The multiprotein complex, dynactin, is an integral part of the cytoplasmic dynein motor and is required for dynein-based motility in vitro and in vivo. In living cells, perturbation of the dynein–dynactin interaction profoundly blocks mitotic spindle assembly, and inhibition or depletion of dynein or dynactin from meiotic or mitotic cell extracts prevents microtubules from focusing into spindles. In interphase cells, perturbation of the dynein–dynactin complex is correlated with an inhibition of ER-to-Golgi movement and reorganization of the Golgi apparatus and the endosome–lysosome system, but the effects on microtubule organization have not previously been defined. To explore this question, we overexpressed a variety of dynactin subunits in cultured fibroblasts. Subunits implicated in dynein binding have effects on both microtubule organization and centrosome integrity. Microtubules are reorganized into unfocused arrays. The pericentriolar components, γ tubulin and dynactin, are lost from centrosomes, but pericentrin localization persists. Microtubule nucleation from centrosomes proceeds relatively normally, but microtubules become disorganized soon thereafter. Overexpression of some, but not all, dynactin subunits also affects endomembrane localization. These data indicate that dynein and dynactin play important roles in microtubule organization at centrosomes in fibroblastic cells and provide new insights into dynactin–cargo interactions.

Most tumor cells are characterized by increased genomic instability and chromosome segregational defects, often associated with hyperamplification of the centrosome and the formation of multipolar spindles. However, extra centrosomes do not always lead to multipolarity. Here, we describe a process of centrosomal clustering that prevented the formation of multipolar spindles in noncancer cells. Noncancer cells needed to overcome this clustering mechanism to allow multipolar spindles to form at a high frequency. The microtubule motor cytoplasmic dynein was a critical part of this coalescing machinery, and in some tumor cells overexpression of the spindle protein NuMA interfered with dynein localization, promoting multipolarity.

Microtubule-associated proteins (MAPs) use particular microtubule-binding domains that allow them to interact with microtubules in a manner specific to their individual cellular functions. Here, we have identified a highly basic microtubule-binding domain in the p150 subunit of dynactin that is only present in the dynactin members of the CAP-Gly family of proteins. Using single-particle microtubule-binding assays, we found that the basic domain of dynactin moves progressively along microtubules in the absence of molecular motors - a process we term 'skating'. In contrast, the previously described CAP-Gly domain of dynactin remains firmly attached to a single point on microtubules. Further analyses showed that microtubule skating is a form of one-dimensional diffusion along the microtubule. To determine the cellular function of the skating phenomenon, dynein and the dynactin microtubule-binding domains were examined in single-molecule motility assays. We found that the basic domain increased dynein processivity fourfold whereas the CAP-Gly domain inhibited dynein motility. Our data show that the ability of the basic domain of dynactin to skate along microtubules is used by dynein to maintain longer interactions for each encounter with microtubules.

Cytoplasmic dynein and dynactin are megadalton-sized multisubunit molecules that function together as a cytoskeletal motor. In the present study, we explore the mechanism of dynein-dynactin binding in vitro and then extend our findings to an in vivo context. Solution binding assays were used to define binding domains in the dynein intermediate chain (IC) and dynactin p150Glued subunit. Transient overexpression of a series of fragments of the dynein IC was used to determine the importance of this subunit for dynein function in mammalian tissue culture cells. Our results suggest that a functional dynein-dynactin interaction is required for proper microtubule organization and for the transport and localization of centrosomal components and endomembrane compartments. The dynein IC fragments have different effects on endomembrane localization, suggesting that different endomembranes may bind dynein via distinct mechanisms. INTRODUCTIONCytoplasmic dynein is a minus end-directed, microtubulebased motor that provides the force for translocation, tension, and organization of cellular components. Each cytoplasmic dynein molecule contains two enzymatically active heads that convert the energy of ATP hydrolysis into mechanical work. These are connected to a basal cargo binding unit (for reviews of dynein structure, see King, 2000a,b). Cytoplasmic dynein requires another multisubunit protein complex, dynactin, for full activity (Gill et al., 1991;Schroer and Sheetz, 1991;Boylan et al., 2000). Dynactin contributes to cytoplasmic dynein function by acting as an "adapter" that expands the range of cargoes dynein can move (for review, see Karki and Holzbaur, 1999) and by increasing motor processivity (King and Schroer, 2000). Dynactin can also act independently of cytoplasmic dynein to anchor microtubules at the centrosome (Quintyne and Schroer, 2002). Dynactin is thought to accomplish these diverse tasks by using its distinct cargo-, cytoplasmic dynein-and microtubulebinding domains.The interaction between dynein and dynactin seems to be tightly regulated, because dynactin and dynein are not always colocalized in cells (for reviews, see Holleran et al., 1998;Karki and Holzbaur, 1999). For example, dynactin is concentrated at centrosomes throughout the cell cycle, whereas dynein accumulates between the time of centriole duplication (mid-S phase) and the onset of mitosis (Quintyne and Schroer, 2002). Both proteins are found at kinetochores, spindle poles, and along spindle microtubules during mitosis, but after mitosis dynein is lost from the centrosome, whereas dynactin remains. Similarly, dynactin, but not dynein, associates with microtubule plus ends (Vaughan et al., 1999;Habermann et al., 2001). Finally, dynein and dynactin colocalize on numerous endomembranes destined to move along microtubules (Habermann et al., 2001) and decrease in concert at the onset of mitosis (Niclas et al., 1996). Apparently, dynein and dynactin can be either associated or separate in cells, which highlights the importance of understanding how dynein...