Recent evidence suggests that the force for poleward movement of chromosomes during mitosis is generated at or close to the kinetochores. Chromosome movement depends on motion relative to microtubules, but the identities of the motors remain uncertain. One candidate for a mitotic motor is dynein, a large multimeric enzyme which can move along microtubules toward their slow growing end. Dyneins were originally found in axonemes of cilia and flagella where they power microtubule sliding. Recently, cytoplasmic dyneins have also been found, and specific antibodies have been raised against them. The cellular localization of dynein has previously been studied with several antibodies raised against flagellar dynein, but the relevance of these data to the distribution of cytoplasmic dynein is not known. Antibodies raised against cytoplasmic dyneins have shown localization of dynein antigens to the mitotic spindles in Caenorhabditis elegans embryos (Lye et al., personal communication) and punctate cytoplasmic structures in Dictyostelium amoebae. Using antibodies that recognize subunits of cytoplasmic dyneins, we show here that during mitosis, cytoplasmic dynein antigens concentrate near the kinetochores, centrosomes and spindle fibres of HeLa and PtK1 cells, whereas at interphase they are distributed throughout the cytoplasm. This is consistent with the hypothesis that cytoplasmic dynein is a mitotic motor.

Abstract. We describe two dynein heavy chain (DHC)-like polypeptides (DHCs 2 and 3) that are distinct from the heavy chain of conventional cytoplasmic dynein (DHC1) but are expressed in a variety of mammalian cells that lack axonemes. DHC2 is a distant member of the "cytoplasmic" branch of the dynein phylogenetic tree, while DHC3 shares more sequence similarity with dynein-like polypeptides that have been thought to be axonemal. Each cytoplasmic dynein is associated with distinct cellular organelles. DHC2 is localized predomi-

To understand the molecular basis of microtubule-associated motility during mitosis, the mechanochemical factors that generate the relevant motile force must be identified. Myosin, the ATPase that interacts with actin to produce the force for muscle contraction and other forms of cell motility, is believed to be involved in cytokinesis but not in mitosis. Dynein, the mechanochemical enzyme that drives microtubule sliding in eukaryotic cilia and flagella, has been identified in the cytoplasm of sea urchin eggs, but the evidence that it is involved in cytoplasmic microtubule-based motility (rather than serving as a precursor for embryonic cilia) is equivocal. Microtubule-associated ATPases have been prepared from other tissues, but their role in cytoplasmic motility is also unknown. Recent work on axoplasmic transport, however, has led to the identification of a novel mechanochemical protein called kinesin, which is thought to generate the force for moving vesicles along axonal microtubules. These results suggest that kinesin may also be a mechanochemical factor for non-axoplasmic forms of microtubule-based motility, such as mitosis. We describe here the identification and isolation of a kinesin-like protein from the cytoplasm of sea urchin eggs. We present evidence that this protein is localized in the mitotic spindle, and propose that it may be a mechanochemical factor for some form of motility associated with the mitotic spindle.

The diversity of dynein's functions in mammalian cells is a manifestation of both the existence of multiple dynein heavy chain isoforms and an extensive set of associated protein subunits. In this study, we have identified and characterized a novel subunit of the mammalian cytoplasmic dynein 2 complex. The sequence similarity between this 33-kDa subunit and the light intermediate chains (LICs) of cytoplasmic dynein 1 suggests that this protein is a dynein 2 LIC (D2LIC). D2LIC contains a P-loop motif near its NH 2 terminus, and it shares a short region of similarity to the yeast GTPases Spg1p and Tem1p. The D2LIC subunit interacts specifically with DHC2 (or cDhc1b) in both reciprocal immunoprecipitations and sedimentation assays. The expression of D2LIC also mirrors that of DHC2 in a variety of tissues. D2LIC colocalizes with DHC2 at the Golgi apparatus throughout the cell cycle. On brefeldin A-induced Golgi fragmentation, a fraction of D2LIC redistributes to the cytoplasm, leaving behind a subset of D2LIC that is localized around the centrosome. Our results suggest that D2LIC is a bona fide subunit of cytoplasmic dynein 2 that may play a role in maintaining Golgi organization by binding cytoplasmic dynein 2 to its Golgi-associated cargo. INTRODUCTIONDyneins are large, multisubunit motor proteins that are involved in a wide range of cellular processes. There are two classes of dyneins: axonemal and cytoplasmic. Axonemal dyneins drive and coordinate motility in cilia and flagella (reviewed in Gibbons, 1995;Porter, 1996), whereas cytoplasmic dyneins contribute to a variety of processes, including vesicle transport, formation and localization of the Golgi complex, mitotic spindle assembly and positioning, nuclear migration, and chromosome movements (reviewed in Holzbaur and Vallee, 1994;Hirokawa et al., 1998).The cytoplasmic dynein family can be subdivided further. Three distinct isoforms of the dynein heavy chain (DHC) have been identified in HeLa cells (Vaisberg et al., 1996) and four in rat testis (Criswell and Asai, 1998). The most extensively studied member of this family is DHC1 (reviewed in Hirokawa, 1998) followed by DHC2, which has now been identified in a variety of cells and tissues (Gibbons et al., 1994;Tanaka et al., 1995;Criswell et al., 1996;Vaisberg et al., 1996;Criswell and Asai, 1998). DHC2 has been localized to the apical cytoplasm of ciliated epithelial cells (Criswell et al., 1996) and to the Golgi apparatus in nonciliated mammalian cells (Vaisberg et al., 1996). Microinjection of DHC2 antibodies resulted in the fragmentation and dispersal of the Golgi apparatus, indicating a role for dynein 2 in the formation and organization of the Golgi complex (Vaisberg et al., 1996). A homologous cytoplasmic dynein 2 heavy chain (cDhc1b) has been identified in Chlamydomonas flagella, where it is involved in the transport of flagellar assembly components (Pazour et al., 1999;Porter et al., 1999) and in Caenorhabditis elegans ciliated sensory neurons, where it is also implicated in intraflagellar transport (W...