Interferometric Scattering Microscopy for the Study of Molecular Motors

Methods Enzymol. 2016;581:517-539. doi: 10.1016/bs.mie.2016.08.016. Epub 2016 Oct 10.

Abstract

Our understanding of molecular motor function has been greatly improved by the development of imaging modalities, which enable real-time observation of their motion at the single-molecule level. Here, we describe the use of a new method, interferometric scattering microscopy, for the investigation of motor protein dynamics by attaching and tracking the motion of metallic nanoparticle labels as small as 20nm diameter. Using myosin-5, kinesin-1, and dynein as examples, we describe the basic assays, labeling strategies, and principles of data analysis. Our approach is relevant not only for motor protein dynamics but also provides a general tool for single-particle tracking with high spatiotemporal precision, which overcomes the limitations of single-molecule fluorescence methods.

Keywords: Dynein; High speed; Interferometric scattering microscopy; Kinesin; Molecular motors; Myosin; Single molecule; Single-particle tracking.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Dyneins / chemistry
  • Dyneins / isolation & purification*
  • Humans
  • Kinesin / chemistry
  • Kinesin / isolation & purification*
  • Microscopy, Fluorescence / methods*
  • Microscopy, Interference / methods
  • Molecular Motor Proteins / chemistry
  • Molecular Motor Proteins / metabolism
  • Myosins / chemistry
  • Myosins / isolation & purification*

Substances

  • KIF5B protein, human
  • Molecular Motor Proteins
  • Myosins
  • Dyneins
  • Kinesin