CLIP-170 homologue and NUDE play overlapping roles in NUDF localization in Aspergillus nidulans

Proteins in the cytoplasmic dynein pathway accumulate at the microtubule plus end, giving the appearance of comets when observed in live cells. The targeting mechanism for NUDF (LIS1/Pac1) of Aspergillus nidulans, a key component of the dynein pathway, has not been clear. Previous studies have demonstrated physical interactions of NUDF/LIS1/Pac1 with both NUDE/NUDEL/Ndl1 and CLIP-170/Bik1. Here, we have identified the A. nidulans CLIP-170 homologue, CLIPA. The clipA deletion did not cause an obvious nuclear distribution phenotype but affected cytoplasmic microtubules in an unexpected manner. Although more microtubules failed to undergo long-range growth toward the hyphal tip at 32 degrees C, those that reached the hyphal tip were less likely to undergo catastrophe. Thus, in addition to acting as a growth-promoting factor, CLIPA also promotes microtubule dynamics. In the absence of CLIPA, green fluorescent protein-labeled cytoplasmic dynein heavy chain, p150(Glued) dynactin, and NUDF were all seen as plus-end comets at 32 degrees C. However, under the same conditions, deletion of both clipA and nudE almost completely abolished NUDF comets, although nudE deletion itself did not cause a dramatic change in NUDF localization. Based on these results, we suggest that CLIPA and NUDE both recruit NUDF to the microtubule plus end. The plus-end localization of CLIPA itself seems to be regulated by different mechanisms under different physiological conditions. Although the KipA kinesin (Kip2/Tea2 homologue) did not affect plus-end localization of CLIPA at 32 degrees C, it was required for enhancing plus-end accumulation of CLIPA at an elevated temperature (42 degrees C).     

The LIS1-related NUDF protein of Aspergillus nidulans interacts with the coiled-coil domain of the NUDE/RO11 protein

The nudF gene of the filamentous fungus Aspergillus nidulans acts in the cytoplasmic dynein/dynactin pathway and is required for distribution of nuclei. NUDF protein, the product of the nudF gene, displays 42% sequence identity with the human protein LIS1 required for neuronal migration. Haploinsufficiency of the LIS1 gene causes a malformation of the human brain known as lissencephaly. We screened for multicopy suppressors of a mutation in the nudF gene. The product of the nudE gene isolated in the screen, NUDE, is a homologue of the nuclear distribution protein RO11 of Neurospora crassa. The highly conserved NH(2)-terminal coiled-coil domain of the NUDE protein suffices for protein function when overexpressed. A similar coiled-coil domain is present in several putative human proteins and in the mitotic phosphoprotein 43 (MP43) of X. laevis. NUDF protein interacts with the Aspergillus NUDE coiled-coil in a yeast two-hybrid system, while human LIS1 interacts with the human homologue of the NUDE/RO11 coiled-coil and also the Xenopus MP43 coiled-coil. In addition, NUDF coprecipitates with an epitope-tagged NUDE. The fact that NUDF and LIS1 interact with the same protein domain strengthens the notion that these two proteins are functionally related.     

Mutations in the heavy chain of cytoplasmic dynein suppress the nudF nuclear migration mutation of Aspergillus nidulans

To identify proteins that interact directly or indirectly with the NUDF protein, which is required for nuclear migration in Aspergillus nidulans, we initiated a screen for extragenic suppressors of the heat-sensitive nudF6 mutation. Suppressor mutations in at least five genes, designated snfA–snfE, caused improved growth and nuclear migration at high temperatures compared to the nudF6 parent. Two snfC mutations mapped near the nudA gene, which encodes the cytoplasmic dynein heavy chain, and could be repaired by transformation with wild-type nudA DNA, demonstrating that they are mutations in nudA. The snfC mutations are bypass suppressors of nudF and genetic evidence indicated that NUDA and NUDF act in the same nuclear migration pathway. Taken together, our data suggests that NUDF affects nuclear migration by acting on the dynein motor system. Received: 4 January 1997 / Accepted: 26 February 1997     

