Role of Dynactin in Endocytic Traffic: Effects of Dynamitin Overexpression and Colocalization with CLIP-170

The flow of material from peripheral, early endosomes to late endosomes requires microtubules and is thought to be facilitated by the minus end-directed motor cytoplasmic dynein and its activator dynactin. The microtubule-binding protein CLIP-170 may also play a role by providing an early link to endosomes. Here, we show that perturbation of dynactin function in vivo affects endosome dynamics and trafficking. Endosome movement, which is normally bidirectional, is completely inhibited. Receptor-mediated uptake and recycling occur normally, but cells are less susceptible to infection by enveloped viruses that require delivery to late endosomes, and they show reduced accumulation of lysosomally targeted probes. Dynactin colocalizes at microtubule plus ends with CLIP-170 in a way that depends on CLIP-170’s putative cargo-binding domain. Overexpression studies using p150(Glued), the microtubule-binding subunit of dynactin, and mutant and wild-type forms of CLIP-170 indicate that CLIP-170 recruits dynactin to microtubule ends. These data suggest a new model for the formation of motile complexes of endosomes and microtubules early in the endocytic pathway

Opposing motor activities are required for the organization of the mammalian mitotic spindle pole

Abstract. We use both in vitro and in vivo approaches to examine the roles of Eg5 (kinesin-related protein), cytoplasmic dynein, and dynactin in the organization of the microtubules and the localization of NuMA (Nuclear protein that associates with the Mitotic A__pparatus) at the polar ends of the mammalian mitotic spindle. Perturbation of the function of Eg5 through either immunodepletion from a cell free system for assembly of mitotic asters or antibody microinjection into cultured cells leads to organized astral microtubule arrays with expanded polar regions in which the minus ends of the microtubules emanate from a ring-like structure that contains NuMA. Conversely, perturbation of the function of cytoplasmic dynein or dynactin through either specific immunodepletion from the cell free system or expression of a dominant negative subunit of dynactin in cultured cells results in the complete lack of organization of microtubules and the failure to efficiently concentrate the NuMA protein despite its association with the microtubules. Simultaneous immunodepletion of these proteins from the cell free system for mitotic aster assembly indicates that the plus end--directed activity of Eg5 antagonizes the minus end--directed activity of cytoplasmic dynein and a minus end--directed activity associated with NuMA during the organization of the microtubules into a morphologic pole. Taken together, these results demonstrate that the unique organization of the minus ends of microtubules and the localization of NuMA at the polar ends of the mammalian mitotic spindle can be accomplished in a centrosome-independent manner by the opposing activities of plus end- and minus end-directed motors. T HE mitotic spindle is a complex microtubule-based structure that is responsible for chromosome segregatio