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  • Review Article
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Moving into the cell: single-molecule studies of molecular motors in complex environments

Nature Reviews Molecular Cell Biology volume 12, pages 163–176 (2011)Cite this article

Subjects

Key Points

  • Single-molecule approaches for studying the dynamic properties of motor proteins have come of age. Recent technical developments allow us to see more details of molecular motions and of the forces that molecules generate.

  • Atomic force microscopy provides the highest available resolution, of about one nanometre, for imaging soft and dynamic motor proteins in action. Recently, significant progress has been made in imaging fragile samples and in high-speed imaging, with rates of up to 25 frames per second being achieved; for example, it is possible to image kinesin motors on top of microtubules with low forces while still being able to resolve single domains of the motor proteins.

  • Fluorescence microscopy is unbeaten in achieving molecular specificity of imaging. Progress has been rapid, especially in single-molecule fluorescence methods. Detectors, which are mostly charged coupled device cameras, are constantly getting more efficient and less noisy. Methods for restricting the sample volume to suppress background noise are becoming increasingly sophisticated. Last, but not least, chemical fluorophores, genetically encoded fluorescent proteins and fluorescent nanoparticles are becoming more versatile, bright and stable.

  • Single-molecule fluorescence experiments in cells remain challenging. Crowding and background fluorescence are difficult to avoid. High hopes are resting on newly developed bright and stable dyes, as well as on fluorescent nanoparticles, especially in the near-infrared spectral range, where cellular background fluorescence is minimal.

  • Optical trapping has been firmly established as a tool of choice when measuring steps or power strokes of motor proteins as well as forces generated by single motors. Optical trapping is being implemented in increasingly sophisticated and powerful ways. The resolution of sub-nanometre steps is possible, time resolution can be as good as microseconds, and controlled forces of piconewtons can be exerted on single molecules in well-controlled geometries.

  • It remains a challenge to apply optical tweezers in cells. Specificity of trapping, as opposed to indiscriminate trapping of various intracellular objects, is hard to achieve, and it is difficult to calibrate force and displacement measurements in cells. Promising developments include the trapping of distinct high-index cellular components, such as lipid droplets, and the use of externally introduced high-index particles, such as gold nanobeads, as well as the exploration of resonantly enhanced trapping.

Abstract

Much has been learned in the past decades about molecular force generation. Single-molecule techniques, such as atomic force microscopy, single-molecule fluorescence microscopy and optical tweezers, have been key in resolving the mechanisms behind the power strokes, 'processive' steps and forces of cytoskeletal motors. However, it remains unclear how single force generators are integrated into composite mechanical machines in cells to generate complex functions such as mitosis, locomotion, intracellular transport or mechanical sensory transduction. Using dynamic single-molecule techniques to track, manipulate and probe cytoskeletal motor proteins will be crucial in providing new insights.

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Figure 1: Cytoskeletal motor structures and functions.
Figure 2: Using AFM to study single motor proteins.
Figure 3: Single-molecule fluorescence microscopy to study motor proteins.
Figure 4: Optical tweezers to study single motor proteins.
Figure 5: Optical tweezers to study movement of dimeric motors and power strokes of single motor heads.
Figure 6: Optical tweezers used to measure force fluctuations of cells.

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Acknowledgements

We thank the Medical Research Council, UK (C.V.) and the Deutsche Forschungsgemeinschaft (Center for Molecular Physiology of the Brain, (CMPB)) (C.F.S.) for financial support.

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  1. Department of Cellular Physiology, Institute of Physiology, Ludwig-Maximilians-Universität München, Schillerstrasse 44, München, 80336, Germany

    Claudia Veigel

  2. Centre for NanoScience, Universität München, Schillerstrasse 44, München, 80336, Germany

    Claudia Veigel

  3. Drittes Physikalisches Institut, Georg-August-Universität Göttingen, Friedrich-Hund-Platz 1, Göttingen, 37077, Germany

    Christoph F. Schmidt

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Glossary

Förster resonance energy transfer

Non-radiative energy transfer from an excited donor fluorophore to an acceptor fluorophore in the immediate vicinity can be used to measure nanometre distances owing to the strong distance-dependence of the effect.

Piezo actuator

A piezoelectric crystal driven by a DC voltage to perform length changes in the Ångström to micrometre range.

Quantum dot

A nanometre-sized fluorescent semiconductor particle, the fluorescent properties of which depend on its shape and size.

Total internal reflection fluorescence

Excitation of fluorescence by the evanescent light field of a laser beam that is totally internally reflected at an interface between a medium of higher and one of lower index of refraction.

Point spread function

The point spread function characterizes the diffractive properties of an imaging system and is equal to the image of a point source of light through this imaging system.

Epi-illumination

Microscope illumination through the objective, commonly used for fluorescence excitation.

Back-focal-plane interferometry

Laser interferometric detection of the displacement of optically trapped particles from the focus; uses imaging of the back focal plane of the microscope condenser, typically onto a quadrant photodiode.

Blinking

The switching of single fluorophores between bright and dark states, which hinders their use in time-resolved experiments.

Polarization interference contrast

A technique that is similar to differential interference contrast and that allows the sensitive detection of slight optical phase changes, induced by local heating owing to an absorbing nanoparticle, by interference with a slightly offset reference beam.

Kymograph

A graphical representation of movement along one axis over time, assembled by laterally adjoining linear slices out of consecutive video frames.

Gaussian bell curve

The most common probability density function of a random variable, defined by

where μ is the mean and σ2 is the variance of the distribution.

Isometric force

The force that is generated by a muscle contracting without changing length.

Trap force

The force that is imposed by optical tweezers on a trapped particle.

Viscous drag

The frictional force on objects moving in a viscous fluid. The viscous drag force is proportional to the velocity of the moving object. For small spherical objects moving slowly through a viscous fluid (that is, at a low Reynolds number) the viscous drag coefficient is β = 6πηr, where r is the Stokes radius of the particle and η the viscosity of the fluid.

Boltzmann constant

This constant relates the average thermal energy of a microscopic particle to temperature.

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Veigel, C., Schmidt, C. Moving into the cell: single-molecule studies of molecular motors in complex environments. Nat Rev Mol Cell Biol 12, 163–176 (2011). https://doi.org/10.1038/nrm3062

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