EUKARYOTIC CELL, Mar. 2007, p. 555–562 Vol. 6, No. 3 1535-9778/07/$08.00ϩ0 doi:10.1128/EC.00266-06 Copyright © 2007, American Society for Microbiology. All Rights Reserved. Aspergillus nidulans Dis1/XMAP215 Protein AlpA Localizes to Spindle Pole Bodies and Microtubule Plus Ends and Contributes to Growth Directionalityᰔ† Cathrin Enke,1,2‡ Nadine Zekert,1‡ Daniel Veith,1,2‡ Carolin Schaaf,1 Sven Konzack,1,2 and Reinhard Fischer1,2* University of Karlsruhe, Applied Microbiology, Hertzstrasse 16, D-76187 Karlsruhe,1 and Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Str., D-35043 Marburg,2 Germany Received 18 August 2006/Accepted 21 December 2006 The dynamics of cytoplasmic microtubules (MTs) is largely controlled by a protein complex at the MT plus end. In Schizosaccharomyces pombe and in filamentous fungi, MT plus end-associated proteins also determine growth directionality. We have characterized the Dis1/XMAP215 family protein AlpA from Aspergillus nidulans and show that it determines MT dynamics as well as hyphal morphology. Green fluorescent protein-tagged AlpA localized to MT-organizing centers (centrosomes) and to MT plus ends. The latter accumulation occurred independently of conventional kinesin or the Kip2-familiy kinesin KipA. alpA deletion strains were viable and only slightly temperature sensitive. Mitosis, nuclear migration, and nuclear positioning were not affected, but hyphae grew in curves rather than straight, which appeared to be an effect of reduced MT growth and dynamics. Microtubules (MTs) are hollow tubes which are generated C terminus. However, all of them harbor a coiled-coil region from microtubule-organizing centers, and they perform multi- instead. XMAP215 proteins have a prominent MT-stabilizing ple structural and dynamic functions in a cell. Although com- function (12). Recently, it was shown nicely in Saccharomyces prising an important part of the cell skeleton, MTs are very cerevisiae that the Dis1/XMAP215 protein Stu2 binds to tubu- dynamic structures, which assemble at one end ␣,␤-tubulin lin heterodimers and associates to the MT plus end, where it dimers, stop growth after some time, undergo a catastrophe appears to be responsible for the loading of ␣,␤-tubulin dimers event, and subsequently shrink. This dynamic instability is to the growing end (1). This activity may explain the Stu2 regulated by a number of different MT-associated proteins stabilization activity of MTs in living cells. (MAPs), one of which was discovered in Xenopus and named Besides the MT stabilization activity of Dis1/XMAP215 pro- XMAP215 (5). Similar proteins, which are meanwhile classi- teins, DdCP224, the Dictyostelium discoideum homologue, is fied in the Dis1/XMAP215 family, exist in eukaryotes from involved in MT-cortex interactions. There is evidence that this yeast to plants and humans (17). Common to all of them is contact is mediated by cortical dynein with which DdCP224 is their association with MTs and the presence of TOG domains able to physically interact (9). and HEAT repeats, which are responsible for interactions with In this paper, we have analyzed the function of the Dis1/ many different associated proteins. One MAP can interact XMAP215-like protein AlpA in Aspergillus nidulans. The pro- through its TOG domains and HEAT repeats with several tein localized at the spindle pole bodies (the fungal homo- other MAPs. The proteins were classified into three different logues of centromeres) and at MT plus ends. Interestingly, groups (17). Members of the first group have four TOG do- deletion of the gene was not lethal, although a drastic reduc- mains, including one to five HEAT repeats within each of tion of the MT array and MT dynamics was observed. Hyphae them, and a conserved C terminus. Human ch-TOG belongs to of an alpA deletion strain grew in curves, suggesting that AlpA the first group together with Xenopus XMAP215, Drosophila is involved in the determination of growth directionality. (Msps), Dictyostelium (DdCP224), and Arabidopsis (MOR1) (Fig. 1). The second group has only one known member from MATERIALS AND METHODS Caenorhabditis elegans (ZYG-9). Members of the third group Strains, plasmids, and culture conditions. Supplemented minimal and com- have only two TOG domains with several HEAT repeats and, plete media for A. nidulans were prepared as described previously, and standard in comparison to group one members, do not have a conserved strain construction procedures were as described by Hill and Ka¨fer (10). A list of A. nidulans strains used in this study is given in Table 1. Standard laboratory Escherichia coli strains (XL1-Blue) were used. Plasmids are listed in Table 2. Light and fluorescence microscopy. For live-cell imaging, cells were grown in * Corresponding author. Mailing address: University of Karlsruhe, glass-bottom dishes (World Precision Instruments, Berlin, Germany) in 4 ml of Applied Microbiology, Hertzstrasse 16, D-76187 Karlsruhe. Phone: minimal medium containing either 2% glycerol (or ethanol) or 2% glucose as a 49 721 608 4630. Fax: 49 721 608 4509. E-mail: