveau moléculaire

Towards the prevention of vascular dysfunctions and the targeted delivery of drugs through the endothelia – TransEndotheliaTunnel

TransEndotheliaTunnel

We have discovered a new mode of opening of transendothelial macroaperture (TEM) tunnels, which is driven by the relaxation of the actomyosin cytoskeleton leading to a cellular dewetting phenomenon. We identified key biochemical and physical parameters withstanding cycles of TEM opening and closure. We aim at identifying molecular players that control extension/resealing of the TEMs. The cellular dewetting hypothesis implies a membrane curvature-sensing mechanism that we propose herein to unveil

General objectives and main issues raised

The key questions raised by our previous results are about: (1) the function of this actomyosin-ring in relationship to the increase of line tension deduced from our theory of cellular dewetting and that accounts for the induction of TEMs of a limited size; (2) how the actomyosin form at a newly curved membrane and (3) how MIM drives the recruitment and activation of Arp2/3 and the effect of the I-BAR domain on membrane tension. <br />We thus propose to target several aims: (1) to identify cellular players influencing TEM maximal size, (2) to characterize the actomyosin ring interacting partners that control the extension and resealing of the TEMs, (3) to unveil how cells sense the membrane curvature and (4) to develop an assay to be used in drug screening approach

Our project is multidisciplinary. It is based on several approaches: imaging (incl. super-resolution photonic microscopy), biophysics (incl. force spectroscopy using atomic force microscopy, laser ablation, micromanipulation (micropipettes and optical tweezers) of giant liposomes and membrane nanotubes) and the use of high-throughput and high-content screening methods in a context of endothelial cells intoxicated by bacterial toxins such as EDIN from S aureus and edema factor from anthrax. The project has also a part of cell physics theory that is refined in function of our experimental findings.

• The endothelium serves as a protective semipermeable barrier in blood vessels and lymphatic vessels. Leukocytes and pathogens can pass directly through the endothelium by opening holes in endothelial cells, known as transendothelial cell macroaperture tunnels (TEMs), which are formed by contact and self-fusion of the apical and basal plasma membranes. These tunnels open transiently. We have made critical progresses in defining physical principles governing the opening of TEMs, the control of their size by a cell autonomous reorganization of actomyosin cytoskeleton coupled to physic theory and started to identify new critical cell regulators controlling their closure and maximal width. We have set-up conditions to screen chemical compounds interfering with the dynamic of TEM opening and closure.
• Cellular dewetting theory predicted that a line tension of uncharacterized origin works at TEM boundaries to limit their widening. We have defined that ezrin enhances line tension along transcellular tunnel edges via NMIIa driven actomyosin cable formation (Common article in revision at Nature communications). By conducting high resolution microscopy approaches, we have unveiled the presence of an actomyosin cable encircling TEMs. We have developed a theoretical cellular dewetting framework to interpret TEM physical parameters that are quantitatively determined by laser ablation experiments. This allowed us to establish the critical role of ezrin and non-muscle myosin II (NMII) in the progressive implementation of line tension. Mechanistically, fluorescence-recovery-after-photobleaching experiments point for the up-stream role of ezrin in stabilizing actin filaments at the edges of TEMs, thereby favoring their crosslinking by NMIIa. Collectively, our findings ascribe to ezrin and NMIIa a critical function of enhancing line tension at the cell boundary surrounding the TEMs by promoting the formation of an actomyosin ring.

We have developed a theoretical cellular dewetting framework to interpret TEM physical parameters that are quantitatively determined by laser ablation experiments. This groundbreaking approach has allowed us to ascribe to ezrin and NMIIa a new critical function of enhancing line tension at the cell boundary surrounding the TEMs by promoting to the formation of an actomyosin ring. Briefly, by combining laser ablation experiments with a physical model derived by analogy with dewetting theory, we provide compelling evidence that TEM size is restricted by the organization of an actomyosin belt encircling TEMs. This belt develops line tension over time, thereby dictating TEM maximal size. Mechanistically, we propose that ezrin, by stabilizing actin filaments around TEMs, favors the NMIIa-dependent bundling of F-actin into a stiff cable. Disruption of this cell-mediated control of TEM size tends to promote an unlimited widening of TEMs and thereby provokes the rupture of endothelial cell integrity. This ascribes to ezrin molecule a function of control of cell tensegrity that contributes to maintain endothelial cell integrity.
Ezrin is one of the most abundant membrane protein. It links actin cytoskeleton to the plasma membrane. Since it is enriched on highly curved structures such as the edge of TEM or filopodia, we have studied whether it has an intrinsic curvature sensor capability. We found that ezrin is not a curvature sensor, which would lead to cell edge instability but can be enriched to highly curved membrane area such as TEM by its interaction with I-BAR domains.
We have succeeded to induce the formation of TEMs in the absence of toxins or drugs, by indentating the cells with an AFM tip. These experiments mimic the action of podosomes during leukocyte diapedesis. It also confirms the protective action of the actin cortex against spontaneous TEM formation in non-intoxicated cells.

