Centrosome assembly is important for mitotic spindle formation and if defective may contribute to genomic instability in cancer. Here we show that in somatic cells centrosome assembly of two proteins involved in microtubule nucleation, pericentrin and ␥ tubulin, is inhibited in the absence of microtubules. A more potent inhibitory effect on centrosome assembly of these proteins is observed after specific disruption of the microtubule motor cytoplasmic dynein by microinjection of dynein antibodies or by overexpression of the dynamitin subunit of the dynein binding complex dynactin. Consistent with these observations is the ability of pericentrin to cosediment with taxol-stabilized microtubules in a dynein-and dynactin-dependent manner. Centrosomes in cells with reduced levels of pericentrin and ␥ tubulin have a diminished capacity to nucleate microtubules. In living cells expressing a green fluorescent protein-pericentrin fusion protein, green fluorescent protein particles containing endogenous pericentrin and ␥ tubulin move along microtubules at speeds of dynein and dock at centrosomes. In Xenopus extracts where ␥ tubulin assembly onto centrioles can occur without microtubules, we find that assembly is enhanced in the presence of microtubules and inhibited by dynein antibodies. From these studies we conclude that pericentrin and ␥ tubulin are novel dynein cargoes that can be transported to centrosomes on microtubules and whose assembly contributes to microtubule nucleation. INTRODUCTIONCentrosomes and other microtubule-organizing centers represent a structurally diverse class of organelles that share the common ability to nucleate and organize microtubules and play an important role in many fundamental cellular processes (for review, see Kellogg et al., 1994;Zimmerman et al., 1999). In interphase cells, centrosome-anchored microtubules serve as tracks for molecular motor-based transport and positioning of vesicles and organelles (see Karki and Holzbaur, 1999). Centrosomes also serve to anchor important regulatory molecules such as protein kinase A, which has been shown to regulate spindle function (Schmidt et al., 1999;Takahashi et al., 1999;Witczak et al., 1999;Diviani et al., 2000; D. Diviani, J. Langeberg, A. Purohit, A. Young, S. Doxsey, and J. Scott, unpublished results). Moreover, an increasing number of molecules that regulate cellular processes such as cell cycle progression and centrosome duplication are localized to centrosomes (see Doxsey, 1998;Zimmerman et al., 1999). In mitotic cells, centrosomes play an important role in the assembly and function of mitotic spindles and thus in the fidelity of chromosome segregation (Merdes and Cleveland, 1997;Waters and Salmon, 1997; see Compton, 1998;Hyman and Karsenti, 1998). In tumor cells, centrosome structure, number, and function are altered, suggesting that centrosome defects may contribute to tumorigenesis as first hypothesized by Boveri (1914) (also see Wilson, 1925;Chial and Winey, 1999;Pihan and Doxsey, 1999;Salisbury et al., 1999).Centrosomes in most...

Pericentrin is a conserved protein of the centrosome involved in microtubule organization. To better understand pericentrin function, we overexpressed the protein in somatic cells and assayed for changes in the composition and function of mitotic spindles and spindle poles. Spindles in pericentrin-overexpressing cells were disorganized and mispositioned, and chromosomes were misaligned and missegregated during cell division, giving rise to aneuploid cells. We unexpectedly found that levels of the molecular motor cytoplasmic dynein were dramatically reduced at spindle poles. Cytoplasmic dynein was diminished at kinetochores also, and the dynein-mediated organization of the Golgi complex was disrupted. Dynein coimmunoprecipitated with overexpressed pericentrin, suggesting that the motor was sequestered in the cytoplasm and was prevented from associating with its cellular targets. Immunoprecipitation of endogenous pericentrin also pulled down cytoplasmic dynein in untransfected cells. To define the basis for this interaction, pericentrin was coexpressed with cytoplasmic dynein heavy (DHCs), intermediate (DICs), and light intermediate (LICs) chains, and the dynamitin and p150Glued subunits of dynactin. Only the LICs coimmunoprecipitated with pericentrin. These results provide the first physiological role for LIC, and they suggest that a pericentrin–dynein interaction in vivo contributes to the assembly, organization, and function of centrosomes and mitotic spindles.

