Filopodia are actin-rich membrane protrusions essential for cell morphogenesis, motility, and cancer invasion. How cells control filopodium initiation on the plasma membrane remains elusive. We performed experiments in cellulo, in vitro, and in silico to unravel the mechanism of filopodium initiation driven by the membrane curvature sensor IRSp53 (insulin receptor substrate protein of 53 kDa). We showed that full-length IRSp53 self-assembles into clusters on membranes depending on PIP 2 . Using well-controlled in vitro reconstitution systems, we demonstrated that IRSp53 clusters recruit the actin polymerase VASP (vasodilator-stimulated phosphoprotein) to assemble actin filaments locally on membranes, leading to the generation of actin-filled membrane protrusions reminiscent of filopodia. By pulling membrane nanotubes from live cells, we observed that IRSp53 can only be enriched and trigger actin assembly in nanotubes at highly dynamic membrane regions. Our work supports a regulation mechanism of IRSp53 in its attributes of curvature sensation and partner recruitment to ensure a precise spatial-temporal control of filopodium initiation.

Filopodia are actin-rich membrane protrusions essential for cell morphogenesis, motility, and cancer invasion. How cells control filopodia initiation on the plasma membrane remains elusive. We performed experiments in cellulo, in vitro and in silico to unravel the mechanism of filopodia initiation driven by the membrane curvature sensor IRSp53. We showed that full-length IRSp53 self-assembles into clusters on membranes depending on PIP2. Using well-controlled in vitro reconstitution systems, we demonstrated that IRSp53 clusters recruit the actin polymerase VASP to assemble actin filaments locally on membranes, leading to the generation of actin-filled membrane protrusions reminiscent of filopodia. By pulling membrane nanotubes from live cells, we observed that IRSp53 can only be enriched and trigger actin assembly in nanotubes at highly dynamic membrane regions. Our work supports a regulation mechanism of IRSp53 in its attributes of curvature sensation and partner recruitment to ensure a precise spatial-temporal control of filopodia initiation.

During cell adhesion, integrins form clusters that transmit mechanical forces to the substrate (mechanotransduction) and regulate biochemical signaling depending on substrate stiffness. In recent years, mechanotransduction studies significantly advanced our understanding of cell adhesion. Most studies were performed on rigid substrates such as glass, while more physiologically relevant fluid membranes have been less explored. In contrast to rigid substrates, integrins' ligands on fluid supported lipid bilayers (SLBs) are mobile and adhesive complexes cannot serve as anchoring points promoting cell spreading. Here, we demonstrate that cells spread on SLBs coated with Invasin, a high-affinity integrin ligand. We show that in contrast to SLBs functionalized with RGD peptides, integrin clusters grow and mature on Invasin-SLBs to a similar extent as on glass. While actomyosin contraction dominates adhesion maturation on stiff substrates, we find that integrin mechanotransduction and cell spreading on fluid SLBs rely on dynein pulling forces along microtubules, perpendicular to membranes, and microtubules pushing on adhesive complexes, respectively. Our findings, supported by a theoretical model, demonstrate a new mechanical role for microtubules in integrin clustering on fluid substrates. These forces may also occur on non-deformable surfaces, but have been overlooked.