Chapter 7 Recombinant p50/Dynamitin as a Tool to Examine the Role of Dynactin in Intracellular Processes

Abstract

Publisher Summary Dynein is a minus-end-directed motor responsible for many intracellular functions. Traditionally, it is difficult to interfere with dynein function and thus test its role in intracellular organization. Recently, a tool has been described, which involves the overexpression of one of the components of dynactin, p50/dynamitin. When overexpressed in cells by transfection, p50/dynamitin disrupts the dynactin complex and therefore the function of dynein in spindle assembly and Golgi organization. Although, it is a valuable tool, in many systems it is not possible to transfect cells. This chapter describes a simple two-step method for the production of large amounts of active p50/dynamitin in bacteria using ammonium sulfate precipitation and subsequent anion-exchange chromatography. The chapter demonstrates that the recombinant protein disrupts the dynactin complex in Xenopus egg extracts. Expressed p50/dynamitin is used to block spindle pole assembly in Xenopus egg extracts and rearrangement of microtubules during neuronal differentiation, and microinjection of p50/dynamitin leads to the dispersion of the Golgi apparatus in mammalian as well as Xenopus tissue culture cells.