Invadopodia are involved in cancer invasion by generating mechanical forces and secreting proteolytic enzymes. It is hypothesized that physical forces applied to integrin, especially α 5 β 1 integrin, are strongly associated with the generation of invadopodial forces and protrusive activity of invadopodia; however, their mechanical relationship remains elusive. Here, a new deoxyribonucleic acid-based force probe is developed for mapping α 5 β 1 integrin tension with single-molecule precision. Using this probe, the formation and maturation of invadopodia depending on integrin type and tension are investigated. In addition, the spatio-temporal analysis of the integrin tension map is performed to measure changes in integrin tension and invadopodial force in each stage of invadopodia development. Consequently, it is found that α 5 β 1 integrin tension above 40 pN is critical not only for adhesion but also for the maturation of invadopodia, eventually leading to the generation of a strong invadopodial force greater than 100 pN through α 5 β 1 integrinligand complexes with actomyosin contractility. This biophysical mechanism underlying the invadopodia formation and mechanical activation may provide new insights into cancer invasion and metastasis.

A major difference between amyloid precursor protein (APP) isoforms (APP695 and APP751) is the existence of a Kunitz type protease inhibitor (KPI) domain which has a significant impact on the homo‐ and hetero‐dimerization of APP isoforms. However, the exact molecular mechanisms of dimer formation remain elusive. To characterize the role of the KPI domain in APP dimerization, we performed a single molecule pull down (SiMPull) assay where homo‐dimerization between tethered APP molecules and soluble APP molecules was highly preferred regardless of the type of APP isoforms, while hetero‐dimerization between tethered APP751 molecules and soluble APP695 molecules was limited. We further investigated the domain level APP‐APP interactions using coarse‐grained models with the Martini force field. Though the model initial ternary complexes (KPI‐E1, KPI‐KPI, KPI‐E2, E1‐E1, E2‐E2, and E1‐E2) generated using HADDOCK (HD) and AlphaFold2 (AF2), the binding free energy profiles and the binding affinities of the domain combinations were investigated via the umbrella sampling with Martini force field. Additionally, membrane‐bound microenvironments at the domain level were modeled. As a result, it was revealed that the KPI domain has a stronger attractive interaction with itself than the E1 and E2 domains, as reported elsewhere. Thus, the KPI domain of APP751 may form additional attractive interactions with E1, E2 and the KPI domain itself, whereas it is absent in APP695. In conclusion, we found that the APP751 homo‐dimer formation is predominant than the homodimerization in APP695, which is facilitated by the presence of the KPI domain.