Nuclear migration plays an important role in the growth and development of many organisms including the multinuclear fungus Aspeus nddans. We have Identified four genes, nudA, nudC, nudF, and nudG, in which temperature-sensitive mutations affect nuclear distribution. In this report, we describe the cloning of the nudA gene by complementatlon of the mutant phenotype by using a chromosomeViI-specific cosmid library. A genomic fragment of nud4 hybridized to an mRNA of -14 kb. Sequencing analysis of nuA revealed four ATP-binding sites that are characteristic of the cytoplasmic dynein heavy chaln. The amino acid sequence of the nudA gene product shows 52% overall identity with the rat brain cytoplasmic dynein heavy chain. Our study provides in vivo evidence that dynein, a microtubule motor molecule, plays a role in the nuclear migration process.In addition to the obvious role of nuclear migration in fusion of pronuclei during fertilization (1), nuclear movement is critical for proper growth and development in both higher and lower eukaryotes. For example, intracellular nuclear migration occurs during brain epithelial development and may mediate epithelial folding (2). Nuclei migrate in muscle cells and form clusters beneath acetylcholine receptors in neuromuscular junctions (3). Nuclei also assemble into tightly packed rows in virus-induced cell syncytia (4). During early embryonic development in Drosophila melanogaster, nuclei migrate from deep within the egg to the cortex prior to cellularization (5). Nuclear movement to an asymmetric position can generate unequal daughter cells and determine cellular polarity (6-8).Although nuclear migration is of general importance in biology, little is known about the force used to generate the movement and the signals that regulate this process. We have initiated a study of nuclear movement using the multinuclear filamentous fungus Aspergillus nidulans as a model system because of its powerful genetics and the ease with which nuclear migration can be observed. In A. nidulans, nuclei migrate actively into the mycelium in a microtubuledependent fashion (9, 10). We have isolated a set of mutants in A. nidulans that are defective in nuclear migration (11). Genetic analysis of these nuclear distribution (nud) XX19 (nudA2, pyrG89, chaAl, nicA2, and/or nicB8), XX8 (nudA4, pyrG89, wA2, chaAl), XX10 (nudAS, pyrG89, wA2, chaAl), and XX24 (pabaAl, yAI) were used.Growth Media and Nuclear Staining. YAG (13) + UU (0.12% uridine and 0.12% uracil) plates were used for colony growth. For nuclear staining, 106 asexual spores (conidia) were inoculated on coverslips in a Petri dish containing 30 ml of YG (YAG without agar) + UU medium. After 7 h of incubation at 440C, the cells were fixed, and the DNA was stained with 4',6-diamidino-2-phenylindole dihydrochloride (12) and photographed using a Zeiss epifluorescence microscope.Cloning and Sequencing of nud4. The chromosome VIIIspecific cosmid library was obtained from the Fungal Genetics Stock Center (Kansas City, KS). DNA isolation and ...

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.

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The ciliate Paramecium tetraurelia presents a powerful system to define the structural basis for dynein functional diversity within a single cell. This analysis will depend on the biochemical resolution of the dynein proteins. As an important first step, the three heavy chains of the ciliary outer arm dynein of paramecium were characterized. Sucrose density gradient centrifugation in a high salt buffer separated the dynein into a 22S species, which contained the alpha and beta heavy chains, and a 12S species, which contained the gamma chain as well as the inner arm dynein heavy chains. Both the 22S and 12S species retained enzymatic latency as indicated by stimulation of MgATPase activity by 0.1% Triton X-100. An unusual ATP-independent V1-like photolysis of only the beta chain provided the basis for estimating that the beta chain contributes almost half of the 22S MgATPase activity that is susceptible to V1 photolysis. The combination of the density gradient separation of the partially dissociated dynein and the ATP-independent V1-like photolysis of only the beta chain led to the unambiguous assignment of the V1 photolytic products to the appropriate parent heavy chains. An estimate of the molecular sizes of the three heavy chains was obtained. The photolytic peptide maps, which define the ATP-binding domains, were determined for the three heavy chains.

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