Probing force in living cells with optical tweezers: from single-molecule mechanics to cell mechanotransduction.

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.

A protocol for single molecule imaging and tracking of processive myosin motors.

Myosin is a large family of actin-based molecular motors, which includes efficient intracellular transporters that move cargoes and material essential for cell's life. Here, we describe protocols for labelling single myosin motors with quantum dots, tracking them in an in vitro reconstituted single-molecule motility assay, acquiring image stacks and analyzing them. We describe the required steps to obtain trajectories of single myosin motors from which fundamental biophysical parameters such as the motor velocity, run length and step size can be derived. We also describe protocols for an ensemble actin gliding assay, which is valuable to test the motor viability and its ensemble properties. The protocols allow probing the effect of changes in nucleotides, ions, and buffer composition on the motor properties and are easily generalizable to track the movements of different motor proteins.

Myosin V fluorescence imaging dataset for single-molecule localization and tracking.

Myosin-5B is one of three members of the myosin-5 family of actin-based molecular motors fundamental in recycling endosome trafficking and collective actin network dynamics. Through single-molecule motility assays, we recently demonstrated that myosin-5B can proceed in 36-nm steps along actin filaments as single motor. By analyzing trajectories of single myosin-5B along actin filaments we showed that its velocity is dependent on ATP concentration, while its run length is independent on ATP concentration, as a landmark of processivity. Here, we share image stacks acquired under total internal reflection fluorescence (TIRF) microscopy and representative trajectories of single myosin-5B molecules labelled with Quantum Dots (QD-myo-5B) moving along actin filaments at different ATP concentrations (0.3-1000 µM). Localization of QD-myo-5B was performed with the PROOF software, which is freely available [1]. The data can be valuable for researchers interested in molecular motors motility, both from an experimental and modeling point of view, as well as to researchers developing single particle tracking algorithms. The data is related to the research article "Dissecting myosin-5B mechanosensitivity and calcium regulation at the single molecule level" Gardini et al., 2015.

Dissecting myosin-5B mechanosensitivity and calcium regulation at the single molecule level.

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.

Kinetic mechanism of initiation by RepD as a part of asymmetric, rolling circle plasmid unwinding.

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.