Reconstituting Functional Microtubule-Barrier Interactions

Autor(en): Taberner, Nuria
Weber, Georges
You, Changjiang 
Dries, Roland
Piehler, Jacob 
Dogterom, Marileen
Herausgeber: Piel, M
Thery, M
Stichwörter: ASSAYS; BINDING; Cell Biology; DYNAMICS; MOTOR; PEPTIDES; PROTEINS; SURFACES; TRACKING
Erscheinungsdatum: 2014
Herausgeber: ELSEVIER ACADEMIC PRESS INC
Journal: MICROPATTERNING IN CELL BIOLOGY, PT B
Methods in Cell Biology
Volumen: 120
Startseite: 69
Seitenende: 90
Zusammenfassung: 
Local interactions between the tips of microtubules and the cell cortex, or other cellular components such as kinetochores, play an important role in essential cellular processes like establishing cell polarity, distribution of organelles, and microtubule aster and chromosome positioning. Here we present two in vitro assays that specifically mimic microtubule-cortex interactions by employing selectively functionalized microfabricated barriers that allow for the immobilization of proteins with a range of affinities We describe the microfabrication process to create gold or glass barriers and the subsequent functionalization of these barriers using self-assembled thiol monolayers or polylysine-poly(ethylene glycol), respectively. Near-permanent attachment of proteins is obtained using biotinylated surfaces combined with streptavidin and biotinylated proteins. Lower affinity interactions, further tunable with the addition of imidazole, are obtained using nickel-nitrilotriacetic acid (Ni(II)-NTA) functionalization combined with his-tagged proteins. Both mono-NTA and tris-NTA compounds are used. We show an assay to reconstitute the ``end-on'' interaction between dynamic microtubule tips and barrier-attached dynein, mimicking the cellular situation at the cortex and at kinetochores. In a second assay, we reconstitute microtubule-based delivery of end-tracking proteins to functionalized barriers, mimicking the transport of cell-end markers to the cell poles in interphase fission yeast cells.
ISBN: 9780124171367
ISSN: 0091679X
DOI: 10.1016/B978-0-12-417136-7.00005-7

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