This is repeated until a filopodium aligns on the pattern ridge which subsequently leads to the assembly of a robust F-actin network and an extensive contact zone with the ridge. This then enables to switch off the Pazopanib dynamic unstable behavior observed in non-aligned filopodia, allowing to filopodium stabilization for hours, and ultimately leading to steady neurite outgrowth. The two distinct filopodial behaviors we observe most likely depend on different levels of coupling between the substrate and the cytoskeleton as proposed in the ����molecular clutch model����. Extensive interaction of aligned Y-27632 dihydrochloride filopodia with the substrate, might allow a much more efficient cytoskeletal coupling than in non-aligned filopodia, leading to constant filopodial protrusion. In this case, the formation of a robust F-actin network might allow to counteract the actin retrograde flow in the aligned filopodium, leading to its stabilization. In unaligned filopodia, less stable substrate-cytoskeletal coupling might occur, due to the limited interaction with the ECM. In this case, filopodia retraction might occur because strong retrograde flow exceeds actin assembly at the filopodium tip. Obviously, in our purely ECM driven system, integrins are the sensors that allow to interprete the line pattern. Consistently, conformationally activated, but unligated integrins have been observed in filopodia of neuronal growth cones and might allow to sense the extent of filopodium contact. One important question is then which signaling events downstream of the integrins allow the formation and maintenance of the robust F-actin network observed in aligned filopodia that allows stabilization of the molecular clutch. A first hint is that this does not occur on the plain substrate, on which each filopodium senses an identical amount of laminin. This suggests that on the line pattern, formation of the robust F-actin network requires integration of spatially regulated adhesive signals from the aligned filopodia at the growth cone tip and from the non-aligned filopodia that are continuously operating on the distal part of the growth cone. However, the signaling events occurring downstream of these receptors remain elusive and our whole-cell measurements of signaling activities certainly could not resolve the precise spatio-temporal regulation of minute pools of signaling molecules in the growth cone that is relevant to this system. Understanding the signal amplification events that allow the formation of this F-actin rich network will therefore requires advanced live cell imaging techniques that allow to resolve their spatio-temporal dynamics in the growth cone.