Open Access

Stefanie D. Pritzl, Jan Lipfert

• Mechanical forces are central to biological function across scales, from whole organisms to individual molecules. At the cellular and subcellular levels, force generation, sensing, and mechanotransduction shape diverse processes including gene expression, morphogenesis, and disease progression. Single-molecule force spectroscopy provides critical insights into these mechanics, with magnetic tweezers (MTs) emerging as a versatile tool with unique advantages. MTs operate across physiologically relevant forces (∼0.01–100 pN) and enable stable, long-duration, and multiplexed measurements without photodamage, making them ideally suited to investigate proteins under near-native conditions. This review highlights the evolution of MT-based protein mechanics, spanning early cell microrheology to recent single-molecule studies. We focus on key developments and applications, including investigations of cytoskeletal, membrane, and motor proteins, force-sensitive cell adhesion complexes,Mechanical forces are central to biological function across scales, from whole organisms to individual molecules. At the cellular and subcellular levels, force generation, sensing, and mechanotransduction shape diverse processes including gene expression, morphogenesis, and disease progression. Single-molecule force spectroscopy provides critical insights into these mechanics, with magnetic tweezers (MTs) emerging as a versatile tool with unique advantages. MTs operate across physiologically relevant forces (∼0.01–100 pN) and enable stable, long-duration, and multiplexed measurements without photodamage, making them ideally suited to investigate proteins under near-native conditions. This review highlights the evolution of MT-based protein mechanics, spanning early cell microrheology to recent single-molecule studies. We focus on key developments and applications, including investigations of cytoskeletal, membrane, and motor proteins, force-sensitive cell adhesion complexes, mechanoresponsive ion channels, and virus-host interactions. Furthermore, we discuss the integration of MTs with fluorescence readouts and emerging in vivo applications, underscoring the expanding role of MTs in decoding the molecular basis of mechanobiology.…