Amit D. Mehta

Myosin-V is a processive actin-based motor

Class-V myosins, one of 15 known classes of actin-based molecular motors, have been implicated in several forms of organelle transport perhaps working with microtubule-based motors such as kinesin,. Such movements may require a motor with mechanochemical properties distinct from those of myosin-II, which operates in large ensembles to drive high-speed motility as in muscle contraction. Based on its function and biochemistry, it has been suggested that myosin-V may be a processive motor, like kinesin,. Processivity means that the motor undergoes multiple catalytic cycles and coupled mechanical advances for each diffusional encounter with its track. This allows single motors to support movement of an organelle along its track. Here we provide direct evidence that myosin-V is indeed a processive actin-based motor that can move in large steps approximating the 36-nm pseudo-repeat of the actin filament.

Fiber optic in vivo imaging in the mammalian nervous system

The compact size, mechanical flexibility, and growing functionality of optical fiber and fiber optic devices are enabling several new modalities for imaging the mammalian nervous system in vivo. Fluorescence microendoscopy is a minimally invasive fiber modality that provides cellular resolution in deep brain areas. Diffuse optical tomography is a non-invasive modality that uses assemblies of fiber optic emitters and detectors on the cranium for volumetric imaging of brain activation. Optical coherence tomography is a sensitive interferometric imaging technique that can be implemented in a variety of fiber based formats and that might allow intrinsic optical detection of brain activity at a high resolution. Miniaturized fiber optic microscopy permits cellular level imaging in the brains of behaving animals. Together, these modalities will enable new uses of imaging in the intact nervous system for both research and clinical applications.

Reflections of a lucid dreamer: optical trap design considerations.

Publisher Summary This chapter discusses the optical trap design considerations. The optical trap technique can be used to constrain and move small particles in solution using a light microscope and laser beam. Trapping size scales and sensitivity are well suited for studying the mechanical properties of single cells, organelles, and even molecules. The chapter describes considerations involved in the planning and implementation of an optical trapping microscope for high-resolution force and displacement measurements of trapped particles. These molecules will bind to and move the actin filament, allowing measurement of their mechanical properties at the single molecule level. The chapter illustrates the observations that these beads with nanometer resolution use active feedback loops to suppress bead diffusion by rapid trap deflection, and observethe specimen by using brightfield and fluorescent imaging simultaneously.

Biomechanics, one molecule at a time.

Single-molecule observation has come of age. Parallel developments of sensitive mechanical probes and single fluorophore detection now fuse into unique combinations, allowing investigators to examine shape and chemical transitions of single molecules with ever increasing precision and finesse. Optical trapping, using focused laser beams to constrain dielectric particles in solution (for reviews see Ref. 1), has emerged as a widely used and versatile tool to examine mechanically interesting proteins and DNA. The associated forces of light on matter can be rendered sufficiently weak that single molecules compete with them. In most applications to date, molecules of interest are attached to uniform dielectric beads, which are trapped and used as handles to configure an appropriate experimental geometry. One can detect bead position with high precision, monitoring biological activity by tracking probe displacement. Such methods allow accurate, quantitative characterization of force and displacement transients driven or experienced by single molecules, providing a unique edge in deciphering the underlying mechanisms and reaction schemes. Several biomolecules have met variants on this theme. Here, we focus attention on three classes: processive motors, nonprocessive motors, and proteins experiencing significant strain.

Single molecule biochemistry using optical tweezers.

The use of optical trapping to create extremely compliant mechanical probes has ushered in a new field of biological inquiry, the mechanical and kinetic study of proteins at the single molecule level. This review focuses on three examples of such study and includes methods of extracting parameters of interest from the raw data such experiments generate.

Single myosin molecule mechanics

Publisher Summary This chapter focuses on single myosin molecule mechanics. In muscle, myosin forms bipolar thick filaments that, together with a host of structural, regulatory and other protein components, interdigitate with actin filaments to form individual contractile units, of which many operate in series and parallel combination. Unlike kinesin, myosin II behaves nonprocessively, implying that it spends only a small fraction of its ATP turnover time in strongly bound contact with an actin filament. A single myosin molecule cannot support continued movement of an actin filament, since the filament diffuses away while the molecule is detached. The speed at which a myosin ensemble propels the movement of an actin filament is limited by the unitary displacement of a single molecule divided by its “strongly bound state time,” or the mean time it spends in an essentially irreversible, force-generating contact. The conventional swinging crossbridge theory for muscle contraction holds that for each ATP hydrolyzed, myosin binds to actin and undergoes a conformational change or working stroke before detaching.

Single Molecule Myosin Mechanics Measured Using Optical Trapping

The idea of using light to manipulate matter was suggested by Hansch and Schawlow in 1975,1 although the roots likely extend to nineteenth century Maxwell ideas, the understanding that light carries momentum. The optical tweeter, using a single focused laser beam to constrain remotely the position of a dielectric particle in aqueous solution, was demonstrated initially by Ash and colleagues in 1986.2 Light cannot match more direct mechanical probes in crude force, but in this weakness lies its greatest strength: exquisite sensitivity to the tiny forces exerted by single cells and even molecules.