Rayan Said - Academia.edu (original) (raw)
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Papers by Rayan Said
bioRxiv (Cold Spring Harbor Laboratory), Dec 5, 2022
The cohesion of tissues requires that cells establish cell-cell junctions. Cells contact each oth... more The cohesion of tissues requires that cells establish cell-cell junctions. Cells contact each other by forming Arp2/3-dependent lamellipodia before they initiate the formation of cadherin-based adherens junctions (AJs). Maturing AJs then assemble actin under force though the formation of a mechanosensitive complex comprising the actinbinding proteins α-catenin, vinculin and VASP, which individually act on the nucleation, elongation and organisation of actin filaments in different ways. However, the activity of the ternary complex that these actin-regulatory proteins form has not been investigated due to the difficulty of assembling this complex in vitro in the absence of force. Here, we first designed mutants of these proteins that interact independently of force. We then studied their activity by combining actin polymerization kinetics in fluorescence spectroscopy with observation of single actin filaments in TIRF microscopy. Our results reveal how α-catenin, vinculin and VASP combine their activities in a complex to inhibit Arp2/3-mediated branching, stimulate the nucleation and elongation of linear actin filaments from profilin-actin and crosslink these filaments into bundles. These findings shed light on the molecular mechanisms by which actin regulators synergistically control the transition of actin architecture and dynamics that accompanies the formation and maturation of AJs.
Focal adhesions (FAs) strengthen their link with the actin cytoskeleton to resist force. Talin-vi... more Focal adhesions (FAs) strengthen their link with the actin cytoskeleton to resist force. Talin-vinculin association could reinforce actin anchoring to FAs by controlling actin polymerization. However, the actin polymerization activity of the talin-vinculin complex is not known because it requires the reconstitution of the mechanical and biochemical activation steps that control the association of talin and vinculin and the exposure of their actin-binding domains. By combining kinetic and binding assays with single actin filament observations in TIRF microscopy, we show that the association of talin and vinculin mutants, mimicking different degrees of activation, results in a variety of activities. In particular, mechanically stretched talin and activated vinculin combine to stimulate actin assembly synergistically through a sequential mechanism in which filaments are nucleated, capped and released to elongate. Our findings suggest a versatile mechanism for the regulation of actin as...
bioRxiv (Cold Spring Harbor Laboratory), Dec 5, 2022
The cohesion of tissues requires that cells establish cell-cell junctions. Cells contact each oth... more The cohesion of tissues requires that cells establish cell-cell junctions. Cells contact each other by forming Arp2/3-dependent lamellipodia before they initiate the formation of cadherin-based adherens junctions (AJs). Maturing AJs then assemble actin under force though the formation of a mechanosensitive complex comprising the actinbinding proteins α-catenin, vinculin and VASP, which individually act on the nucleation, elongation and organisation of actin filaments in different ways. However, the activity of the ternary complex that these actin-regulatory proteins form has not been investigated due to the difficulty of assembling this complex in vitro in the absence of force. Here, we first designed mutants of these proteins that interact independently of force. We then studied their activity by combining actin polymerization kinetics in fluorescence spectroscopy with observation of single actin filaments in TIRF microscopy. Our results reveal how α-catenin, vinculin and VASP combine their activities in a complex to inhibit Arp2/3-mediated branching, stimulate the nucleation and elongation of linear actin filaments from profilin-actin and crosslink these filaments into bundles. These findings shed light on the molecular mechanisms by which actin regulators synergistically control the transition of actin architecture and dynamics that accompanies the formation and maturation of AJs.
Focal adhesions (FAs) strengthen their link with the actin cytoskeleton to resist force. Talin-vi... more Focal adhesions (FAs) strengthen their link with the actin cytoskeleton to resist force. Talin-vinculin association could reinforce actin anchoring to FAs by controlling actin polymerization. However, the actin polymerization activity of the talin-vinculin complex is not known because it requires the reconstitution of the mechanical and biochemical activation steps that control the association of talin and vinculin and the exposure of their actin-binding domains. By combining kinetic and binding assays with single actin filament observations in TIRF microscopy, we show that the association of talin and vinculin mutants, mimicking different degrees of activation, results in a variety of activities. In particular, mechanically stretched talin and activated vinculin combine to stimulate actin assembly synergistically through a sequential mechanism in which filaments are nucleated, capped and released to elongate. Our findings suggest a versatile mechanism for the regulation of actin as...