Molecular Motors
Many functions fundamental for cell life performed by enzymes (for example DNA transcription, protein synthesis, or cellular trafficking) involve production of work and movement. The capability of converting the chemical energy contained in molecules like ATP into mechanical work has earned these enzymes the name “molecular motors” (or “motor proteins”).
Mechanics of non-processive myosin motors
Our data suggest that the decrease in the amplitude of the working stroke observed in muscle fibers is due to premature unbinding of the molecule from actin, thus providing a molecular explanation for the mechanism of regulation of the myosin working stroke in the intact muscle. Experiments conducted on small arrays of myosin molecules reported a load-independent size of the working stroke that seemed to contradict the prior results obtained on muscle fibers. That result is in agreement with the constant working stroke we found when averaging only the long events and is probably a consequence of the inability to detect premature detachment events in those experiments owing to limited time resolution and a different experimental configuration. Our data therefore reconcile the contradictory results found in the literature.
[1] 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)
[2] Capitanio M., Canepari M., Cacciafesta P., Lombardi V., Cicchi R., Maffei M., Pavone F. S., and R. Bottinelli, Two independent mechanical events in the interaction cycle of skeletal muscle myosin with actin, Proc. Natl. Acad. Sci. USA 103, pp. 87-92, DOI: 10.1073/pnas.0506830102 , 2006 January 3. (full text)
[3] Elangovan R., Capitanio M., Melli L., Pavone F.S., Lombardi V., and G. Piazzesi, An integrated in vitro and in situ study of Kinetics of myosin II from frog skeletal muscle, J. Physiol , 590 (5), pp. 1227-1242, DOI: 10.1113/jphysiol.2011.222984, 2012, February 5. (full text)v
Skeletal muscle myosin is one of the most challenging motors due to its non-processive nature and very rapid kinetics. During the years, we have developed different approaches to investigate the molecular mechanisms of this motor: in-vitro motility assay and single fiber mechanics [3], optical tweezers [2], and, more recently, our ultra-fast force-clamp laser tweezers [1].
Our method revealed a complex mechanism of regulation of the myosin working stroke by force and previously undetectable fast detachment pathways. In particular, a premature (< 5 ms) dissociation pathway becomes more populated as the force is increased, resulting in a working stroke that decreased with load.
Tracking movements of processive myosin motors
Mechanosensitive proteins including members of the myosin superfamily respond to the spatial distribution, direction, strength, and duration of forces with changed mechanoenzymatic properties. Among all myosins, myosin-5B has been recently shown to be involved in many fundamental cellular processes such as postsynaptic plasticity, epithelial cell polarization, and rearrangement of the actin network responsible for precise nucleus positioning in oocytes. However, themolecular mechanisms underlying its motility and its regulation by calcium and by external forces were largely unexplored. Thanks to a combination ofadvanced single-molecule tools we could set the first characterization of the biophysical properties of this fundamental molecular motor. Through fluorescence-based in vitro motility assay we studied the processivity of myosin-5B motors under unloaded conditions. We demonstrated that myosin-5B moves processively in 36 nm steps on individual actin filaments as single motor. We then investigate load-dependence of myosin-5B movements with ultrafast force-clamp spectroscopy, a sub-millisecond and sub-nanometer resolution technique based on laser tweezers, thus finding that myosin-5B step size, velocity, and run length are strongly mechanosensitive. Resistive forces progressively decrease myosin-5B velocity and run length up to stall at about 2 pN, where forward and backward stepping reach equilibrium. The motor directionality is reversed for forces >2 pN. On the other hand, assistive forces moderately affect myosin velocity, but strongly accelerate the detachment of the motor from the actin filament. We found that myosin-5B, although processive, is not as strong as myosin-5A as a single motor and probably evolved to efficiently transport cargoes in ensembles. Finally, we showed that Ca2+ finely regulates myosin-5B motility in vitro by uncoupling the mechanical and enzymatic activity of the motor, giving insight into its transport function in neuronal cells. Our study provides mechanistic insight into the molecular basis underlying myosin-5B-based transport and offers a detailed model of its trafficking function in the crowded actin cytoskeleton of mammalian cells.