16] Microtubule-mediated golgi capture by semiintact Chinese hamster ovary cells

Abstract

This chapter explains the establishment of a functional assay that detects the interaction of isolated Golgi complexes with the microtubule-based cytoskeleton. Semiintact, but not intact, Chinese hamster ovary (CHO) cells are able to capture Golgi complexes by a process that requires ATP hydrolysis, cytosolic and peripheral membrane proteins, intact microtubules, and cytoplasmic dynein. Semiintact cells provide a native microtubule organizing center (MTOC) and other cytoplasmic components that may be important for physiologically relevant organelle-microtubule interactions. The association of wild-type Golgi complexes with the mutant CHO cells is measured after differential centrifugation: semiintact cells are sedimented through a sucrose cushion, and cell-associated Golgi complexes are detected by assaying the pellet for GlcNAc transferase I enzyme activity. Moreover, Golgi complexes bear peripherally associated proteins on their surfaces that mediate this process. Perhaps it is these proteins that couple dynein to the Golgi complex. The Golgi capture assay provides the first functional assay that will permit the elucidation of the mechanism by which the Golgi complex is able to bind to, and move along, microtubules in a cell cycle-regulated manner.