Migration of nuclei throughout the mycelium is essential for the growth and differentiation of filamentous fungi. In Aspergillus nidulans, the nudlA gene, which is involved in nuclear migration, encodes a cytoplasmic dynein heavy chain. In this paper we use antibodies to characterize the AspergiUus cytoplasmic dynein heavy chain (ACDHC) and to show that the ACDHC is concentrated at the growing tip of the fungal mycelium. We demonstrate that four temperaturesensitive mutations in the nudA gene result in a striking decrease in ACDHC protein. Cytoplasmic dynein has been implicated in nuclear division in animal cells. Because the temperature-sensitive nudA mutants are able to grow slowly with occasional nuclei found in the mycelium and are able to undergo nuclear division, we have created a deletion/disruption nudA mutation and a tightly downregulated nud4 mutation. These mutants exhibit a phenotype very similar to that of the temperature-sensitive nudA4 mutants with respect to growth, nuclear distribution, and nuclear division. This suggests that there are redundant backup motor proteins for both nuclear migration and nuclear division.Cytoplasmic dynein is a minus-end-directed, microtubuledependent motor protein that has been functionally implicated in organelle motility, mitosis in mammalian cells, and nuclear migration in fungi (1). In the filamentous fungus Aspergillus nidulans, several temperature-sensitive (ts) nuclear distribution (nud) mutants, including the nudA, nudF, and nudC mutants, have been isolated and their genes have been cloned by complementation of the mutant phenotype. nudF encodes a 49-kDa WD-repeat protein, NUDF, whose amino acid sequence is 42% identical to that of the human LIS1 protein, which is required for neuronal migration during brain development (2). nudC encodes a 22-kDa protein whose function is connected with that of the NUDF protein (2, 3). nudAl encodes a cytoplasmic dynein heavy chain (CDHC) (4). Four recessive ts mutations of the nudA gene have been identified. All four block nuclear migration into the mycelium but have no apparent effect on nuclear division. Since cytoplasmic dynein plays a role in mitosis in higher eukaryotes, why the nudA mutations have no effect on mitosis in A. nidulans poses an interesting problem. As the nud phenotype by definition specifies clusters of nuclei, the nud mutations may have been selected as mutations that affect nuclear migration but that specifically do not affect nuclear division. Thus, the possibility exists that a complete loss of nudA function might give rise to a mitotic block similar to that seen in mammalian cells after injection of dynein antibody.To address this problem and to initiate the biochemical analysis of the A. nidulans cytoplasmic dynein complex, we have made a strain (AnudA) in which the four ATP-binding sites of the heavy chain are deleted and another strain (alcA(p)::nudA) in which the only copy of the nudA gene is under the control of the inducible/repressible alcA promoter. We have generated an antibody agai...
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.
Microtubule motors, such as the minus end-directed motor, cytoplasmic dynein, play an important role in maintaining the integrity, intracellular location, and function of the Golgi apparatus, as well as in the translocation of membrane between the endoplasmic reticulum and Golgi apparatus. We have immunolocalised conventional cytoplasmic dynein heavy chain to the Golgi apparatus in cultured vertebrate cells. In addition, we present evidence that cytoplasmic dynein heavy chain cycles constitutively between the endoplasmic reticulum and Golgi apparatus: it colocalises partially with the intermediate compartment, it is found on nocodazole-induced peripheral Golgi elements and, most strikingly, on Brefeldin A-induced tubules that are moving towards microtubule plus ends. The direction of movement of membrane between the endoplasmic reticulum and Golgi apparatus is therefore unlikely to be regulated by controlling motor-membrane interactions: rather, the motors probably remain bound throughout the whole cycle, with their activity being modulated instead. We also report that the overexpression of p50/dynamitin results in the loss of cytoplasmic dynein heavy chain from the membrane of peripheral Golgi elements. These results explain how dynamitin overexpression causes the inhibition of endoplasmic reticulum-to-Golgi transport complex movement towards the centrosomal region, and support the general model that an intact dynactin complex is required for cytoplasmic dynein binding to all cargoes.
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