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© The Rockefeller University Press, 0021-9525/1997//469 $5.00
The Journal of Cell Biology, Volume 139, Number 2, , 1997 469-484

Article

Overexpression of the Dynamitin (p50) Subunit of the Dynactin Complex Disrupts Dynein-dependent Maintenance of Membrane Organelle Distribution



Janis K. Burkhardt*,{ddagger}, Christophe J. Echeverri§,||, Tommy Nilsson{ddagger}, and Richard B. Vallee§,||

* The University of Chicago, Department of Pathology, Chicago, Illinois 60637; {ddagger} European Molecular Biology Laboratory, 69012 Heidelberg, Germany; § Cell Biology Group, Worcester Foundation for Biomedical Research, Shrewsbury, Massachusetts 01545; and || University of Massachusetts Graduate School of Biomedical Sciences, Department of Cell Biology, Worcester, Massachusetts 01655

Dynactin is a multisubunit complex that plays an accessory role in cytoplasmic dynein function. Overexpression in mammalian cells of one dynactin subunit, dynamitin, disrupts the complex, resulting in dissociation of cytoplasmic dynein from prometaphase kinetochores, with consequent perturbation of mitosis (Echeverri, C.J., B.M. Paschal, K.T. Vaughan, 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 dynein to kinetochores and, potentially, to membrane organelles. The current study reports on the dynamitin interphase phenotype. In dynamitin-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 to the cell periphery. This redistribution was disrupted by nocodazole, implicating an underlying plus end–directed microtubule motor activity. The Golgi stack, monitored using sialyltransferase, galactosyltransferase, and N-acetylglucosaminyltransferase I, was dramatically disrupted into scattered structures that colocalized with components of the intermediate compartment (ERGIC-53 and ERD-2). The disrupted Golgi elements were revealed by EM to represent short stacks similar to those formed by microtubule-depolymerizing agents. Golgi-to-ER traffic of stack markers induced by brefeldin A was not inhibited by dynamitin overexpression. Time-lapse observations of dynamitin-overexpressing cells recovering from brefeldin A treatment revealed that the scattered Golgi elements do not undergo microtubule-based transport as seen in control cells, but rather, remain stationary at or near their ER exit sites. These results indicate that dynactin is specifically required for ongoing centripetal movement of endocytic organelles and components of the intermediate compartment. Results similar to those of dynamitin overexpression were obtained by microinjection with antidynein intermediate chain antibody, consistent with a role for dynactin in mediating interactions of cytoplasmic dynein with specific membrane organelles. These results suggest that dynamitin plays a pivotal role in regulating organelle movement at the level of motor–cargo binding.

Abbreviations used in this paper: BFA, brefeldin A; DIC, dynein intermediate chain; GFP, green fluorescent protein; LAMP, lysosome-associated membrane protein; MTOC, microtubule-organizing center; NAGT-I, β1,2 N-acetylglucosaminyltransferase I; ST, {alpha}2,6 sialyltransferase; TfR; transferrin receptor; VSV-G, vesicular stomatitis virus G protein.

Address all correspondence to Janis K. Burkhardt, Department of Pathology, The University of Chicago, 5841 S. Maryland Ave., MC1089, Chicago, IL 60637. Tel.: (773) 834-0869. Fax: (773) 702-3701. e-mail: jburkhar{at}flowcity.bsd.uchicago.edu


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