Understanding cellular force transmission dynamics is crucial in mechanobiology. We developed the DNA-based ForceChrono probe to measure force magnitude, duration, and loading rates at the single-molecule level within living cells. The ForceChrono probe circumvents the limitations of in vitro single-molecule force spectroscopy by enabling direct measurements within the dynamic cellular environment. Our findings reveal integrin force loading rates of 0.5-2 pN/s and durations ranging from tens of seconds in nascent adhesions to approximately 100 s in mature focal adhesions. The probe\'s robust and reversible design allows for continuous monitoring of these dynamic changes as cells undergo morphological transformations. Additionally, by analyzing how mutations, deletions, or pharmacological interventions affect these parameters, we can deduce the functional roles of specific proteins or domains in cellular mechanotransduction. The ForceChrono probe provides detailed insights into the dynamics of mechanical forces, advancing our understanding of cellular mechanics and the molecular mechanisms of mechanotransduction.

The geometric shape of a cell is strongly influenced by the cytoskeleton, which, in turn, is regulated by integrin-mediated cell-extracellular matrix (ECM) interactions. To investigate the mechanical role of integrin in the geometrical interplay between cells and the ECM, we proposed a single-cell micropatterning technique combined with molecular tension fluorescence microscopy (MTFM), which allows us to characterize the mechanical properties of cells with prescribed geometries. Our results show that the curvature is a key geometric cue for cells to differentiate shapes in a membrane-tension- and actomyosin-dependent manner. Specifically, curvatures affect the size of focal adhesions (FAs) and induce a curvature-dependent density and spatial distribution of strong integrins. In addition, we found that the integrin subunit β1 plays a critical role in the detection of geometric information. Overall, the integration of MTFM and single-cell micropatterning offers a robust approach for investigating the nexus between mechanical cues and cellular responses, holding potential for advancing our understanding of mechanobiology.

Macrophages are a type of immune cell that helps eliminate pathogens and diseased cells. Recent research has shown that macrophages can sense mechanical cues from potential targets to perform effective phagocytosis, but the mechanisms behind it remain unclear. In this study, we used DNA-based tension probes to study the role of integrin-mediated forces in FcγR-mediated phagocytosis. The results showed that when the phagocytic receptor FcγR is activated, the force-bearing integrins create a \"mechanical barrier\" that physically excludes the phosphatase CD45 and facilitates phagocytosis. However, if the integrin-mediated forces are physically restricted at lower levels or if the macrophage is on a soft matrix, CD45 exclusion is significantly reduced. Moreover, CD47-SIRPα \"don\'t eat me\" signaling can reduce CD45 segregation by inhibiting the mechanical stability of the integrin barrier. These findings demonstrate how macrophages use molecular forces to identify physical properties and combine them with biochemical signals from phagocytic receptors to guide phagocytosis.