To understand the kinetic and mechanical aspects of how filopodia shaft adhesions grow and mature, and how this synchronizes with the protrusion of lamellipodia, we decided to visualize the formation of filopodia adhesions with Tyrphostin AG 1296 respect to the clustering of b3-integrin receptors in the filopodia membrane. The b3-integrin is one of the major transmembrane proteins in the formation of earliest cell-substrate adhesions. While a5b1 integrins specifically recognize fibronectin and the clustering of a5b1 integrins determines the strength of cell-substrate adhesions, avb3 integrins mediate the Triflusal rigidity sensing at the cell leading edge and, together with talin, enable early mechanotransduction events. To better understand how filopodia shaft adhesions are formed and get incorporated in the lamellum, we monitored the substrate adhesion and spreading of rat embryonic fibroblasts that were stably transfected with b3-integrin-EGFP. This b3-integrin-EGFP construct has previously been validated carefully and utilized to study the kinetics of integrin clustering, as well as for studies detailing the mechanism of how the interaction of talin with b3integrin enables talin activation and reciprocal integrin clustering. Here we were particularly interested in characterizing the growth dynamics of filopodia shaft b3-integrin clusters in response to the local dynamics of protrusion and retraction of lamellipodia. In addition, the impact of lamellipodia dynamics on the characteristics of cytoskeleton organization at filopodia adhesion was examined. Since the cycles of lamellipodium protrusion and retraction are more pronounced on rigid than on soft polyacrylamide gels coated with FN, we finally asked how these different cyclic motions of lamellipodia due to substrate rigidity might affect the life cycle of filopodia shaft adhesions. To quantify the fluorescence intensity of filopodia b3-integrinEGFP clusters as a function of time, the average fluorescence intensity per pixel of filopodia b3-integrin-EGFP clusters was measured. It rapidly increased when first contacting the advancing and later the pausing lamellipodium.