Neurospora crassa ro-10 and ro-11 genes encode novel proteins required for nuclear distribution

Movement and distribution of nuclei in fungi have been shown to be dependent on cytoplasmic microtubules and the microtubule-associated motor cytoplasmic dynein. We have isolated hundreds of Neurospora crassa mutants, known as ropy, that are defective in nuclear distribution. Three of the ro genes, ro-1, ro-3 and ro-4, have been shown to encode subunits of either cytoplasmic dynein or the dynein activator complex, dynactin. In this report, we describe the isolation and initial characterization of two additional ro genes, ro-10 and ro-11. ro-10 and ro-11 are non-essential genes that encode novel 24 kDa and 75 kDa proteins respectively. Both ro-10 and ro-11 mutants retain the ability to generate long cytoplasmic microtubule tracks, suggesting that the nuclear distribution defect is not caused by a gross defect in the microtubule cytoskeleton. RO10, as well as RO4 (actin-related protein ARP1, the most abundant subunit of dynactin), appears to be required for the stability of RO3 (p150Glued), the largest subunit of dynactin. We propose that ro-10 mutants lack proper nuclear distribution, because RO10 is either a subunit of dynactin and required for dynactin activity or essential for assembly of the dynactin complex. ro-11 mutations have no effect on RO1 or RO3 levels and have only a very slight effect on the localization pattern of cytoplasmic dynein and dynactin. The role of RO11 in the movement and distribution of nuclei in N. crassa hyphae remains unknown.     

Microscopic analysis of Neurospora ropy mutants defective in nuclear distribution.

Movement and distribution of nuclei in fungi has been shown to be dependent on microtubules and the microtubule-associated motor cytoplasmic dynein. Neurospora crassa mutants known as ropy are defective in nuclear distribution. We have shown that three of the ro genes, ro-1, ro-3, and ro-4, encode subunits of either cytoplasmic dynein or the dynein activator complex, dynactin. Three other ro genes, ro-7, ro-10, and ro-11, are required for the integrity or localization of cytoplasmic dynein or dynactin. In this report, we describe a microscopic analysis of N. crassa ro mutants. Our results reveal that defects in germination of conidia, placement of septa, and mitochondrial morphology are typical of ro mutants. Two classes of cytoplasmic microtubules are identified in wild-type and ro mutants. One class of microtubules has no obvious association with nuclei while the other class of microtubules connects spindle pole bodies of adjacent nuclei. The possible role of internuclear microtubule tracts in the movement and distribution of nuclei is discussed.     

p150Glued, the largest subunit of the dynactin complex, is nonessential in Neurospora but required for nuclear distribution.

Dynactin is a multisubunit complex that is required for cytoplasmic dynein, a minus-end-directed, microtubule-associated motor, to efficiently transport vesicles along microtubules in vitro. p150Glued, the largest subunit of dynactin, has been identified in vertebrates and Drosophila and recently has been shown to interact with cytoplasmic dynein intermediate chains in vitro. The mechanism by which dynactin facilitates cytoplasmic dynein-dependent vesicle transport is unknown. We have devised a genetic screen for cytoplasmic dynein/dynactin mutants in the filamentous fungus Neurospora crassa. In this paper, we report that one of these mutants, ro-3, defines a gene encoding an apparent homologue of p150Glued, and we provide genetic evidence that cytoplasmic dynein and dynactin interact in vivo. The major structural features of vertebrate and Drosophila p150Glued, a microtubule-binding site at the N-terminus and two large alpha-helical coiled-coil regions contained within the distal two-thirds of the polypeptide, are conserved in Ro3. Drosophila p150Glued is essential for viability; however, ro-3 null mutants are viable, indicating that dynactin is not an essential complex in N. crassa. We show that N. crassa cytoplasmic dynein and dynactin mutants have abnormal nuclear distribution but retain the ability to organize cytoplasmic microtubules and actin in anucleate hyphae.     