The Aspergillus cytoplasmic dynein heavy chain and NUDF localize to microtubule ends and affect microtubule dynamics

Cytoplasmic dynein is a multisubunit, minus end-directed microtubule motor that uses dynactin as an accessory complex to perform various in vivo functions including vesicle transport, spindle assembly, and nuclear distribution [1]. We previously showed that in the filamentous fungus Aspergillus nidulans, a GFP-tagged cytoplasmic dynein heavy chain (NUDA) forms comet-like structures that exhibited microtubule-dependent movement toward and back from the hyphal tip [2]. Here we demonstrate that another protein in the NUDA pathway, NUDF, which is homologous to the human LIS1 protein involved in brain development [3, 4], also exhibits such dynamic behavior. Both NUDA and NUDF are located at the ends of microtubules, and this observation suggests that the observed dynamic behavior is due to their association with the dynamic microtubule ends. To address whether NUDA and NUDF play a role in regulating microtubule dynamics in vivo, we constructed a GFP-labeled alpha-tubulin strain and used it to compare microtubule dynamics in vivo in wild-type A. nidulans versus temperature-sensitive loss-of-function mutants of nudA and nudF. The mutants showed a lower frequency of microtubule catastrophe, a lower rate of shrinkage during catastrophe, and a lower frequency of rescue. The microtubules in the mutant cells also paused longer at the hyphal tip than wild-type microtubules. These results indicate that cytoplasmic dynein and the LIS1 homolog NUDF affect microtubule dynamics in vivo.     

An Alpha Tubulin Mutation Suppresses Nuclear Migration Mutations in Aspergillus Nidulans

Microtubules and cytoplasmic dynein, a microtubule-dependent motor, are required for nuclei to move along the hyphae of filamentous fungi. Nuclear migration in Aspergillus nidulans is blocked by heat-sensitive (hs(-)) mutations in the nudA gene, which encodes dynein heavy chain, and the nudF gene, which encodes a G protein β-subunit-like protein. Hs(-) mutations in the nudC and nudG genes also prevent nuclear migration. We have isolated extragenic suppressor mutations that reverse the hs(-) phenotypes caused by these mutations. Here we show that one nudF suppressor also suppresses hs(-) mutations in nudA, nudC, and nudG and deletions in nudA and nudF. This suppressor mutation is in the tubA alpha tubulin gene, and its characteristics suggest that it destabilizes microtubules. The mutation alters microtubule staining and confers sensitivity to cold and benomyl, two treatments that destabilize microtubules. Treatment with low concentrations of benomyl also suppresses the hs(-) nudA, nudC, nudF, and nudG mutations and the nudA and nudF deletions. Suppression of the hs(-) nudA mutation and the nudA deletion is especially interesting because these strains lack active dynein heavy chain. Together, these results suggest that microtubule destabilization allows nuclei to migrate even in the absence of cytoplasmic dynein motor function.     

Mutations in the heavy chain of cytoplasmic dynein suppress the nudF nuclear migration mutation of Aspergillus nidulans

To identify proteins that interact directly or indirectly with the NUDF protein, which is required for nuclear migration in Aspergillus nidulans, we initiated a screen for extragenic suppressors of the heat-sensitive nudF6 mutation. Suppressor mutations in at least five genes, designated snfA–snfE, caused improved growth and nuclear migration at high temperatures compared to the nudF6 parent. Two snfC mutations mapped near the nudA gene, which encodes the cytoplasmic dynein heavy chain, and could be repaired by transformation with wild-type nudA DNA, demonstrating that they are mutations in nudA. The snfC mutations are bypass suppressors of nudF and genetic evidence indicated that NUDA and NUDF act in the same nuclear migration pathway. Taken together, our data suggests that NUDF affects nuclear migration by acting on the dynein motor system.     

