Structure and mechanics of composite cytoskeletal networks – CoCyNet

Cytoskeletal networks are largely responsible for the mechanical properties of living cells, and are composed of three types of interconnected biopolymers: actin, intermediate filaments (IFs) and microtubules. Among those, actin filaments and microtubules form dynamic networks that can quickly assemble and disassemble in response to their environment, while they are not mechanically resistant and tend to break or disassemble at moderate strains. On the contrary, vimentin, which we will use as a model to study IFs in this proposal, forms long-lived networks with slow assembly/disassembly, but with remarkable mechanical properties as vimentin filaments are highly stretchable and resist breakage. Although actin and vimentin have different structural and physical properties, there is growing evidence that they work in coordination to control cellular functions such as cell migration, cell division or mechano-sensing. For example, vimentin filaments control the assembly of actin stress fibers (SFs) which are connected to integrin adhesion, and they orient traction forces that drive cell migration, indicating that the vimentin network acts as a load-bearing superstructure capable of integrating and reorienting actin-based forces. The dynamic coupling of vimentin with actin has also been associated with limitation of cell damage during processes involving large deformations, which takes place during embryonic development, cancer metastasis or would healing. The interplay between vimentin and actin is therefore crucial for the protection of cells against external forces.

While the regulation of cellular functions by biochemical signaling has been known for a long time, it is becoming increasingly clear that they are also regulated by mechanical force arising from the extracellular environment, the plasma membrane or the cytoskeleton. The regulation of actin binding proteins functions by mechanical forces has been extensively studied, but whether similar mechanisms also exist for vimentin binding proteins or cross-linkers of actin/vimentin remains less explored.

Physical actin/vimentin crosstalk could originate from three different types of interaction: (i) a potential direct binding; (ii) steric effects; (iii) connection via cross-linkers. Importantly and consistent with a functional crosstalk, some of the actin/vimentin cross-linkers are already known to crosslink actin filaments such as myosinIIB, filaminA, plectin or fimbrin, suggesting that they are crucial in the modulation of both networks kinetics and morphologies. However, very few studies have addressed the interplay between vimentin and actin at the molecular level, and none involves its regulation by force. Here, we hypothesize that the coupling between actin and vimentin networks is responsible for emergent physical properties which are fundamental for many cellular functions, such as sustaining large deformations or large scale force generation.

The goal of the project is to elucidate how the dynamic morphology of composite cytoskeletal networks is determined by their molecular components and how it is regulated by mechanical cues. We want to understand how actin and vimentin networks regulate each other’s properties, either directly or through specific passive and active cross-linkers, and how the crosstalk between them is modulated under external stresses. We will study the structure and dynamics of actin/vimentin networks and their regulation by force in contexts of increasing complexity: from single filaments and bundles reconstituted in vitro (WP1 without external force, WP2 with force) to cellular networks in live cells (WP3). We expect to further understand how cells integrate actin cytoskeleton and vimentin mechano-transduction to ensure adequate mechanical responses during cellular functions, and unravel novel mechanisms involved in mechano-sensing.