Studying Kinesin Motors by Optical 3D-Nanometry in Gliding Motility Assays

Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany.
Methods in cell biology (Impact Factor: 1.42). 12/2010; 95(C):247-71. DOI: 10.1016/S0091-679X(10)95014-0
Source: PubMed


Recent developments in optical microscopy and nanometer tracking have facilitated our understanding of microtubules and their associated proteins. Using fluorescence microscopy, dynamic interactions are now routinely observed in vitro on the level of single molecules, mainly using a geometry in which labeled motors move on surface-immobilized microtubules. Yet, we think that the historically older gliding geometry, in which motor proteins bound to a substrate surface drive the motion microtubules, offers some unique advantages. (1) Motility can be precisely followed by coupling multiple fluorophores and/or single bright labels to the surface of microtubules without disturbing the activity of the motor proteins. (2) The number of motor proteins involved in active transport can be determined by several strategies. (3) Multimotor studies can be performed over a wide range of motor densities. These advantages allow for studying cooperativity of processive as well as nonprocessive motors. Moreover, the gliding geometry has proven to be most promising for nanotechnological applications of motor proteins operating in synthetic environments. In this chapter we review recent methods related to gliding motility assays in conjunction with 3D-nanometry. In particular, we aim to provide practical advice on how to set up gliding assays, how to acquire high-precision data from microtubules and attached quantum dots, and how to analyze data by 3D-nanometer tracking.

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Available from: Cécile Leduc, Oct 15, 2014
    • "Microtubule assembly rates were determined by measuring light scattering in a bulk phase turbidity assay (Gaskin et al., 1974). Microtubule properties after co-polymerization were visualized and tested using a microtubule gliding motility assay (Nitzsche et al., 2010). "
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    • "The study of protein dynamics is essential for the understanding the multi-molecular complexes at subcellular levels [27]. For understanding of microtubules and their associated proteins, dynamic interactions are routinely observed in vitro on the level of single molecules, mainly using a geometry in which labeled motors move on surface-immobilized microtubules [28]. Machan and Hof discussed several key questions of lateral mobility investigation in planar lipid membranes, including the influence of membrane and aqueous phase composition, choice of a fluorescent tracer molecule, frictional coupling between the two membrane leaflets and between membrane and solid support [29]. "
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