long nondividing multinucleated hyphae and a particular positioning of nuclei even during mitosis is not required. Nuclear migration in A. gossypii consists of frequent and independent oscillations (short-range back-and-forth move- ments) and bypassings (1 nucleus overtakes 1 or a few nuclei) with a net nuclear movement of nuclei toward the growing hyphal tips (Alberti-Segui et al., 2001; Gladfelter and Berman, 2009; Lang et al., 2010a,b). SPBs are the only MTOCs in A. gossypii. They are embed- ded in the nuclear envelope and can nucleate up to six cMTs of variable length and variable orientation at their outer plaques (Lang et al., 2010a). A. gossypii cMTs are up to 10 times longer than budding yeast cMTs (Tirnauer et al., 1999; Lang et al., 2010a). Using microtubule (MT)-destabilizing drugs, it has been shown that the overall forward movement of nuclei does not required cMTs but is rather a cotransport of nuclei with the cytoplasmic stream, whereas nuclear oscillation and bypassing are cMT-dependent processes (Alberti-Segui et al., 2001; Lang et al., 2010a,b). However, what exactly generates those nuclear movements is still poorly understood. It has been hypothesized that intrinsic dynamics of cMTs and/or MT- motor proteins could be responsible for them. Because orthologues of all proteins involved in cMT dy- namics and the control of nuclear migration in budding yeast are conserved in A. gossypii, we were wondering why can cMTs grow so long in A. gossypii. Hyphae of A. gossypii are much longer than budding yeast cells, and one could argue that cMT length had to adapt to hyphal length to transport vesicles to the growing tips. However, this hypothesis can be excluded because, like in budding yeast, and in contrast to other ﬁlamentous fungi or mammalian cells, cMTs are not required for cell growth in A. gossypii (Alberti-Segui et al., 2001; Wendland and Walther, 2005; Kohli et al., 2008). To understand the cellular role of long cMTs in A. gossypii, we asked how does the control of cMT length inﬂuence nuclear oscillation and bypassing events. We investigated the MT polymerization and depolymerization rates and fre- quencies of cMTs and studied the role of the tip MT binding protein Bik1 and the kinesins Kip2 (Kinesin-7) and Kip3 (Kinesin-8) in cMT dynamics. We then asked whether cMTs interact with speciﬁc cortical sites and how cMTs interact with the cell cortex to induce nuclear movements. Are nuclear movements triggered by cMT pushing, pulling, sliding, or shrinking forces? The results we present here con- tribute to the understanding of how A. gossypii and budding yeast, with basically the same components, adapted nuclear migration to speciﬁc cellular needs. MATERIALS AND METHODS A. gossypii Media and Growth Conditions A. gossypii media and culturing protocols are described in Ayad-Durieux et al. (2000) and Wendland et al. (2000). Strains were grown in Ashbya full medium (AFM medium: 1% bactopeptone, 1% yeast extract, 2% glucose, and 0.1% myo- inositol) at 30°C. Transformants derived from A. gossypii reference strains were selected on AFM plates containing 200 mg/ml G418 (geneticin; ForMedium Ltd., Hunstanton, Norfolk, England) or 50 g/ml ClonNAT (Werner BioAgents, Jena, Germany). To test the effect of benomyl on growth, AFM plates were prepared by adding 33–132 M benomyl (Sigma-Aldrich, St. Louis, MO; dissolved in di- methyl sulfoxide [DMSO]) directly into the medium before pouring the plates. Strain Construction A. gossypii transformation protocols are described in Ayad-Durieux et al. (2000) and Wendland et al. (2000). All strains constructed in this study were derived from reference strains expressing either a histone H4-green ﬂuores- cent protein (GFP) fusion (ASG46 strain: ADE2-HHF1-yeGFP; Gladfelter et al., 2006) or an exogenous GFP-tubulin1 fusion (ADE2-GFP-AgTUB1; Lang et al., 2010a). A. gossypii deletion mutants were made by a polymerase chain reac- tion (PCR)-based one-step gene-targeting approach (Wendland et al., 2000). PCR were performed using standard methods with Taq polymerase from Vol. 21, November 1, 2010 Cytoplasmic MTs Behavior in A. gossypii Roche Diagnostics (Mannheim, Germany), and oligonucleotides were synthe- sized at Microsynth (Balgach, Switzerland). Oligonucleotide primers are listed in Supplemental Table S1. For gene deletions, A. gossypii cells were transformed with PCR products