title: Reconstitution of cortical Dynein function.
abstract: Cytoplasmic dynein is a major microtubule (MT)-associated motor in nearly all eukaryotic cells. A subpopulation of dyneins associates with the cell cortex and the interaction of this cortical dynein with MTs helps to drive processes such as nuclear migration, mitotic spindle orientation, and cytoskeletal reorientation during wound healing. In this chapter, we describe three types of assays in which interactions between cortical dynein and MTs are reconstituted in vitro at increasing levels of complexity. In the first 1D assay, MTs, nucleated from a centrosome attached to a surface, grow against dynein-coated gold barriers. In this assay configuration, the interactions between MTs and dynein attached to a barrier can be studied in great detail. In the second and third assays, a freely moving dynamic aster is placed in either a 2D microfabricated chamber or a 3D water-in-oil emulsion droplet, with dynein-coated boundaries. These assays can be used to study how cortical dynein positions centrosomes. Finally, we discuss future possibilities for increasing the complexity of these reconstituted systems.
authors.affiliation: FOM Institute AMOLF, Science Park, Amsterdam, The Netherlands.
authors.forename: Sophie
authors.initials: S
authors.lastname: Roth
authors.affiliation: Fas Center for Systems Biology, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Liedewij
authors.initials: L
authors.lastname: Laan
authors.affiliation: FOM Institute AMOLF, Science Park, Amsterdam, The Netherlands. Electronic address: dogterom@amolf.nl.
authors.forename: Marileen
authors.initials: M
authors.lastname: Dogterom
mesh_terms: D000818:Animals; D018385:Centrosome / Q000737:chemistry / Q000378:metabolism; D004398:Dyneins / Q000032:analysis / Q000378:metabolism*; D004867:Equipment Design; D006046:Gold / Q000737:chemistry; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D013438:Sulfhydryl Compounds; D006046:Gold; D004398:Dyneins
keywords: Centrosomes; Cortical dynein; Gold-thiol chemistry; Microfabricated chambers; Microfluidics; Microtubules; w/o emulsion droplet