Null Mutants of the Neurospora Actin-related Protein 1 Pointed-End Complex Show Distinct Phenotypes

Dynactin is a multisubunit complex that regulates the activities of cytoplasmic dynein, a microtubule-associated motor. Actin-related protein 1 (Arp1) is the most abundant subunit of dynactin, and it forms a short filament to which additional subunits associate. An Arp1 filament pointed-end--binding subcomplex has been identified that consists of p62, p25, p27, and Arp11 subunits. The functional roles of these subunits have not been determined. Recently, we reported the cloning of an apparent homologue of mammalian Arp11 from the filamentous fungus Neurospora crassa. Here, we report that N. crassa ro-2 and ro-12 genes encode the respective p62 and p25 subunits of the pointed-end complex. Characterization of Delta ro-2, Delta ro-7, and Delta ro-12 mutants reveals that each has a distinct phenotype. All three mutants have reduced in vivo vesicle trafficking and have defects in vacuole distribution. We showed previously that in vivo dynactin function is required for high-level dynein ATPase activity, and we find that all three mutants have low dynein ATPase activity. Surprisingly, Delta ro-12 differs from Delta ro-2 and Delta ro-7 and other previously characterized dynein/dynactin mutants in that it has normal nuclear distribution. Each of the mutants shows a distinct dynein/dynactin localization pattern. All three mutants also show stronger dynein/dynactin-membrane interaction relative to wild type, suggesting that the Arp1 pointed-end complex may regulate interaction of dynactin with membranous cargoes.     

A Neurospora crassa Arp1 mutation affecting cytoplasmic dynein and dynactin localization

Of the actin-related proteins, Arp1 is the most similar to conventional actin, and functions solely as a component of the multisubunit complex dynactin. Dynactin has been identified as an activator of the microtubule-associated motor cytoplasmic dynein. The role of Arp1 within dynactin is two-fold: (1) it serves as a structural scaffold protein for other dynactin subunits; and (2) it has been proposed to link dynactin, and thereby dynein, with membranous cargo via interaction with spectrin. Using the filamentous fungus Neurospora crassa, we have identified genes encoding subunits of cytoplasmic dynein and dynactin. In this study, we describe a genetic screen for N. crassa Arp1 (ro-4) mutants that are defective for dynactin function. We report that the ro-4(E8) mutant is unusual in that it shows alterations in the localization of cytoplasmic dynein and dynactin and in microtubule organization. In the mutant, dynein/dynactin complexes co-localize with bundled microtubules at hyphal tips. Given that dynein transports membranous cargo from hyphal tips to distal regions, the cytoplasmic dynein and dynactin complexes that accumulate along microtubule tracts at hyphal tips in the ro-4(E8) mutant may have either reduced motor activity or be delayed for activation of motor activity following cargo binding.     

A fungal actin-related protein involved in nuclear migration

The ro-4 mutant of the filamentous fungus Neurospora crassa forms distinctive colonies in which hyphae grow into rope-like aggregates. This unusual morphology coincides with a defect in hyphal nuclear migration. The ro-4 gene was cloned from a cosmid library by complementation of the closely linked pab-2 gene. The deduced 380 amino acid protein is most similar to the vertebrate actin-related protein/centractin. The R04 protein is not essential for cell viability, and new strains created by inducing point mutations at the ro-4 locus have a phenotype which is very similar to that of the original mutant. This study provides genetic evidence that an actin-related protein plays a role in nuclear motility. Since nuclear motility is believed to be a microtubule-dependent process, the ro-4 gene product may function as a component of the dynactin complex which activates force generation by cytoplasmic dynein.     

The LIS1-related protein NUDF of Aspergillus nidulans and its interaction partner NUDE bind directly to specific subunits of dynein and dynactin and to alpha- and gamma-tubulin

The NUDF protein of Aspergillus nidulans, which is required for nuclear migration through the fungal mycelium, closely resembles the LIS1 protein required for migration of neurons to the cerebral cortex in humans. Genetic experiments suggested that NUDF influences nuclear migration by affecting cytoplasmic dynein. NUDF interacts with another protein, NUDE, which also affects nuclear migration in A. nidulans. Interactions among LIS1, NUDE, dynein, and gamma-tubulin have been demonstrated in animal cells. In this paper we examine the interactions of the A. nidulans NUDF and NUDE proteins with components of dynein, dynactin, and with alpha- and gamma-tubulin. We show that NUDF binds directly to alpha- and gamma-tubulin and to the first P-loop of the cytoplasmic dynein heavy chain, whereas NUDE binds directly to alpha- and gamma-tubulin, to NUDK (actin-related protein 1), and to the NUDG dynein LC8 light chain. The data suggest a direct role for NUDF in regulation of the dynein heavy chain and an effect on other dynein/dynactin subunits via NUDE. The interactions between NUDE, NUDF, and gamma-tubulin suggest that this protein may also be involved in the regulation of dynein function. Additive interactions between NUDE and dynein and dynactin subunits suggest that NUDE acts as a scaffolding factor between components.     