Point mutations in the stem region and the fourth AAA domain of cytoplasmic dynein heavy chain partially suppress the phenotype of NUDF/LIS1 loss in Aspergillus nidulans

Cytoplasmic dynein performs multiple cellular tasks but its regulation remains unclear. The dynein heavy chain has a N-terminal stem that binds to other subunits and a C-terminal motor unit that contains six AAA (ATPase associated with cellular activities) domains and a microtubule-binding site located between AAA4 and AAA5. In Aspergillus nidulans, NUDF (a LIS1 homolog) functions in the dynein pathway, and two nudF6 partial suppressors were mapped to the nudA dynein heavy chain locus. Here we identified these two mutations. The nudAL1098F mutation resides in the stem region, and nudAR3086C is in the end of AAA4. These mutations partially suppress the phenotype of nudF deletion but do not suppress the phenotype exhibited by mutants of dynein intermediate chain and Arp1. Surprisingly, the stronger DeltanudF suppressor, nudAR3086C, causes an obvious decrease in the basal level of dynein's ATPase activity and an increase in dynein's distribution along microtubules. Thus, suppression of the DeltanudF phenotype may result from mechanisms other than simply the enhancement of dynein's ATPase activity. The fact that a mutation in the end of AAA4 negatively regulates dynein's ATPase activity but partially compensates for NUDF loss indicates the importance of the AAA4 domain in dynein regulation in vivo.     

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.     

Extragenic Suppressors of Nudc3, a Mutation That Blocks Nuclear Migration in Aspergillus Nidulans

Nuclear migration plays an important role in the growth and development of many organisms including the filamentous fungus Aspergillus nidulans. We have cloned three genes from A. nidulans, nudA, nudC, and nudF, in which mutations affect nuclear migration. The nudA gene encodes the heavy chain of cytoplasmic dynein. The nudC gene encodes a 22-kD protein. The nudF gene was identified as an extracopy suppressor of the temperature sensitive (ts(-)) nudC3 mutation. The nudC3 mutation substantially decreases the intracellular concentration of the nudF protein at restrictive temperature. This is restored toward the normal level by an extra copy of nudF. To identify other genes whose products interact directly or indirectly with the NUDC protein, we have isolated a set of extragenic suppressors of the nudC3 temperature-sensitive mutation. Genetic analysis of 16 such extragenic suppressors showed them to represent nine different genes, designated sncA-sncI (for suppressor of nudC). sncA-sncH were either dominant or semidominant in diploids homozygous for nudC3 and heterozygous for the snc mutations. All of the suppressors reversed the ts(-) phenotype of nudC3 by restoring the intracellular concentration of the NUDF protein.     

Deletion of nudC, a nuclear migration gene of Aspergillus nidulans, causes morphological and cell wall abnormalities and is lethal.

Nuclear migration is required for normal development in both higher and lower eukaryotes. In fungi this process is mediated by cytoplasmic dynein. It is believed that this motor protein is anchored to the cell membrane and moves nuclei by capturing and pulling on spindle pole body microtubules. To date, four genes have been identified and shown to be required for this process in Aspergillus nidulans. The nudA and nudG genes, respectively, encode the heavy and light chains of cytoplasmic dynein, and the nudF and nudC gene products encode proteins of 49 and 22 kDa. The precise biochemical functions of the nudF and nudC genes have not yet been identified. In this report we further investigate NUDC protein function by deleting the nudC gene. Surprisingly, although deletion of nudA and nudF affect nuclear migration, deletion of nudC profoundly affected the morphology and composition of the cell wall. Spores of the strain deleted for nudC grew spherically and lysed. The thickness of the cell wall was increased in the deletion mutant and wall polymer composition was abnormal. This phenotype could be repressed by growth on osmotically buffered medium at low temperature. Similar, but less severe, effects were also noted in a strain depleted for NUDC by down-regulation. These results suggest a possible relationship between fungal cell wall biosynthesis and nuclear migration.     

