Rafael Tapia Rojo

Abstract: Mechanical forces are a crucial cellular cue that guides several physiological processes, including migration, division, and development. Understanding how cells detect and interpret forces from their environment requires measuring the dynamics of the key force-sensing proteins under biologically meaningful forces—typically only of a few pN—and over timescales comparable to a protein’s lifetime inside the cell—which can be of several minutes or even hours. Current single-molecule force spectroscopy instruments fail in manipulating very low forces and suffer from large mechanical drift, which restricts measurements to just a few minutes. These instrumental shortcomings limit our present understanding of how protein mechanosensors work inside the cell. Here, I will present our latest development in magnetic tweezers force spectroscopy instrumentation, which, by implementing a magnetic tape head as the force-generating device, shows unique force control and stability, allowing to monitor the dynamics of a single protein under physiologically-relevant forces during several days. Thanks to these new capabilities, we have measured the folding dynamics of the talin protein—a key protein mechanosensor working in focal adhesions—under force conditions resembling those in the cell. First, we explored the response of talin upon complex force signals, such as mechanical noise and force oscillations. Our data demonstrate that talin works as a mechanical bandpass, undergoing stochastic resonance over a narrow frequency range while ignoring random force fluctuations. Second, thanks to the high stability of our technique, we measured talin dynamics over very long timescales, up to several uninterrupted days. We demonstrate that talin, an uncomplicated two-state folder when observed for a few minutes, shows gradual conformational complexity when the measuring time is extended to hours or even days. These examples illustrate the ability of magnetic tweezers to understand how single proteins respond to physiologically-relevant forces, which opens the possibility to directly address a large number of questions of critical biological relevance.