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 20 nm 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.
|Original language||English (US)|
|Title of host publication||Methods in Enzymology|
|Publisher||Academic Press Inc.|
|Number of pages||23|
|State||Published - 2016|
|Name||Methods in Enzymology|
Bibliographical noteFunding Information:
P.K. is supported by an ERC starting grant (NanoScope). J.A. was supported by a Marie Curie Fellowship (330215). K.J.M. and W.O.H. were supported by the NIH R01 GM076476. L.G.L. and Y.E.G. were supported by NIH Grants P01 GM087253 and R01 GM086352. L.G.L was supported by the NIH training grant T32- GM008275. J.R.S. and Y.T. were supported by funds from the NHLBI Intramural Research Program.
- High speed
- Interferometric scattering microscopy
- Molecular motors
- Single molecule
- Single-particle tracking