Dynamics of cytoplasmic dynein in living cells and the effect of a mutation in the dynactin complex actin-related protein Arp1

Cytoplasmic dynein is a minus-end-directed microtubule motor that participates in multiple cellular activities such as organelle transport and mitotic spindle assembly [1]. To study the dynamic behavior of cytoplasmic dynein in the filamentous fungus Aspergillus nidulans, we replaced the gene for the cytoplasmic dynein heavy chain, nudA, with a gene encoding a green fluorescent protein (GFP)-tagged chimera, GFP-nudA. The GFP-NUDA fusion protein is fully functional in vivo: strains expressing only the GFP-tagged nudA grow as well as wild-type strains. Fluorescence microscopy showed GFP-NUDA to be in comet-like structures that moved in the hyphae toward the growing tip. Retrograde movement of some GFP-NUDA comets after they arrived at the tip was also observed. These dynamics of GFP-NUDA were not observed in cells treated with a microtubule-destabilizing drug, benomyl, suggesting they are microtubule-dependent. The rate of GFP-NUDA tip-ward movement is similar to the rate of cytoplasmic microtubule polymerization toward the hyphal tip, suggesting that GFP-NUDA is associated and moving with the polymerizing ends of microtubules. A mutation in actin-related protein Arp1 of the dynactin complex abolishes the presence of these dynamic GFP-NUDA structures near the hyphal tip, suggesting a targeting role of the dynactin complex.     

Cytoplasmic dynein intermediate chain and heavy chain are dependent upon each other for microtubule end localization in Aspergillus nidulans

The multisubunit microtubule motor, cytoplasmic dynein, targets to various subcellular locations in eukaryotic cells for various functions. The cytoplasmic dynein heavy chain (HC) contains the microtubule binding and ATP binding sites for motor function, whereas the intermediate chain (IC) is implicated in the in vivo targeting of the HC. Concerning any targeting event, it is not known whether the IC has to form a complex with the HC for targeting or whether the IC can target to a site independently of the HC. In the filamentous fungus Aspergillus nidulans, the dynein HC is localized to the ends of microtubules near the hyphal tip. In this study, we demonstrate that our newly identified dynein IC in A. nidulans is also localized to microtubule ends and is required for HC's localization to microtubule ends in living cells. With the combination of two reagents, an HC loss-of function mutant and the green fluorescent protein (GFP)-fused IC that retains its function, we show that the IC's localization to microtubule ends also requires HC, suggesting that cytoplasmic dynein HC-IC complex formation is important for microtubule end targeting. In addition, we show that the HC localization is not apparently altered in the deletion mutant of NUDF, a LIS1-like protein that interacts directly with the ATP-binding domain of the HC. Our study suggests that, although HC-IC association is important for the targeting of dynein to microtubule ends, other essential components, such as NUDF, may interact with the targeted dynein complex to produce full motor activities in vivo.     

Extragenic Suppressors of a Dynein Mutation That Blocks Nuclear Migration in Aspergillus Nidulans

Cytoplasmic dynein is a large molecular weight protein complex that functions as a microtubule-dependent, negative, end-directed ``motor.' Mutations in nudA, which encodes the heavy chain of cytoplasmic dynein, inhibit nuclear migration in Aspergillus nidulans. This paper describes the selection and characterization of extragenic suppressors of the nudA1 mutation preparatory to the identification of other proteins that interact directly or indirectly with the cytoplasmic dynein heavy chain. To facilitate future cloning of the suppressor genes, we have searched particularly for extragenic suppressor mutations that also convey a selectable phenotype, such as cold or dimethyl sulfoxide sensitivity. Genetic analysis of 16 revertants has defined at least five extragenic suppressors of nudA1 (snaA-E). All the sna mutations except one were recessive in diploids homozygous for nudA1 and heterozygous for sna mutations. To characterize the nuclear migration phenotype in the sna mutants, conidia of one representative of each complementation group were germinated, fixed and nuclei stained. The sna mutants display partial suppression of the nudA1 nuclear migration defect. Although conidiophores were produced in the sna mutants, they failed to develop normally and to produce spores. Examination of the nudA1,sna conidiophores under the microscope showed that nuclear migration into the metulae and phialides was defective.     