The light intermediate chains (LICs) of cytoplasmic dynein consist of multiple isoforms, which undergo post-translational modification to produce a large number of species separable by two-dimensional electrophoresis and which we have proposed to represent at least two gene products. Recently, we demonstrated the first known function for the LICs: binding to the centrosomal protein, pericentrin, which represents a novel, non-dynactin-based cargo-binding mechanism. Here we report the cloning of rat LIC1, which is approximately 75% homologous to rat LIC2 and also contains a P-loop consensus sequence. We compared LIC1 and LIC2 for the ability to interact with pericentrin, and found that only LIC1 will bind. A functional P-loop sequence is not required for this interaction. We have mapped the interaction to the central region of both LIC1 and pericentrin. Using recombinant LICs, we found that they form homooligomers, but not heterooligomers, and exhibit mutually exclusive binding to the heavy chain. Additionally, overexpressed pericentrin is seen to interact with endogenous LIC1 exclusively. Together these results demonstrate the existence of two subclasses of cytoplasmic dynein: LIC1-containing dynein, and LIC2-containing dynein, only the former of which is involved in pericentrin association with dynein.Cytoplasmic dynein is a large, multi-subunit complex (1), which functions as a molecular motor that moves cellular components toward the minus ends of microtubules and determines the distribution of many vesicular organelles (2). Cytoplasmic dynein has also been found to be involved in many aspects of mitosis, where it is found at the kinetochore, spindle poles, and cell cortex (3-7).The cytoplasmic dynein complex is composed of four subunit classes: the heavy (HCs), 1 intermediate (ICs), light intermediate (LICs), and light chains (LCs). The dynein heavy chains are large (532 kDa) polypeptides that contain four ATPase domains and are responsible for microtubule binding and catalytic activity (2). The intermediate chains are a diverse set of subunits derived by alternative splicing from two different genes (8). The ICs have been found to be responsible for the interaction of the dynein complex with a second complex called dynactin, which is required for dynein-based motility, by directly binding to the p150Glued dynactin subunit (8, 9). Dynactin is thought to be involved in linking dynein to various organelles in the cell; thus the intermediate chains have been proposed to have an important function in dynein targeting (7). The LCs are a diverse family of low and very low molecular weight subunits (10 -12); a role in subcellular targeting has been proposed (13).The HCs, ICs, and LCs all have homologous counterparts in flagellar and ciliary forms of dynein. The LICs, however, are unique to cytoplasmic dynein. They contain a P-loop consensus sequence of unknown function (14, 15). Two-dimensional electrophoresis of both rat and chicken LICs reveals numerous LIC species, at least some of which result from phosphorylati...

Tpr is a 270-kD coiled-coil protein localized to intranuclear filaments of the nuclear pore complex (NPC). The mechanism by which Tpr contributes to the structure and function of the nuclear pore is currently unknown. To gain insight into Tpr function, we expressed the full-length protein and several subdomains in mammalian cell lines and examined their effects on nuclear pore function. Through this analysis, we identified an NH2-terminal domain that was sufficient for association with the nucleoplasmic aspect of the NPC. In addition, we unexpectedly found that the acidic COOH terminus was efficiently transported into the nuclear interior, an event that was apparently mediated by a putative nuclear localization sequence. Ectopic expression of the full-length Tpr caused a dramatic accumulation of poly(A)+ RNA within the nucleus. Similar results were observed with domains that localized to the NPC and the nuclear interior. In contrast, expression of these proteins did not appear to affect nuclear import. These data are consistent with a model in which Tpr is tethered to intranuclear filaments of the NPC by its coiled coil domain leaving the acidic COOH terminus free to interact with soluble transport factors and mediate export of macromolecules from the nucleus.