Regulation of cytoplasmic dynein function in vivo by the Drosophila Glued complex

The Drosophila Glued gene product shares sequence homology with the p150 component of vertebrate dynactin. Dynactin is a multiprotein complex that stimulates cytoplasmic dynein-mediated vesicle motility in vitro. In this report, we present biochemical, cytological, and genetic evidence that demonstrates a functional similarity between the Drosophila Glued complex and vertebrate dynactin. We show that, similar to the vertebrate homologues in dynactin, the Glued polypeptides are components of a 20S complex. Our biochemical studies further reveal differential expression of the Glued polypeptides, all of which copurify as microtubule-associated proteins. In our analysis of the Glued polypeptides encoded by the dominant mutation, Glued, we identify a truncated polypeptide that fails to assemble into the wild-type 20S complex, but retains the ability to copurify with microtubules. The spatial and temporal distribution of the Glued complex during oogenesis is shown by immunocytochemistry methods to be identical to the pattern previously described for cytoplasmic dynein. Significantly, the pattern of Glued distribution in oogenesis is dependent on dynein function, as well as several other gene products known to be required for proper dynein localization. In genetic complementation studies, we find that certain mutations in the cytoplasmic dynein heavy chain gene Dhc64C act as dominant suppressors or enhancers of the rough eye phenotype of the dominant Glued mutation. Furthermore, we show that a mutation that was previously isolated as a suppressor of the Glued mutation is an allele of Dhc64C. Together with the observed dependency of Glued localization on dynein function, these genetic interactions demonstrate a functional association between the Drosophila dynein motor and Glued complexes.     

Live imaging of Drosophila brain neuroblasts reveals a role for Lis1/dynactin in spindle assembly and mitotic checkpoint control

Lis1 is required for nuclear migration in fungi, cell cycle progression in mammals, and the formation of a folded cerebral cortex in humans. Lis1 binds dynactin and the dynein motor complex, but the role of Lis1 in many dynein/dynactin-dependent processes is not clearly understood. Here we generate and/or characterize mutants for Drosophila Lis1 and a dynactin subunit, Glued, to investigate the role of Lis1/dynactin in mitotic checkpoint function. In addition, we develop an improved time-lapse video microscopy technique that allows live imaging of GFP-Lis1, GFP-Rod checkpoint protein, green fluorescent protein (GFP)-labeled chromosomes, or GFP-labeled mitotic spindle dynamics in neuroblasts within whole larval brain explants. Our mutant analyses show that Lis1/dynactin have at least two independent functions during mitosis: first promoting centrosome separation and bipolar spindle assembly during prophase/prometaphase, and subsequently generating interkinetochore tension and transporting checkpoint proteins off kinetochores during metaphase, thus promoting timely anaphase onset. Furthermore, we show that Lis1/dynactin/dynein physically associate and colocalize on centrosomes, spindle MTs, and kinetochores, and that regulation of Lis1/dynactin kinetochore localization in Drosophila differs from both Caenorhabditis elegans and mammals. We conclude that Lis1/dynactin act together to regulate multiple, independent functions in mitotic cells, including spindle formation and cell cycle checkpoint release.     