The nuclear migration protein NUDF/LIS1 forms a complex with NUDC and BNFA at spindle pole bodies

Nuclear migration depends on microtubules, the dynein motor complex, and regulatory components like LIS1 and NUDC. We sought to identify new binding partners of the fungal LIS1 homolog NUDF to clarify its function in dynein regulation. We therefore analyzed the association between NUDF and NUDC in Aspergillus nidulans. NUDF and NUDC directly interacted in yeast two-hybrid experiments via NUDF's WD40 domain. NUDC-green fluorescent protein (NUDC-GFP) was localized to immobile dots in the cytoplasm and at the hyphal cortex, some of which were spindle pole bodies (SPBs). We showed by bimolecular fluorescence complementation microscopy that NUDC directly interacted with NUDF at SPBs at different stages of the cell cycle. Applying tandem affinity purification, we isolated the NUDF-associated protein BNFA (for binding to NUDF). BNFA was dispensable for growth and for nuclear migration. GFP-BNFA fusions localized to SPBs at different stages of the cell cycle. This localization depended on NUDF, since the loss of NUDF resulted in the cytoplasmic accumulation of BNFA. BNFA did not bind to NUDC in a yeast two-hybrid assay. These results show that the conserved NUDF and NUDC proteins play a concerted role at SPBs at different stages of the cell cycle and that NUDF recruits additional proteins specifically to the dynein complex at SPBs.     

Accumulation of cytoplasmic dynein and dynactin at microtubule plus ends in Aspergillus nidulans is kinesin dependent

The mechanism(s) by which microtubule plus-end tracking proteins are targeted is unknown. In the filamentous fungus Aspergillus nidulans, both cytoplasmic dynein and NUDF, the homolog of the LIS1 protein, localize to microtubule plus ends as comet-like structures. Herein, we show that NUDM, the p150 subunit of dynactin, also forms dynamic comet-like structures at microtubule plus ends. By examining proteins tagged with green fluorescent protein in different loss-of-function mutants, we demonstrate that dynactin and cytoplasmic dynein require each other for microtubule plus-end accumulation, and the presence of cytoplasmic dynein is also important for NUDF's plus-end accumulation. Interestingly, deletion of NUDF increases the overall accumulation of dynein and dynactin at plus ends, suggesting that NUDF may facilitate minus-end-directed dynein movement. Finally, we demonstrate that a conventional kinesin, KINA, is required for the microtubule plus-end accumulation of cytoplasmic dynein and dynactin, but not of NUDF.     

Kinesin-1 and Cytoplasmic Dynein Act Sequentially to Move the Meiotic Spindle to the Oocyte Cortex in Caenorhabditis elegans

During female meiosis in animals, the meiotic spindle is attached to the egg cortex by one pole during anaphase to allow selective disposal of half the chromosomes in a polar body. In Caenorhabditis elegans, this anaphase spindle position is achieved sequentially through kinesin-1–dependent early translocation followed by anaphase-promoting complex (APC)-dependent spindle rotation. Partial depletion of cytoplasmic dynein heavy chain by RNA interference blocked spindle rotation without affecting early translocation. Dynein depletion also blocked the APC-dependent late translocation that occurs in kinesin-1–depleted embryos. Time-lapse imaging of green fluorescent protein-tagged dynein heavy chain as well as immunofluorescence with dynein-specific antibodies revealed that dynein starts to accumulate at spindle poles just before the initiation of rotation or late translocation. Accumulation of dynein at poles was kinesin-1 independent and APC dependent, just like dynein driven spindle movements. This represents a case of kinesin-1/dynein coordination in which these two motors of opposite polarity act sequentially and independently on a cargo to move it in the same direction.     

NudF, a nuclear migration gene in Aspergillus nidulans, is similar to the human LIS-1 gene required for neuronal migration.

During a study of the genetics of nuclear migration in the filamentous fungus Aspergillus nidulans, we cloned a gene, nudF, which is required for nuclear migration during vegetative growth as well as development. The NUDF protein level is controlled by another protein NUDC, and extra copies of the nudF gene can suppress the nudC3 mutation. nudF encodes a protein with 42% sequence identity to the human LIS-1 (Miller-Dieker lissencephaly-1) gene, which is required for proper neuronal migration during brain development. This strong similarity suggests that the LIS-1 gene product may have a function similar to that of NUDF and supports previous findings to suggest that nuclear migration may play a role in neuronal migration.     

