Nanometer-resolution microtubule polymerization assays using optical tweezers and microfabricated barriers.
Microtubule (MT) polymerization dynamics, which are crucial to eukaryotic life and are the target of important anticancer agents, result from the addition and loss of 8-nm-long tubulin-dimer subunits. Addition and loss of one or a few subunits cannot be observed at the spatiotemporal resolution of conventional microscopy, and requires development of approaches with higher resolution. Here we describe an assay in which one end of an MT abuts a barrier, and MT length changes are coupled to the movement of an optically trapped bead, the motion of which is tracked with high resolution. We detail assay execution, including preparation of the experimental chamber and orientation of the MT against the barrier. We describe design requirements for the experimental apparatus and barriers, and preparation of materials including stable, biotinylated MT seeds from which growth is initiated and NeutrAvidin-coated beads. Finally, we discuss advantages of moving the optical trap such that it applies a constant force (force clamping), detection limits, the importance of high temporal resolution, data analysis, and potential sources of experimental artifacts.
Charlebois
Blake D
BD
Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan 48109, USA.
Schek
Henry T
HT
Hunt
Alan J
AJ
D000818:Animals; D019411:Clinical Laboratory Techniques; D004867:Equipment Design / Q000295:instrumentation / Q000379:methods; D004868:Equipment Failure; D006801:Humans; D057230:Limit of Detection; D055616:Microtechnology / Q000295:instrumentation* / Q000379:methods*; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism*; D049329:Nanostructures / Q000032:analysis / Q000737:chemistry; D052898:Optical Tweezers / Q000706:statistics & numerical data; D055503:Protein Multimerization