Role of dynein, dynactin, and CLIP-170 interactions in LIS1 kinetochore function

Mutations in the human LIS1 gene cause type I lissencephaly, a severe brain developmental disease involving gross disorganization of cortical neurons. In lower eukaryotes, LIS1 participates in cytoplasmic dynein-mediated nuclear migration. We previously reported that mammalian LIS1 functions in cell division and coimmunoprecipitates with cytoplasmic dynein and dynactin. We also localized LIS1 to the cell cortex and kinetochores of mitotic cells, known sites of dynein action. We now find that the COOH-terminal WD repeat region of LIS1 is sufficient for kinetochore targeting. Overexpression of this domain or full-length LIS1 displaces CLIP-170 from this site without affecting dynein and other kinetochore markers. The NH2-terminal self-association domain of LIS1 displaces endogenous LIS1 from the kinetochore, with no effect on CLIP-170, dynein, and dynactin. Displacement of the latter proteins by dynamitin overexpression, however, removes LIS1, suggesting that LIS1 binds to the kinetochore through the motor protein complexes and may interact with them directly. We find that of 12 distinct dynein and dynactin subunits, the dynein heavy and intermediate chains, as well as dynamitin, interact with the WD repeat region of LIS1 in coexpression/coimmunoprecipitation and two-hybrid assays. Within the heavy chain, interactions are with the first AAA repeat, a site strongly implicated in motor function, and the NH2-terminal cargo-binding region. Together, our data suggest a novel role for LIS1 in mediating CLIP-170-dynein interactions and in coordinating dynein cargo-binding and motor activities.     

动力蛋白激活蛋白亚单位Dynamitin具有ATP酶活性的生物化学特征

动力蛋白激活蛋白(dynactin) 是一个与胞浆内动力蛋白的功能相关的多亚基复合物.动力蛋白（dynein）为向微管负端运输的马达蛋白,其多种功能包括细胞核迁移、有丝分裂纺锤体定位以及细胞间期和有丝分裂的细胞骨架再组装.Dynamitin,是一个50 kD的动力蛋白激活蛋白亚单位, 对于稳定动力蛋白激活蛋白复合物是非常重要的.为研究这种稳定性机制,分析了dynamitin的序列,并揭示dynamitin的一些DNA序列与ATP酶的Walker A 和 Walker B 序列具有同源性.纯化的谷胱甘肽巯基转移酶标签蛋白dynamitin和无此标签的蛋白dynamitin都特异性显示了ATP酶活性.DNA序列Walker A的失活突变可废除dynamitin蛋白的ATP酶活性,而Walker B 序列无此作用.因此,突变实验进一步证实dynamitin蛋白的ATP酶活性.ATP酶活性的动力学研究结果表明Km为 125.78μmol/L和 Kcat 为7.4 min-1     

Clustering of nuclei in multinucleated hyphae is prevented by dynein-driven bidirectional nuclear movements and microtubule growth control in Ashbya gossypii

During filamentous fungus development, multinucleated hyphae employ a system for long-range nuclear migration to maintain an equal nuclear density. A decade ago the microtubule motor dynein was shown to play a central role in this process. Previous studies with Ashbya gossypii revealed extensive bidirectional movements and bypassings of nuclei, an autonomous cytoplasmic microtubule (cMT) cytoskeleton emanating from each nucleus, and pulling of nuclei by sliding of cMTs along the cortex. Here, we show that dynein is the sole motor for bidirectional movements and bypassing because these movements are concomitantly decreased in mutants carrying truncations of the dynein heavy-chain DYN1 promoter. The dynactin component Jnm1, the accessory proteins Dyn2 and Ndl1, and the potential dynein cortical anchor Num1 are also involved in the dynamic distribution of nuclei. In their absence, nuclei aggregate to different degrees, whereby the mutants with dense nuclear clusters grow extremely long cMTs. As in budding yeast, we found that dynein is delivered to cMT plus ends, and its activity or processivity is probably controlled by dynactin and Num1. Together with its role in powering nuclear movements, we propose that dynein also plays (directly or indirectly) a role in the control of cMT length. Those combined dynein actions prevent nuclear clustering in A. gossypii and thus reveal a novel cellular role for dynein.     

Cytoplasmic dynein ATPase activity is regulated by dynactin-dependent phosphorylation

