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.
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 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-offunction 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-enddirected 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. INTRODUCTIONIn eukaryotic cells, the microtubule cytoskeleton is essential for cell cycle progression, the establishment of cell polarity, and cell migration. In most interphase cells, microtubules are polarized in such a way that the minus ends are located at the microtubule organizing center near the nucleus while the plus ends extend to the periphery. Microtubule plus ends are very dynamic, constantly exploring the cytoplasmic space with alternate growing and shrinking phases (reviewed by Desai and Mitchison, 1997;Gundersen, 2002). Kinesin superfamily members and cytoplasmic dynein together play important roles in intracellular trafficking of various proteins, vesicles, and organelles. Because conventional kinesin is a plus-end-directed motor and dynein is a minus-end-directed motor, it is thought that conventional kinesin is responsible for moving materials from the cell body toward cell periphery, whereas cytoplasmic dynein moves materials from the cell periphery back to the cell body (reviewed by Goldstein and Yang, 2000).We have shown that cytoplasmic dynein is required for the proper distribution of nuclei within the hyphae of the filamentous fungus Aspergillus nidulans (reviewed by Morris et al., 1998b). Cytoplasmic dynein is a complex that consists of heavy chains (HCs), intermediate chains (ICs), light intermediate chains, and light chains (reviewed by King, 2000;Tynan et al., 2000). The HC contains the motor domain (reviewed by Asai and Koonce, 2001), and the other chains may target the motor to various cargoes (Steffen et al., 1997;Tai et al., 1999;Young et al., 2000). The function of cytoplasmic dynein requires dynactin (reviewed by Holleran et al., 1998;Ma et al., 1999;Roghi and Allan, 1999;King and Schroer, 2000;Kumar et al., 2000), a complex that contains multiple subunits, including the 150-kDa dynactin protein and the actin-related proteins Arp1 and Arp11 (reviewed by Holleran et al., 1998;Eckley et al., 1999). Our genetic analyses of nuclear distribution identified multiple nuclear d...
HookA is a novel linker protein that binds to endosomes and to dynein–dynactin and promotes dynein–early endosome interaction in Aspergillus.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.