The “8-kD” Cytoplasmic Dynein Light Chain Is Required for Nuclear Migration and for Dynein Heavy Chain Localization in Aspergillus nidulans

The heavy chain of cytoplasmic dynein is required for nuclear migration in Aspergillus nidulans and other fungi. Here we report on a new gene required for nuclear migration, nudG, which encodes a homologue of the “8-kD” cytoplasmic dynein light chain (CDLC). We demonstrate that the temperature sensitive nudG8 mutation inhibits nuclear migration and growth at restrictive temperature. This mutation also inhibits asexual and sexual sporulation, decreases the intracellular concentration of the nudG CDLC protein and causes the cytoplasmic dynein heavy chain to be absent from the mycelial tip, where it is normally located in wild-type mycelia. Coimmunoprecipitation experiments with antibodies against the cytoplasmic dynein heavy chain (CDHC) and the nudG CDLC demonstrated that some fraction of the cytoplasmic dynein light chain is in a protein complex with the CDHC. Sucrose gradient sedimentation analysis, however, showed that not all of the NUDG protein is complexed with the heavy chain. A double mutant carrying a cytoplasmic dynein heavy chain deletion plus a temperature-sensitive nudG mutation grew no more slowly at restrictive temperature than a strain with only the CDHC deletion. This result demonstrates that the effect of the nudG mutation on nuclear migration and growth is mediated through an interaction with the CDHC rather than with some other molecule (e.g., myosin-V) with which the 8-kD CDLC might theoretically interact.     

Interaction mapping of a dynein heavy chain. Identification of dimerization and intermediate-chain binding domains.

Cytoplasmic dynein is a multisubunit microtubule-based motor protein that is involved in several eukaryotic cell motilities. Two dynein heavy chains each form a motor domain that connects to a common cargo-binding tail. Although this tail domain is composed of multiple polypeptides, subunit organization within this region is poorly understood. Here we present an in vitro dissection of the tail-forming region of the dynein heavy chain from Dictyostelium. Our work identifies a sequence important for dimerization and for binding the dynein intermediate chain. The core of this motif localizes within an approximately 150-amino acid region that is strongly conserved among other cytoplasmic dyneins. This level of conservation does not extend to the axonemal dynein heavy chains, suggesting functional differences between the two. Dimerization appears to occur through a different mechanism than the heavy chain-intermediate chain interaction. We corroborate the in vitro interactions with in vivo expression of heavy chain fragments in Dictyostelium. Fragments lacking the interaction domain express well, without an obvious phenotype. On the other hand, the region crucial for both interactions appears to be lethal when overexpressed.     

The dynein heavy chain family

Dynein is the large molecular motor that translocates to the (-) ends of microtubules. Dynein was first isolated from Tetrahymena cilia four decades ago. The analysis of the primary structure of the dynein heavy chain and the discovery that many organisms express multiple dynein heavy chains have led to two insights. One, dynein, whose motor domain comprises six AAA modules and two potential mechanical levers, generates movement by a mechanism that is fundamentally different than that which underlies the motion of myosin and kinesin. And two, organisms with cilia or flagella express approximately 14 different dynein heavy chain genes, each gene encodes a distinct dynein protein isoform, and each isoform appears to be functionally specialized. Sequence comparisons demonstrate that functionally equivalent isoforms of dynein heavy chains are well conserved across species. Alignments of portions of the motor domain result in seven clusters: (i) cytoplasmic dynein Dyhl; (ii) cytoplasmic dynein Dyh2; (iii) axonemal outer arm dynein alpha; (iv) outer arm dyneins beta and gamma; (v) inner arm dynein 1alpha; (vi) inner arm dynein 1beta; and (vii) a group of apparently single-headed inner arm dyneins. Some of the dynein groups contained more than one representative from a single organism, suggesting that these may be tissue-specific variants.