Cytoplasmic dynein is a microtubule-associated motor that utilizes ATP hydrolysis to conduct minus-end directed transport of various organelles. Dynactin is a multisubunit complex that has been proposed to both link dynein with cargo and activate dynein motor function. The mechanisms by which dynactin regulates dynein activity are not clear. In this study, we examine the role of dynactin in regulating dynein ATPase activity. We show that dynein-microtubule binding and ATP-dependent release of dynein from microtubules are reduced in dynactin null mutants, Deltaro-3 (p150(Glued)) and Deltaro-4 (Arp1), relative to wild-type. The dynein-microtubule binding activity, but not the ATP-dependent release of dynein from microtubules, is restored by in vitro mixing of extracts from dynein and dynactin mutants. Dynein produced in a Deltaro-3 mutant has approximately 8-fold reduced ATPase activity relative to dynein isolated from wild-type. However, dynein ATPase activity from wild-type is not reduced when dynactin is separated from dynein, suggesting that dynein produced in a dynactin mutant is inactivated. Treatment of dynein isolated from the Deltaro-3 mutant with lambda protein phosphatase restores the ATPase activity to near wild-type levels. The reduced dynein ATPase activity observed in dynactin null mutants is mainly due to altered affinity for ATP. Radiolabeling experiments revealed that low molecular mass proteins, particularly 20- and 8-kDa proteins, that immunoprecipitate with dynein heavy chain are hyperphosphorylated in the dynactin mutant and dephosphorylated upon lambda protein phosphatase treatment. The results suggest that cytoplasmic dynein ATPase activity is regulated by dynactin-dependent phosphorylation of dynein light chains.     

Dynactin's pointed-end complex is a cargo-targeting module

Dynactin is an essential part of the cytoplasmic dynein motor that enhances motor processivity and serves as an adaptor that allows dynein to bind cargoes. Much is known about dynactin''s interaction with dynein and microtubules, but how it associates with its diverse complement of subcellular binding partners remains mysterious. It has been suggested that cargo specification involves a group of subunits referred to as the “pointed-end complex.” We used chemical cross-linking, RNA interference, and protein overexpression to characterize interactions within the pointed-end complex and explore how it contributes to dynactin''s interactions with endomembranes. The Arp11 subunit, which caps one end of dynactin''s Arp1 filament, and p62, which binds Arp11 and Arp1, are necessary for dynactin stability. These subunits also allow dynactin to bind the nuclear envelope prior to mitosis. p27 and p25, by contrast, are peripheral components that can be removed without any obvious impact on dynactin integrity. Dynactin lacking these subunits shows reduced membrane binding. Depletion of p27 and p25 results in impaired early and recycling endosome movement, but late endosome movement is unaffected, and mitotic spindles appear normal. We conclude that the pointed-end complex is a bipartite structural domain that stabilizes dynactin and supports its binding to different subcellular structures.     

Interaction of the p62 subunit of dynactin with Arp1 and the cortical actin cytoskeleton

Targeting of the minus-end directed microtubule motor cytoplasmic dynein to a wide array of intracellular substrates appears to be mediated by an accessory factor known as dynactin [1-4]. Dynactin is a multi-subunit complex that contains a short actin-related protein 1 (Arp 1) filament with capZ at the barbed end and p62 at the pointed end [5]. The location of the p62 subunit and the proposed role for dynactin as a multifunctional targeting complex raise the possibility of a dual role for p62 in dynein targeting and in Arp1 pointed-end capping. In order to gain further insight into the role of p62 in dynactin function, we have cloned cDNAs that encode two full-length isoforms of the protein from rat brain. We found that p62 is homologous to the nuclear migration protein Ropy-2 from Neurospora [6]; both proteins contain a zinc-binding motif that resembles the LIM domain of several other cytoskeletal proteins [7]. Overexpression of p62 in cultured mammalian cells revealed colocalization with cortical actin, stress fibers, and focal adhesion sites, sites of potential interaction between microtubules and the cell cortex [8,9]. The p62 protein also colocalized with polymers of overexpressed wild-type or barbed-end-mutant Arp1, but not with a pointed-end mutant. Deletion of the LIM domain abolished targeting of p62 to focal-adhesion sites but did not interfere with binding of p62 to actin or Arp1. These data implicate p62 in Arp1 pointed-end binding and suggest additional roles in linking dynein and dynactin to the cortical cytoskeleton.     