reinhard.fischer carbon source. Medium was supplemented with pyridoxine, p-aminobenzoic @bio.uni-karlsruhe.de. acid, biotin, arginine, uracil, or uridine depending on the auxotrophy of the † Supplemental material for this article may be found at http://ec strains. Cells were incubated at room temperature for 1 to 2 days, and images .asm.org/. were captured using an Axiophot microscope (Zeiss, Jena, Germany), a Plan- ‡ These authors contributed equally. apochromatic 63ϫ or 100ϫ oil immersion objective lens, and an HBO50 Hg ᰔ Published ahead of print on 19 January 2007. lamp. Alternatively, a Zeiss AxioImager Z1 with the latest AxioVision software 555 556 ENKE ET AL. EUKARYOT.CELL 4.2) and centrifugation. From the supernatant, total DNA was precipitated with isopropanol, and the pellet was washed twice with 70% ethanol, air dried, resuspended in TE buffer, and stored at 4°C. Southern hybridizations were performed according to the DIG Application Manual for Filter Hybridization (Roche Applied Science, Technical Resources; Roche Diagnostics GmbH, Mannheim, Germany). Deletion of alpA and construction of a ⌬alpA/⌬kipA double mutant. The alpA flanking regions were amplified by PCR using genomic DNA and the primers alpA_LB_fwd (5Ј-TCAAGGGCAGAGAGGGATGCAATC-3Ј) and alpA_ LB_rev_Sfi (5Ј-CGGCCATCTAGGCCTGCGGAAGGTGGCGATG-3Ј) for the upstream region of alpA and alpA_RB_fwd_Sfi (5Ј-CGGCCTGAGTGGCCTG TACGGTCAACTTTAGG-3Ј) and alpA_RB_rev (5Ј-GAGTTCGCTAAGCTC CTCAGTGCCATC-3Ј) for the downstream region and cloned into pCR2.1- TOPO to generate pAT1 and pAT2, respectively (the Sfi restriction sites are underlined in the primer sequences). In a three-fragment ligation, the pyr4 gene from plasmid pCS1 was ligated between the two alpA-flanking regions, resulting in vector pAT3. The vector pAT3 was digested with restriction enzyme KpnI, and the linearized plasmid was transformed into the uracil/uridin-auxotrophic strain TNO2A3. Among six transformants, analyzed by PCR, five displayed homolo- gous integration of the deletion cassette at the alpA locus. As primers for the indicative PCR, we used oligonucleotides derived from the pyr4 gene: pyr4-5Ј (5Ј-GGTTGAGGAAGCAGTCGAGAGC-3Ј) and pyr4-3Ј (5Ј-CTCGAGGACG AGCCGC-3Ј) and the alpA external primers alpA_5Ј-outside (5Ј-TACCCTAA GGTCACTACG-3Ј) and alpA_3Ј-outside (5Ј-AGATGGGTGTTCCTTACG- 3Ј). Two of the ⌬alpA strains (SCS13a and SCS13b) were also analyzed by Southern blotting (data not shown). In both strains, uracil/uridine prototrophy was linked to the alpA deletion, as shown by crossing them with uracil/uridin- auxotrophic alpA wild-type strains (data not shown). To generate a ⌬alpA/⌬kipA double mutant, we crossed the kipA deletion strain SSK44 with the deletion strain of alpA (SCS13). Heterokaryon formation was forced on MM, where none of the parent strains can grow alone. Progeny strains were screened by PCR and Southern blotting for the double deletion (data not shown). Bioinformatics. Protein sequences were aligned using vector NTI software FIG. 1. AlpA belongs to the third group of the Dis1/XMAP215 (Invitrogen), MegAlign, and ClustalW software (http://www.embl-heidelberg family. (A) Two TOG domains, eight HEAT repeats, and a coiled-coil .de). TOG domains and heat repeats of AlpA were identified using “REP” from region were identified, which are common to all class three members, the ExPASy database. including S. pombe Alp14 and Dis1, which is the Dis1/XMAP215 family-founding protein, and S. cerevisiae Stu2. Members of the first group include human ch-TOG, Xenopus XMAP215, Drosophila mela- RESULTS nogaster Msps, D. discoideum DdCP224, and Arabidopsis thaliana MOR1. So far, there is only one known group two member, namely Identification of a Dis1/XMAP215 family protein in A. nidu- ZYG-9 of Caenorhabditis elegans. (B) Phylogenetic analysis of S. lans. To characterize the role of the MT plus end complex for pombe Alp14 (Sp) homologues with S. cerevisiae (Sc), A. nidulans (An), A. fumigatus (Af), and A. oryzae (Ao). Accession numbers are indi- polarized growth, we searched the A. nidulans database with cated. the Schizosaccharomyces pombe Alp14 protein sequence (4) (http://www.broad.mit.edu). The putative homologue AlpA (An5521.2) is a 96.4-kDa protein comprised of 891 amino acid (v 4.5) was used. Fluorescence was observed using standard Zeiss filter combi- residues. The open reading frame is disrupted by three short nations no. 09 (fluorescein isothiocyanate, green fluorescent protein [GFP]) and introns, 70 bp, 72 bp, and 72 bp in size. The intron-exon no. 15 (DsRed). Images were collected and analyzed with a Hamamatsu Orca borders were confirmed by reverse transcription-PCR of small ER II camera system and the Wasabi software (version 1.2) or Zeiss Axiocam cDNAs, subsequent sequencing, and comparison with the se- and AxioVision software. Time-lapse series were obtained with an automated Wasabi program that acquires series of images with 2- or 5-s intervals, 0.1- or quence of genomic DNA. Protein analysis revealed eight 0.75-s exposure time, and about 100 exposures in a sequence.