Mechanotransduction
Force has a fundamental role in a wide array of biological processes. For example, it modulates enzymatic activity, induces structural changes in proteins and nucleic acids, alters kinetics of molecular bonds, regulates motion of molecular motors, and has a role in mechanical transduction and sensory functions. At the molecular level, these processes are ultimately related to the capacity of force to modulate lifetimes of molecular interactions and transition rates in biochemical reaction cycles that involve motion.
Single molecule studies of mechanotransductor proteins
In our lab, we use ultrafast force-clamp spectroscopy, a single-molecule technique based on laser tweezers, to investigate interactions between mechanosensitive proteins with sub-millisecond and sub-nanometer resolution [1]. Mechanical signals occurring at the interface between cell membrane and extracellular matrix and at intercellular junctions trigger specific biochemical signals that regulate cell growth, development and differentiation. α-catenin plays a key role in adherens junctions by acting as a physical linker between the actin cytoskeleton and the E-cadherin-β-catenin complex at the cell membrane. To mimic the mechanical response of α-catenin inside and outside adherens junctions, we studied the interaction between single α/β-catenin heterodimers and single α-catenin homodimers with actin filaments in a range of physiological forces (1-17 pN). Outside the cadherin–catenin complex, α-catenin has been shown to regulate actin dynamics and bind phosphoinositide-activated membranes to promote adhesion and migration. Although previous studies indicate that α-catenin links the cadherin-catenin complex to actin under force, the results are controversial due to the low affinity of α/β-catenin heterodimers to actin. Our experiments show that a single α-catenin molecule resists force on actin only outside the cadherin–catenin complex. However, α-catenin function in adherens junctions critically depends on the accumulation of multiple complexes at the adhesion site to support cell-cell connection under physiological mechanical loads (Arbore et al., bioRxiv 2020.04.15.035527).
[1] Arbore, C., Sergides, M., Gardini L., Pavone F. S. and M. Capitanio,“α-catenin regulates cell junction fluidity by cooperative mechanosensing”, bioRxiv 2020.04.15.035527; doi: https://doi.org/10.1101/2020.04.15.035527
[2] Capitanio M., Canepari M., Maffei M., Beneventi D., Monico C., Vanzi F., Bottinelli R., and F. S. Pavone, Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke, Nature Methods 9, 1013–1019 (2012) full text, cover page, author's file
Single molecule studies of mechanotransductor proteins
Single molecule studies of mechanotransductor proteins
We recently developed an innovative setup combining optical manipulation and fluorescence microscopy at the single molecule level. An infrared laser allows trapping of dielectric microspheres used to mechanically manipulate cells. With such a method forces can be applied and measured simultaneously on biological systems. In living cells, direct measurements of forces in the piconewton range can be performed by using genetically encoded FRET-based tension sensors. Combination of the sensors with force-spectroscopy techniques is a promising approach to investigate how mechanical stimuli propagate from the external environment into the cell. Several FRET sensor proteins have been developed recently including cytoskeletal and adhesion proteins (Guo et al. 2014). In our lab, we have recently characterized and calibrated F-actin and alpha-actinin sensors in single HEK cells. We found that F-actin and alpha-actinin tension increase in the focal adhesion complexes. The potential applications of our method in biological and medical related topics are wide and go beyond the study of the basic mechanotransduction mechanisms: in the near future, our studies will be further extended to the human induced pluripotent stem cells (hiPSC) in order to explore cell differentiation.