title: Studying kinesin motors by optical 3D-nanometry in gliding motility assays.
abstract: 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.
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany.
authors.forename: Bert
authors.initials: B
authors.lastname: Nitzsche
authors.forename: Volker
authors.initials: V
authors.lastname: Bormuth
authors.forename: Corina
authors.initials: C
authors.lastname: Bräuer
authors.forename: Jonathon
authors.initials: J
authors.lastname: Howard
authors.forename: Leonid
authors.initials: L
authors.lastname: Ionov
authors.forename: Jacob
authors.initials: J
authors.lastname: Kerssemakers
authors.forename: Till
authors.initials: T
authors.lastname: Korten
authors.forename: Cecile
authors.initials: C
authors.lastname: Leduc
authors.forename: Felix
authors.initials: F
authors.lastname: Ruhnow
authors.forename: Stefan
authors.initials: S
authors.lastname: Diez
mesh_terms: D000818:Animals; D019411:Clinical Laboratory Techniques; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods; D016547:Kinesins / Q000737:chemistry* / Q000378:metabolism*; D008954:Models, Biological; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000378:metabolism; D009068:Movement / Q000502:physiology*; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods; D045663:Quantum Dots; D013499:Surface Properties
chemical_list: D020409:Molecular Motor Proteins; D016547:Kinesins