ampliﬁed with the pAG140 (GEN3) or pAG100 (NAT) templates and the “gene name”-del5/del3 oligonucleotide pairs, which contained 45-base pair homology upstream and downstream of the open reading frames. The pri- mary transformation produces heterokaryon cells, which have a mixture of “wild-type” (WT) and transformed nuclei. Transformed heterokaryons were veriﬁed with oligonucleotide pairs gene name-VER5/G2.2 and gene name- VER3/G3.3 for GEN3 cassettes (gene name-VER5/V2*NAT1 and gene name- VER3/V3*NAT1 for NAT cassettes; Supplemental Table 1). Three homokary- ons (obtained after sporulation of 3 independent veriﬁed transformants) were characterized for each mutant. For the Bik1-Cherry fusion, a Cherry-NAT-tagging cassette was generated by PCR using as a template the pAGT211 plasmid (Kaufmann, 2009) and the primers BIK1-DEL3 and BIK1-TAG (homologous to the 45 base pairs up- stream of the AgBIK1 stop codon). The resulting PCR product was directly transformed into GFP-Tub1 A. gossypii cells. Veriﬁcations of the heterokary- ons and homokaryons were performed as described above with the primer pairs BIK1-VER3/V3*NAT1. Immunoﬂuorescence and Time-Lapse Microscopy The microscope used was an Axioplan 2 imaging microscope equipped with the objectives Plan-Apochromat 100 1.40-numerical aperture oil differential interference contrast (DIC) and Plan-Apochromat 63 1.40-numerical aper- ture oil DIC (Carl Zeiss, Feldbach, Switzerland) and appropriate ﬁlters (Carl Zeiss and Chroma Technology, Brattleboro, VT). The light source for ﬂuores- cence microscopy was a Polychrome V monochromator (TILL Photonics, Gra¨felﬁng, Germany). Images were acquired at room temperature using a CoolSNAP HQ cooled charge-coupled device camera (Photometrics, Tucson, AZ) with MetaMorph 6.2r6 software (Molecular Devices, Sunnyvale, CA). The distance between two planes in stack acquisitions was set to 1 m for H4-GFP movies and to 0.3 m for anti-tubulin stainings. Brightness and contrast were adjusted using MetaMorph’s “scale image” command. Stacks were deconvolved with MetaMorph’s “2-D deconvolution” module and ﬂat- tened by maximum projection with the “stack arithmetic” function. The Bik1-Cherry GFP-Tub1 movies were 1Z plane movies treated with Flatten Background/Kernel/Equalize light functions. Images were colored and over- laid using MetaMorph’s “overlay images” command. Time-lapse picture series were processed as described above and converted into QuickTime MPEG-4 movies (QuickTime Player Pro; Apple Computer, Cupertino, CA). Immunoﬂuorescence stainings were performed as described previously (Ayad-Durieux et al., 2000; Gladfelter et al., 2006). Rat anti- -tubulin (YOL1/ 34; Serotec, Oxford, United Kingdom) was used at a 1:50 dilution and Alexa Fluor 568 goat anti-rat immunoglobulin G (Invitrogen, Carlsbad, CA) at a 1:200 dilution. For time-lapse acquisition, small pieces of 2-d-old mycelium were cultured on agarose slides as described previously (Lang et al., 2010a). RESULTS While Growing, A. gossypii cMTs Accumulate Bik1 at Their End It has been shown recently that cMTs in A. gossypii are all nucleated from SPB embedded in the nuclear envelope and that SPBs can nucleate up to six cMTs. Short cMTs ( 5 m) spread from SPBs in all directions and long cMTs (up to 25 m) align along the polarity axis in the direction of the hyphal tip, or opposite to it (Lang et al., 2010a). Due to the high complexity of cMT organization in multinucleated hy- phae, the low ﬂuorescence of GFP-labeled MTs and the need to restrict movies to 1Z plane for a better time resolution, it has been so far difﬁcult to carefully measure cMT dynamics in A. gossypii. cMTs apparently pausing or shrinking could actually grow or slide in another focal planes. In budding yeast, the Bik1 protein associates with ends of growing, shrinking, and pausing cMTs (Carvalho et al., 2004). We therefore investigated cMT dynamics in A. gossypii by using time-lapse microscopy with cells expressing GFP-Tub1 ( - tubulin) from an extra gene copy and Bik1-Cherry from the endogenous gene locus. This strain was fully functional because it grew with the same rate that the GFP-Tub1 strain and did not display the characteristic bik1 phenotype (see below). The amino acid sequence of AgBik1 (497 aa) shares 37.0% identity with the Bik1 protein (440 aa) of budding yeast. The N-terminal CAP-Gly MT-binding domain of 3681