Nudel modulates kinetochore association and function of cytoplasmic dynein in M phase

The microtubule-based motor cytoplasmic dynein/dynactin is a force generator at the kinetochore. It also transports proteins away from kinetochores to spindle poles. Regulation of such diverse functions, however, is poorly understood. We have previously shown that Nudel is critical for dynein-mediated protein transport, whereas mitosin, a kinetochore protein that binds Nudel, is involved in retention of kinetochore dynein/dynactin against microtubule-dependent stripping. Here we demonstrate that Nudel is required for robust localization of dynein/dynactin at the kinetochore. It localizes to kinetochores after nuclear envelope breakdown, depending mostly ( approximately 78%) on mitosin and slightly on dynein/dynactin. Depletion of Nudel by RNA interference (RNAi) or overexpression of its mutant incapable of binding either Lis1 or dynein heavy chain abolishes the kinetochore protein transport and mitotic progression. Similar to mitosin RNAi, Nudel RNAi also leads to increased stripping of kinetochore dynein/dynactin in the presence of microtubules. Taking together, our results suggest a dual role of kinetochore Nudel: it activates dynein-mediated protein transport and, when interacting with both mitosin and dynein, stabilizes kinetochore dynein/dynactin against microtubule-dependent stripping to facilitate the force generation function of the motor.     

Cytoplasmic dynein, the dynactin complex, and kinesin are interdependent and essential for fast axonal transport

In axons, organelles move away from (anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and anterograde transport. We have tested the functions of the cytoplasmic dynein heavy chain (cDhc64C) and the p150(Glued) (Glued) component of the dynactin complex with the use of genetic techniques in Drosophila. cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150(Glued) were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued or cDhc64C mutations were stronger than those between Glued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport.     

Analysis of dynactin subcomplexes reveals a novel actin-related protein associated with the arp1 minifilament pointed end.

The multisubunit protein, dynactin, is a critical component of the cytoplasmic dynein motor machinery. Dynactin contains two distinct structural domains: a projecting sidearm that interacts with dynein and an actin-like minifilament backbone that is thought to bind cargo. Here, we use biochemical, ultrastructural, and molecular cloning techniques to obtain a comprehensive picture of dynactin composition and structure. Treatment of purified dynactin with recombinant dynamitin yields two assemblies: the actin-related protein, Arp1, minifilament and the p150(Glued) sidearm. Both contain dynamitin. Treatment of dynactin with the chaotropic salt, potassium iodide, completely depolymerizes the Arp1 minifilament to reveal multiple protein complexes that contain the remaining dynactin subunits. The shoulder/sidearm complex contains p150(Glued), dynamitin, and p24 subunits and is ultrastructurally similar to dynactin's flexible projecting sidearm. The dynactin shoulder complex, which contains dynamitin and p24, is an elongated, flexible assembly that may link the shoulder/sidearm complex to the Arp1 minifilament. Pointed-end complex contains p62, p27, and p25 subunits, plus a novel actin-related protein, Arp11. p62, p27, and p25 contain predicted cargo-binding motifs, while the Arp11 sequence suggests a pointed-end capping activity. These isolated dynactin subdomains will be useful tools for further analysis of dynactin assembly and function.     

Bni1p regulates microtubule-dependent nuclear migration through the actin cytoskeleton in Saccharomyces cerevisiae

The RHO1 gene encodes a yeast homolog of the mammalian RhoA protein. Rho1p is localized to the growth sites and is required for bud formation. We have recently shown that Bni1p is one of the potential downstream target molecules of Rho1p. The BNI1 gene is implicated in cytokinesis and the establishment of cell polarity in Saccharomyces cerevisiae but is not essential for cell viability. In this study, we screened for mutations that were synthetically lethal in combination with a bni1 mutation and isolated two genes. They were the previously identified PAC1 and NIP100 genes, both of which are implicated in nuclear migration in S. cerevisiae. Pac1p is a homolog of human LIS1, which is required for brain development, whereas Nip100p is a homolog of rat p150(Glued), a component of the dynein-activated dynactin complex. Disruption of BNI1 in either the pac1 or nip100 mutant resulted in an enhanced defect in nuclear migration, leading to the formation of binucleate mother cells. The arp1 bni1 mutant showed a synthetic lethal phenotype while the cin8 bni1 mutant did not, suggesting that Bni1p functions in a kinesin pathway but not in the dynein pathway. Cells of the pac1 bni1 and nip100 bni1 mutants exhibited a random distribution of cortical actin patches. Cells of the pac1 act1-4 mutant showed temperature-sensitive growth and a nuclear migration defect. These results indicate that Bni1p regulates microtubule-dependent nuclear migration through the actin cytoskeleton. Bni1p lacking the Rho-binding region did not suppress the pac1 bni1 growth defect, suggesting a requirement for the Rho1p-Bni1p interaction in microtubule function.