Fluorescence microscopy assays on chemically functionalized surfaces for quantitative imaging of microtubule, motor, and +TIP dynamics.
Microtubule cytoskeleton function depends on the dynamic interplay of microtubules and various microtubule-binding proteins. To gain an understanding of cytoskeleton function at the molecular level, it is important to measure quantitatively how cytoskeletal proteins interact with each other in space and time. Here we describe fluorescence microscopy-based in vitro assays on chemically functionalized glass slides for the study of several aspects of microtubule cytoskeleton dynamics: single motor movements, dynamic microtubule plus-end tracking, antiparallel microtubule sliding by microtubule-crosslinking motors, and microtubule gliding by surface-immobilized motors. The combination of a passivating polyethylene glycol layer on the glass with covalently attached functional groups for selective protein capturing ensures excellent control of the surface properties and good preservation of protein activities in these assays. Common to all assays is that they can be performed in the presence of high concentrations of soluble proteins or even cell extract, which in combination with total internal reflection fluorescence microscopy allows the study of complex protein mixtures that were previously not accessible to quantitative imaging in vitro.
Bieling
Peter
P
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Telley
Ivo A
IA
Hentrich
Christian
C
Piehler
Jacob
J
Surrey
Thomas
T
D000818:Animals; D020099:Coated Materials, Biocompatible / Q000737:chemistry / Q000378:metabolism; D005898:Glass / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000378:metabolism / Q000493:pharmacokinetics*; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D008954:Models, Biological; D020409:Molecular Motor Proteins / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics*; D009068:Movement / Q000502:physiology*; D011092:Polyethylene Glycols / Q000737:chemistry / Q000378:metabolism; D013499:Surface Properties
D020099:Coated Materials, Biocompatible; D008869:Microtubule-Associated Proteins; D020409:Molecular Motor Proteins; D011092:Polyethylene Glycols