Abstract. To understand the interactions between the microtubule-based motor protein kinesin and intracellular components, we have expressed the kinesin heavy chain and its different domains in CV-1 monkey kidney epithelial cells and examined their distributions by immunofluorescence microscopy. For this study, we cloned and sequenced cDNAs encoding a kinesin heavy chain from a human placental library. The human kinesin heavy chain exhibits a high level of sequence identity to the previously cloned invertebrate kinesin heavy chains; homologies between the COOHterminal domain of human and invertebrate kinesins and the nonmotor domain of the Aspergillus kinesinlike protein bimC were also found. The gene encoding the human kinesin heavy chain also contains a small upstream open reading frame in a G-C rich 5' untranslated region, features that are associated with transla-

Abstract. Cytoplasmic dynein is a minus end-directed microtubule motor that performs distinct functions in interphase and mitosis. In interphase, dynein transports organelles along microtubules, whereas in metaphase this motor has been implicated in mitotic spindle formation and orientation as well as chromosome segregation. The manner in which dynein activity is regulated during the cell cycle, however, has not been resolved. In this study, we have examined the mechanism by which organelle transport is controlled by the cell cycle in extracts of Xenopus laevis eggs. Here, we show that photocleavage of the dynein heavy chain dramatically inhibits minus end-directed organelle transport and that purified dynein restores this motility, indicating that dynein is the predominant minus end-directed membrane motor in Xenopus egg extracts. By measuring the amount of dynein associated with isolated membranes, we find that cytoplasmic dynein and its activator dynactin detach from the membrane surface in metaphase extracts. The sevenfold decrease in membrane-associated dynein correlated well with the eightfold reduction in minus end-directed membrane transport observed in metaphase versus interphase extracts. Although dynein heavy or intermediate chain phosphorylation did not change in a cell cycle-dependent manner, the dynein light intermediate chain incorporated ~12-fold more radiolabeled phosphate in metaphase than in interphase extracts. These studies suggest that cell cycledependent phosphorylation of cytoplasmic dynein may regulate organelle transport by modulating the association of this motor with membranes.variety of membranous organelles in the cytoplasm are transported along a polarized microtubule array to specific destinations within the cell. The complexity of this membrane traffic must require precise means of regulating both the levels and directionality of such microtubule-based motility. Physiological regulation of organelle transport has been demonstrated most clearly in fish chromatophores, where a cAMP-dependent kinase pathway (Lynch et al., 1986a, b;Rozdzial and Haimo, 1986) is involved in the plus end-directed dispersion of pigment granules along microtubules. Conversely, dephosphorylation mediated by the phosphatase calcineurin (Thaler and Haimo, 1990) and an increase in intracellular Ca 2+ (Kotz and McNiven, 1994) have been implicated in the minus end-directed aggregation of these particles. Phosphorylation may control organelle transport in other cells as well, since the protein phosphatase inhibitor okadaic acid enhances minus end-directed ER tubule transport in frog egg extracts (Allan, 1995), bidirectional transport in mammalian cells in culture (Hamm-Alvarez et al., 1993), and plus end-directed lytic granule movements in T cell ex-

. Kinesin, a microtubule-based forcegenerating molecule, is thought to translocate organelles along microtubules. To examine the function of kinesin in neurons, we sought to suppress kinesin heavy chain (KHC) expression in cultured hippocampal neurons using antisense oligonucleotides and study the phenotype of these KHC "null" cells. Two different antisense oligonucleotides complementary to the KHC sequence reduced the protein levels of the heavy chain by greater than 95% within 24 h after application and produced identical phenotypes . After inhibition of KHC expression for 24 or 48 h, neurons extended an array of neurites often with one neurite longer than the others ; however, the length of all these neurites was significantly reduced . Inhibition of KHC expression also altered the distribution of GAP-43 and synapsin I, two proteins thought to be transported in astNESIN, a microtubule-based motor protein, is a heterotetramer consisting of two 110-130 kD heavy chains and two 60-70 kD light chains (Bloom et al ., 1988 ;Kuznetsov et al., 1988) . The primary structure of the heavy chain is conserved in four widely divergent species : Drosophila (Yang et al ., 1989), squid (Kosik et al ., 1990), sea urchin (Wright et al., 1991), and human (Navone et al ., in press) . Purified kinesin generates ATP-dependent microtubule gliding after being adsorbed onto glass coverslips (Vale et al., 1985a ;Scholey et al., 1985 ;Porter et al ., 1987) or induces the transport of latex beads along microtubules after adsorption onto the bead surface (Vale et al ., 1985a). In experiments with purified bovine chromaffin granules, the addition of kinesin and ATP was sufficient to induce translocation ofthe granules along microtubules (Urrutia et al ., 1991) . Kinesin-coated objects move unidirectionally towards the plus end of microtubules (Vale et al ., 1985b ;Porter et al ., 1987). This direction corresponds to anterograde transport in neurons, since axonal microtubules have their plus ends oriented distally (Filliatreau and sociation with membranous organelles. These proteins, which are normally localized at the tips of growing neurites, were confined to the cell body in antisensetreated cells. Treatment of the cells with the corresponding sense oligonucleotides affected neither the distribution of GAP-43 and synapsin I, nor the length of neurites . A full recovery of neurite length occurred after removal of the antisense oligonucleotides from the medium . These data indicate that KHC plays a role in the anterograde translocation of vesicles containing GAP-43 and synapsin I. A deficiency in vesicle delivery may also explain the inhibition of neurite outgrowth . Despite the inhibition of KHC and the failure of GAP43 and synapsin I to move out of the cell body, hippocampal neurons can extend processes and acquire an asymmetric morphology.

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Kinesin is a microtubule-based motor protein involved in intracellular organelle transport. Neurons are characterized by the presence of at least two isoforms of conventional kinesin: ubiquitous kinesin, expressed in all cells and tissues, and neuronal kinesin, whose pattern of expression is confined to neuronal cells. In order to investigate whether the two kinesin motors, which are encoded by different genes, may play distinct biological roles in neurons, we studied their expression during neuronal differentiation. Human neuroblastoma SH-SY5Y and IMR32 cells and rat phaeochromocytoma PC12 cells were used as an in vitro system for neuronal differentiation and were induced to differentiate in the presence of retinoic acid, a combination of dibutyryl cAMP and 5-bromodeoxyuridine, and nerve growth factor respectively. The expression level of each kinesin isoform was evaluated by quantitative immunoblot before and after pharmacological treatment. We found that in all cell types the expression level of neuronal kinesin, but not of ubiquitous kinesin, is stimulated during differentiation. In particular, SH-SY5Y cells show a 4.5-fold, IMR32 cells a 3-fold and PC12 cells a 7-fold increase in the level of expression of neuronal kinesin. By Northern blot analysis we found that the selective increase in the expression of neuronal kinesin is paralleled by an increase in its mRNA, indicating that there is a transcriptional control of the expression of this kinesin isoform during differentiation of neuroblastoma and PC12 cells. Our results suggest that these cells represent an adequate model to study the function of conventional kinesin and its isoforms.