Characterization and localization of the cytoplasmic dynein heavy chain in Aspergillus nidulans.

Abstract

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...

Cytoplasmic Dynein Light Intermediate Chain Is Required for Discrete Aspects of Mitosis inCaenorhabditis elegans

“…Similar results were observed in Drosophila heavy chain mutants with additional observations suggesting dynein is required to maintain centrosome association with the nuclear envelope (Robinson et al, 1999). In both Aspergillus nidulans and Neurospora crassa, dynein heavy chain mutations reveal a role for the complex in nuclear migration (Plamann et al, 1994;Xiang et al, 1995). Other recent work suggests dynein may mediate microtubule binding at the kinetochore (Wordeman and Mitchison, 1995).…”

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

“…Because cytoplasmic dynein is involved in vesicle migration in other organisms, it would not be unreasonable to suppose that nudC exerts its wall effect by affecting a dynein-dependent migration of vesicles containing essential materials to the wall. This is unlikely (unless A. nidulans has an as yet uncharacterized dynein heavy chain) because the nudA disruption/deletion mutants (and all other known nud mutants) have no effect on cell shape or wall morphology (Beckwith et al, 1995b;Xiang et al, 1995b). Nor did we observe any abnormality of vesicle or vacuole distribution in the spherical AnudC cells.…”

“…We therefore have suggested they may play a regulatory role in the nuclear migration process (Xiang et al, 1995a). The cytoplasmic dynein heavy chain has been located at the growing tips of the mycelium by immunocytochemistry (Xiang et al, 1995b). We have been unable to localize the NUDC and NUDF proteins.…”

Dynein and dynactin deficiencies affect the formation and function of the Spitzenkörper and distort hyphal morphogenesis of Neurospora crassa

“…The effects of these mutations on nuclear distribution and mitosis have been previously documented for this and other fungi (Xiang et al, 1994(Xiang et al, , 1995Plamann et al, 1994 ;Tinsley et al, 1996 ;Inoue et al, 1998). Apparently, the reduction in the overall movement of intracellular components along the microtubular cytoskeleton, caused by dynein\ dynactin deficiencies, had a general effect on all cytoplasmic activities and hence led to an overall reduction in growth rate.…”