OVEREXPRESSION OF THE DYNAMITIN (P50) SUBUNIT OF THE DYNACTIN COMPLEXDISRUPTS DYNEIN-DEPENDENT MAINTENANCE OF MEMBRANE ORGANELLE DISTRIBUTION

Dynactin is a multisubunit complex that plays an accessory role in cyt oplasmic dynein function. Overexpression in mammalian cells of one dyn actin subunit, dynamitin, disrupts the complex, resulting in dissociat ion of cytoplasmic dynein from prometaphase kinetochores, with consequ ent perturbation of mitosis (Echeverri, C.J., B.M. Paschal, K.T. Vaugh an, and R.B. Vallee. 1996. J. Cell Biol. 132:617-634). Based on these results, dynactin was proposed to play a role in linking cytoplasmic d ynein to kinetochores and, potentially, to membrane organelles. The cu rrent study reports on the dynamitin interphase phenotype. In dynamiti n-overexpressing cells, early endosomes (labeled with antitransferrin receptor), as well as late endosomes and lysosomes (labeled with anti- lysosome-associated membrane protein-1 [LAMP-1]), were redistributed t o the cell periphery. This redistribution was disrupted by nocodazole, implicating an underlying plus end-directed microtubule motor activit y. The Golgi stack, monitored using sialyltransferase, galactosyltrans ferase, and N-acetylglucosaminyltransferase I, was dramatically disrup ted into scattered structures that colocalized with components of the intermediate compartment (ERGIC-53 and ERD-2). The disrupted Golgi ele ments were revealed by EM to represent short stacks similar to those f ormed by microtubule-depolymerizing agents. Golgi-to-ER traffic of sla ck markers induced by brefeldin A was not inhibited by dynamitin overe xpression. Time-lapse observations of dynamitin-overexpressing cells r ecovering from brefeldin A treatment revealed that the scattered Golgi elements do not undergo microtubule-based transport as seen in contro l cells, but rather, remain stationary at or near their ER exit sites. These results indicate that dynactin is specifically required for ong oing centripetal movement of endocytic organelles and components of th e intermediate compartment. Results similar to those of dynamitin over expression were obtained by microinjection with antidynein intermediat e chain antibody, consistent with a role for dynactin in mediating int eractions of cytoplasmic dynein with specific membrane organelles. The se results suggest that dynamitin plays a pivotal role in regulating o rganelle movement at the level of motor-cargo binding.