The invention of optical tweezers more than three decades ago has opened new avenues in the study of the mechanical properties of biological molecules and cells. Quantitative force measurements still represent a challenging task in living cells due to the complexity of the cellular environment. Here, we review different methodologies to quantitatively measure the mechanical properties of living cells, the strength of adhesion/receptor bonds, and the active force produced during intracellular transport, cell adhesion, and migration. We discuss experimental strategies to attain proper calibration of optical tweezers and molecular resolution in living cells. Finally, we show recent studies on the transduction of mechanical stimuli into biomolecular and genetic signals that play a critical role in cell health and disease.

Myosin-5B is one of three members of the myosin-5 family of actin-based molecular motors. Despite its fundamental role in recycling endosome trafficking and in collective actin network dynamics, the molecular mechanisms underlying its motility are inherently unknown. Here we combine single-molecule imaging and high-speed laser tweezers to dissect the mechanoenzymatic properties of myosin-5B. We show that a single myosin-5B moves processively in 36-nm steps, stalls at ~2 pN resistive forces, and reverses its directionality at forces >2 pN. Interestingly, myosin-5B mechanosensitivity differs from that of myosin-5A, while it is strikingly similar to kinesin-1. In particular, myosin-5B run length is markedly and asymmetrically sensitive to force, a property that might be central to motor ensemble coordination. Furthermore, we show that Ca2+ does not affect the enzymatic activity of the motor unit, but abolishes myosin-5B processivity through calmodulin dissociation, providing important insights into the regulation of postsynaptic cargoes trafficking in neuronal cells.

Abstractα-catenin is a crucial protein at cell junctions that provides connection between the actin cytoskeleton and the cell membrane. At adherens junctions (AJs), α-catenin forms heterodimers with β-catenin that are believed to resist force on F-actin. Outside AJs, α-catenin forms homodimers that regulates F-actin organization and directly connect the cell membrane to the actin cytoskeleton, but their mechanosensitive properties are inherently unknown. By using ultra-fast laser tweezers we found that a single α-β-catenin heterodimer does not resist force but instead slips along F-actin in the direction of force. Conversely, the action of 5 to 10 α-β-catenin heterodimers together with force applied toward F-actin pointed end engaged a molecular switch in α-catenin, which unfolded and strongly bound F-actin as a cooperative catch bond. Similarly, an α-catenin homodimer formed an asymmetric catch bond with F-actin triggered by protein unfolding under force. Our data suggest that α-catenin clustering together with intracellular tension engage a fluid-to-solid phase transition at the membrane-cytoskeleton interface.

Some bacterial plasmids carry antibiotic resistance genes and replicate by an asymmetric, rolling circle mechanism, in which replication of the two strands is not concurrent. Initiation of this replication occurs via an initiator protein that nicks one DNA strand at the double-stranded origin of replication. In this work, RepD protein from the staphylococcal plasmid pC221 carries this function and allows PcrA helicase to bind and begin unwinding the plasmid DNA. This work uses whole plasmid constructs as well as oligonucleotide-based mimics of parts of the origin to examine the initiation reaction. It investigates the phenomenon that nicking, although required to open a single-stranded region at the origin and so allow PcrA to bind, is not required for another function of RepD, namely to increase the processivity of PcrA, allowing it to unwind plasmid lengths of DNA. A kinetic mechanism of RepD initiation is presented, showing rapid binding of the origin DNA. The rate of nicking varies with the structure of the DNA but can occur with a rate constant of >25 s –1 at 30 °C. The equilibrium constant of the nicking reaction, which involves a transesterification to form a phosphotyrosine bond within the RepD active site, is close to unity.

Cell adhesions dynamically tune their mechanical properties during tissue development and homeostasis. Fluid connections required for cell mobility can switch to solid links to maintain the mechanical rigidity of epithelial layers 1,2 . Changes in the composition and clustering of adhesion molecules have been proposed to modulate cell junction fluidity, but the underlying mechanisms are unclear 3,4 . acatenin has been shown to play a fundamental role in different adhesion sites. At adherens cell-cell junctions (AJ), a-catenin localizes in cadherin-catenin complexes, where it provides a mechanical link between b-catenin and the actin cytoskeleton 5 . However, its function is controversial owing to the low affinity between actin and the a-b-catenin heterodimer 6 . Outside AJ, a-catenin binds itself to form homodimers that connect the cell membrane to the actin cytoskeleton to promote adhesion and migration, but its mechanosensitive properties are inherently unknown 7,8 . Here, using ultra-fast laser tweezers 9 we show that a single mammalian a-catenin molecule displays very different force-bearing properties depending on whether it is associated to b-catenin or not. We found that a single a-b-catenin heterodimer slips along an actin filament in the direction of force, while a single a-catenin homodimer forms a strong asymmetric catch-bond with actin, in which the bond lifetime increases, and the protein unfolds with force. Importantly, assemblies of multiple ab-catenin heterodimers show force-bearing and unfolding properties similar to the acatenin homodimer. Our results indicate that, outside AJ, single a-catenin homodimers act as a mechanical link with the actin cytoskeleton that resists force efficiently. Nonetheless, inside AJ, a-catenin's capability to hold cell-cell connections under physiological loads critically depends on the recruitment of multiple (5-10) complexes. Our data support a molecular model in which a-catenin clustering and intercellular tension engage a fluid-to-solid phase transition at the membranecytoskeleton interface.In any living cell, an array of mechanotransductor proteins responds to mechanical cues to trigger complex molecular signaling, driving cell morphology and gene expression profiles 10 . Among these proteins, a-catenin has been reported to act as a mechanosensor that regulates adherens junctions (AJ) in response to mechanical cues from neighboring cells in a tissue 5 . It is widely accepted that a-catenin in AJ binds b-catenin, which, in turn, connects to the cytoplasmatic portion of E-cadherin to constitute the cadherin-catenin complex (CCC). Since a-catenin also binds actin, a-catenin has been indicated as a major candidate for providing a link between the CCC and the actin cytoskeleton. This molecular link is required