Reconstituting functional microtubule-barrier interactions.
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.
FOM Institute AMOLF, Amsterdam, The Netherlands.
Núria
N
Taberner
FOM Institute AMOLF, Amsterdam, The Netherlands.
Georges
G
Weber
Universität Osnabrück, Osnabrück, Germany.
Changjiang
C
You
FOM Institute AMOLF, Amsterdam, The Netherlands.
Roland
R
Dries
Universität Osnabrück, Osnabrück, Germany.
Jacob
J
Piehler
FOM Institute AMOLF, Amsterdam, The Netherlands.
Marileen
M
Dogterom
D000818:Animals; D019921:Biotinylation / Q000187:drug effects; D002417:Cattle; D004398:Dyneins / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D005898:Glass; D006046:Gold / Q000378:metabolism; D055767:Immobilized Proteins / Q000378:metabolism; D055616:Microtechnology / Q000379:methods*; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D009942:Organometallic Compounds / Q000494:pharmacology; D058105:Polymerization / Q000187:drug effects; D012710:Serum Albumin, Bovine / Q000378:metabolism; D034421:Sus scrofa
D055767:Immobilized Proteins; D009942:Organometallic Compounds; D012710:Serum Albumin, Bovine; C088321:nickel nitrilotriacetic acid; D006046:Gold; D004398:Dyneins; D009571:Nitrilotriacetic Acid
Dynein; Functionalized barriers; Gold chemistry; In vitro reconstitution; Microfabrication; Microtubule end-tracking proteins; Microtubules; Ni(II)-NTA functionalization