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Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of ...

LENS, European Laboratory for Non-Linear Spectroscopy

Physics Department, University of Florence

Here, we describe protocols for three-dimensional tracking of single quantum dot-conjugated molecules with nanometer accuracy in living cells using conventional fluorescence microscopy. The technique exploits out-of-focus images of single emitters combined with an automated pattern-recognition open-source software that fits the images with proper model functions to extract the emitter coordinates. We describe protocols for targeting quantum dots to both membrane components and cytosolic proteins.

Three-Dimensional Tracking of Quantum Dot-Conjugated Molecules in Living Cells.

Mechanical transitions in molecular motors often occur on a submillisecond time scale and rapidly follow binding of the motor with its cytoskeletal filament. Interactions of nonprocessive molecular motors with their filament can be brief and last for few milliseconds or fraction of milliseconds. The investigation of such rapid events and their load dependence requires specialized single-molecule tools. Ultrafast force-clamp spectroscopy is a constant-force optical tweezers technique that allows probing such rapid mechanical transitions and submillisecond kinetics of biomolecular interactions, which can be particularly valuable for the study of nonprocessive motors, single heads of processive motors, or stepping dynamics of processive motors. Here we describe a step-by-step protocol for the application of ultrafast force-clamp spectroscopy to myosin motors. We give indications on optimizing the optical tweezers setup, biological constructs, and data analysis to reach a temporal resolution of few tens of microseconds combined with subnanometer spatial resolution. The protocol can be easily generalized to other families of motor proteins.

High-Speed Optical Tweezers for the Study of Single Molecular Motors.

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 Ca 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.

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

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.

M. Capitanio

LENS, European Laboratory for Non-Linear Spectroscopy

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy