Roth Sophie, Laan Liedewij, Dogterom Marileen
FOM Institute AMOLF, Science Park, Amsterdam, The Netherlands.
Fas Center for Systems Biology, Harvard University, Cambridge, Massachusetts, USA.
Methods Enzymol. 2014;540:205-30. doi: 10.1016/B978-0-12-397924-7.00012-1.
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.
胞质动力蛋白是几乎所有真核细胞中一种主要的与微管（MT）相关的马达蛋白。动力蛋白的一个亚群与细胞皮层相关联，这种皮层动力蛋白与微管的相互作用有助于驱动诸如核迁移、有丝分裂纺锤体定向以及伤口愈合过程中的细胞骨架重新定向等过程。在本章中，我们描述了三种类型的实验，其中皮层动力蛋白与微管之间的相互作用在体外以不断增加的复杂程度进行重构。在第一个一维实验中，从附着于表面的中心体上成核的微管，朝着包被有动力蛋白的金屏障生长。在这种实验配置中，可以非常详细地研究微管与附着在屏障上的动力蛋白之间的相互作用。在第二个和第三个实验中，将一个自由移动的动态星体置于二维微加工腔室或三维油包水乳剂液滴中，其边界包被有动力蛋白。这些实验可用于研究皮层动力蛋白如何定位中心体。最后，我们讨论了增加这些重构系统复杂性的未来可能性。
