ABSTRACT
Cytoplasmic dynein-1 is a large protein complex composed of multiple subunits and has a two-headed structure. This chapter investigates the organization of the tail domain by single particle analysis and gold nanoparticle labelling, and explores its subdomain structure and location of subunits. Cytoplasmic dynein-1 was originally isolated from mammalian brain extract as a microtubule motor protein. Since then, cytoplasmic dynein was found in almost all eukaryotes from fungi to mammals. The chapter aims to explore the organization of the subunits in the dynein tail by determining the positions of the N- and C-termini of the subunits. It shows that streptavidin-binding peptide-tagged recombinant dynein subunits can be purified as the dynein complex associated with other endogenous subunits from HEK293 cells, and that recombinant dynein complexes preserve motor activities. The chapter argues that the His-tagged subunit in the dynactin complex can be specifically labeled by nickel nitrilotriacetic acid nanogold.
