Fluorescence Microscopy Assays on Chemically Functionalized Surfaces for Quantitative Imaging of Microtubule, Motor, and plus TIP Dynamics

Autor(en): Bieling, Peter
Telley, Ivo A.
Hentrich, Christian
Piehler, Jacob 
Surrey, Thomas
Herausgeber: Wilson, L
Correia, JJ
Stichwörter: Cell Biology; CYCLE; DYNEIN; EGG EXTRACT SPINDLES; END TRACKING; HISTIDINE-TAGGED PROTEINS; IN-VITRO; MOTILITY; MOVEMENT; PHOSPHORYLATION; SINGLE KINESIN MOLECULES
Erscheinungsdatum: 2010
Herausgeber: ELSEVIER ACADEMIC PRESS INC
Journal: MICROTUBULES, IN VITRO: MICROTUBULES, IN VITRO
Methods in Cell Biology
Volumen: 95
Startseite: 555
Seitenende: 580
Zusammenfassung: 
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.
ISBN: 9780123748157
ISSN: 0091679X
DOI: 10.1016/S0091-679X(10)95028-0

Show full item record

Page view(s)

4
Last Week
0
Last month
0
checked on May 27, 2024

Google ScholarTM

Check

Altmetric