Contractile actin cables induced by Bacillus anthracis lethal toxin depedent on the histone acetylation machinery (Rolando, Stefani, Doye, Acosta, Visvikis, Yevick, Buchrieser, Mettouchi, Bassereau Lemichez) Cytoskeleton 2015 542:56.

-RSp53 senses negative membrane curvature and phase separates along memebrane tubes (Prévost, Zhao, Manzi, Lemichez, Lappalainen, Callan-Jones, Bassereau) Nat. Commun 2015 15,6:8529.

-Ezrin powers line tension via NMIIa-driven actomyosin cable formation along transcellular tunnel edges (Stefani, Gonzalez-Rodriguez, Senju, Doye, Efimova, Lipuma, Hamaoui, Maddugoda, Cochet-Escartin, Prévost, Janel, Lafont, Svitkina, Lappalainen, Bassereau, Lemichez) Nat. Commun. (in revision)

Force-induced transcellular tunnel formation in endothelial cells (Ng, Webster, Stefani C, Schmid, Lemichez, Bassereau, Fletcher) Mol. Biol. Cell (submitted)

Microbial pathogenesis meets biomechanics (Charles-Orszag, Lemichez, Tran Van Nhieu, Duménil) Curr Opin Cell Biol 38:31-7 (2016)

We have recently discovered a new mode of transient opening of large transendothelial macroaperture (TEM) tunnels, which is driven by the reduction of actomyosin cytoskeleton contractibility under the dependence of a new peculiar form of dewetting phenomenon, that we termed "cellular dewetting". Cells are able to limit the opening of TEMs, in particular with the formation of an actin ring around the TEMs, and promote their closure by extension of membrane waves for tunnel resealing. Our previous work identified key biochemical and physical parameters withstanding cycles of TEM opening and closure. These mechanisms involve molecular players controlling extension and resealing of the TEM tunnels. Our preliminary results identify some of these players that however need to be validated as well as the signaling pathways there are part of. On the other hand, the cellular dewetting hypothesis that we have introduced implies a membrane curvature-sensing mechanism that we propose herein to unveil using in vitro biophysics approaches. Moreover, we propose to screen for key druggable regulators of opening and closure of TEMs with the aim of controlling the endothelium barrier. This mode of transient opening of tunnels is exploited by leukocytes for their transcellular diapedesis and usurped by some pathogens to promote metastatic infection. We have strong genetic evidence that TEM-tunnel inducing toxins are invasive factors of Staphylococcus aureus. Absence of TEM closure triggers hemorrage. This pioneering work paves the way, beyond the scope of the infection field, for comprehensive biology to characterize new molecular basis of idiopathic bleeding and renal dysfunctions and is instrumental in providing a better understandng of the comprehensive system biology analysis of allele variants in patients suffering from deep-vein thrombosis for developing diagnostic tools to estimate risk of vascular diseases. Also, we shall be able to develop new strategies to modulate the endothelium barrier function.

These objectives will be addressed by an interdisciplinary group of scientists with complementary expertise in imaging, cell signaling, cell mechanics, vascular biology, microbiology, biophysics, clinics and computer science. F Lafont (Coordinator) has expertise in host-pathogen interactions and cell mechanics using Atomic Force Microscopy coupled to fluorescence microscopy. E Lemichez (Partner 2) has a renowned experience in bacterial toxin mode of action and associated cell signaling. P Bassereau (Partner 3) is a world expert in cellular physics, especially on the design and physical analysis of model membrane that mimic some cell functions.

We will obtain significant results to be published in high impact factor peer-reviewed scientific journals, to be patented and possibly sustain activity of spin-off. Our results will have impact on vascular dysfunctions. Our highly innovative project, by targeting crucial conceptual gaps, will thus allow addressing important questions beyond the scope of our study. Our results may highlight some molecular mechanisms that could be involved in basic phenomena with wide spectrum of outcomes on several human diseases linked to vascular dysfunctions, bacteremia, metastatic infections, hemorrhages edema, idiopathic forms of provoked and spontaneous bleeding and renal failure.

Project coordination

Frank LAFONT (Centre d'Infection et d'Immunité de Lille)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CNRS Centre d'Infection et d'Immunité de Lille
INSERM INSERM DR PACA ET CORSE
INSTITUT CURIE - SECT DE RECHERCHE
Unité Toxines Bactériennes

Help of the ANR 347,896 euros
Beginning and duration of the scientific project: September 2015 - 36 Months

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