Spatio-Temporal and Mechanical Control of Motile Structures
Team Leader : Gregory Giannone
Grégory Giannone explores the spatiotemporal and mechanical mechanisms driving the dynamics of structures and proteins regulating cell motility
Cells have the ability to adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on neighboring cells and extracellular matrices cells control their environment, shape and movement. This is true from integrin-based adhesive structures of migrating cells to synapses of neurons. Those adhesive structures are the converging zones integrating biochemical and biomechanical signals arising from the extracellular space and the actin cytoskeleton. Thus, the life-cycle of adhesive and cytoskeletal structures are involved in critical cellular functions such as migration, proliferation and differentiation, and regulate cell behavior in many physiological responses such as development. Alterations of adhesive and cytoskeletal organizations contribute to pathologies including cancer, but also cognitive disorders.
At the molecular, sub-cellular, and cellular levels, cell shaping and motility proceed through cycles lasting from seconds to minutes. During those cycles, critical proteins undergo stochastic motions and transient interactions that are essential to their functions. Regulation of these interactions by forces is at the base of mechano-transduction events controlling cell behavior. Therefore, to understand the molecular mechanisms controlling the life cycle of motile structures, it is crucial to study the position and dynamics of proteins but also their interactions and how mechanical forces control these molecular events.
Our goal is to decipher at the molecular level the spatiotemporal and mechanical mechanisms which control the architecture and dynamics of motile structures including integrin-based AS, the lamellipodium and dendritic spines. Exploration of these new dimensions requires an innovative and multidisciplinary approach combining cell biology, biophysics, biomechanics and advanced optical microscopy techniques including super-resolution microscopy, single protein tracking and quantitative image analysis.
We are developing three specific axes: 1/ Integrin adhesion 2/ Actin in dendritic spines 3/ Super-resolution developments
Deciphering mechano-biology using super-resolution microscopy
Cell stretching is amplified by active actin remodeling to deform and recruit proteins in mechano-sensitive structures Detection and conversion of mechanical forces into biochemical signals control cell functions during physiological and pathological processes. Mechano-sensing is based on protein deformations and reorganizations, yet the molecular mechanisms are still unclear. Using a cell stretching device compatible with super-resolution microscopy (SRM) and single protein tracking (SPT), we explored the nanoscale deformations and reorganizations of individual proteins inside mechano-sensitive structures. We achieved SRM after live stretching on intermediate filaments, microtubules and integrin adhesions. Simultaneous SPT and stretching showed that while integrins follow the elastic deformation of the substrate, actin filaments and talin also displayed lagged and transient inelastic responses associated with active acto-myosin remodeling and talin deformations. Capturing acute reorganizations of single-molecule during stretching showed that force-dependent vinculin recruitment is delayed and depends on the maturation of integrin adhesions. Thus, cells respond to external forces by amplifying transiently and locally cytoskeleton displacements enabling protein deformation and recruitment in mechano-sensitive structures.
Authors: Sophie Massou*, Filipe Nunes Vicente*, Franziska Wetzel*, Amine Mehidi, Dan Strehle, Cecile Leduc, Raphaël Voituriez, Olivier Rossier, Pierre, Nassoy and Gregory Giannone
* First co-authors
Orré T., Rossier O. and Giannone G. in Nature Communications - May 2021
Focal adhesions (FAs) initiate chemical and mechanical signals involved in cell polarity, migration, proliferation and differentiation. Super-resolution microscopy revealed that FAs are organized at the nanoscale into functional layers from the lower plasma membrane to the upper actin cytoskeleton. Yet, how FAs proteins are guided into specific nano-layers to promote interaction with given targets is unknown. Using single protein tracking, super-resolution microscopy and functional assays, we link the molecular behavior and 3D nanoscale localization of kindlin with its function in integrin activation inside FAs. We show that immobilization of integrins in FAs depends on interaction with kindlin. Unlike talin, kindlin displays free diffusion along the plasma membrane outside and inside FAs. We demonstrate that the kindlin Pleckstrin Homology domain promotes membrane diffusion and localization to the membrane-proximal integrin nano-layer, necessary for kindlin enrichment and function in FAs. Using kindlin-deficient cells, we show that kindlin membrane localization and diffusion are crucial for integrin activation, cell spreading and FAs formation. Thus, kindlin uses a different route than talin to reach and activate integrins, providing a possible molecular basis for their complementarity during integrin activation.
Mechanical control of actin regulators during cell migration - Nature Cell Biology, Nov. 2021
Actin filaments generate mechanical forces that drive membrane movements during trafficking, endocytosis and cell migration. Reciprocally, adaptations of actin networks to forces regulate their assembly and architecture. Yet, a demonstration of forces acting on actin regulators at actin assembly sites in cells is missing. Here we show that local forces arising from actin filament elongation mechanically control WAVE regulatory complex (WRC) dynamics and function, that is, Arp2/3 complex activation in the lamellipodium. Single-protein tracking revealed WRC lateral movements along the lamellipodium tip, driven by elongation of actin filaments and correlating with WRC turnover. The use of optical tweezers to mechanically manipulate functional WRC showed that piconewton forces, as generated by single-filament elongation, dissociated WRC from the lamellipodium tip. WRC activation correlated with its trapping, dwell time and the binding strength at the lamellipodium tip. WRC crosslinking, hindering its mechanical dissociation, increased WRC dwell time and Arp2/3-dependent membrane protrusion. Thus, forces generated by individual actin filaments on their regulators can mechanically tune their turnover and hence activity during cell migration.
Authors: Amine Mehidi, Frieda Kage, Zeynep Karatas, Maureen Cercy, Matthias Schaks, Anna Polesskaya, Matthieu Sainlos, Alexis Gautreau, Olivier Rossier, Klemens Rottner and Grégory Giannone
Forces generated by lamellipodial actin filament elongation regulate the WAVE complex during cell migration
Nature Cell Biology, 23, pages 1148–1162 (2021). https://www.nature.com/articles/s41556-021-00786-8
*Contact IINS: Grégory Giannone
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