Molecular Biology of the Cell Vol. 21, 3680 –3692, November 1, 2010 Dynamics of Multiple Nuclei in Ashbya gossypii Hyphae Depend on the Control of Cytoplasmic Microtubules Length by Bik1, Kip2, Kip3, and Not on a Capture/Shrinkage Mechanism Sandrine Grava and Peter Philippsen Department of Molecular Microbiology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland Submitted June 21, 2010; Revised August 19, 2010; Accepted September 2, 2010 Monitoring Editor: Kerry S. Bloom Ashbya gossypii has a budding yeast-like genome but grows exclusively as multinucleated hyphae. In contrast to budding yeast where positioning of nuclei at the bud neck is a major function of cytoplasmic microtubules (cMTs), A. gossypii nuclei are constantly in motion and positioning is not an issue. To investigate the role of cMTs in nuclear oscillation and bypassing, we constructed mutants potentially affecting cMT lengths. Hyphae lacking the plus ( )end marker Bik1 or the kinesin Kip2 cannot polymerize long cMTs and lose wild-type nuclear movements. Interestingly, hyphae lacking the kinesin Kip3 display longer cMTs concomitant with increased nuclear oscillation and bypassing. Polymerization and depolymerization rates of cMTs are 3 times higher in A. gossypii than in budding yeast and cMT catastrophes are rare. Growing cMTs slide along the hyphal cortex and exert pulling forces on nuclei. Surprisingly, a capture/shrinkage mechanism seems to be absent in A. gossypii. cMTs reaching a hyphal tip do not shrink, and cMT ends accumulate in hyphal tips. Thus, differences in cMT dynamics and length control between budding yeast and A. gossypii are key elements in the adaptation of the cMT cytoskeleton to much longer cells and much higher degrees of nuclear mobilities. INTRODUCTION Cytoplasmic microtubules (cMTs) have been shown to play an essential role in nuclear movement and positioning in many organisms, i.e., during yeast cell division, embryo development in Caenorhabditis elegans, and Drosophila melanogaster neuron development (Ahringer, 2003). Two important features are re- quired for cMTs to control nuclear movement and positioning: 1) their high dynamicity and 2) their ability to interact with the cell cortex. cMTs are intrinsically polar polymers with a slow- growing minus ( )end associated with the microtubule orga- nizing center (MTOC) and a fast-growing plus ( )end (also called tip) oriented peripherically (Desai and Mitchison, 1997; Carvalho et al., 2003). The cMT ends are highly dynamic structures that undergo phases of polymerization and depoly- merization, leading to frequent collisions with the cell cortex. The overall length of a cMT depends on the balance of growth, shrinkage, and pause phases termed dynamic instability. This balance seems to be mainly controlled by kinesin motor pro- teins and end-tracking proteins ( TIPS). This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E10 – 06 – 0527) on September 15, 2010. Address correspondence to: Peter Philippsen (peter.philippsen@ unibas.ch). Abbreviations used: cMT, cytoplasmic microtubule; SPB, spindle pole body. © 2010 S. Grava and P. Philippsen. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Com- mons License (http://creativecommons.org/licenses/by-nc-sa/3.0). 3680 Fungal and metazoan members of the kinesin-8, kinesin- 13, and kinesin-14 family have been shown to directly reg- ulate cMT dynamics by promoting their depolymerization (Moore and Wordeman, 2004; Sproul et al., 2005; Gupta et al., 2006; Varga et al., 2006). The TIPS speciﬁcally associate with the end of cMTs (Galjart and Perez, 2003). Many of them have important roles in controlling end dynamics by promoting cMT stability (Brunner and Nurse, 2000; Kom- arova et al., 2002). The mammalian TIP protein CLIP-170 and its budding yeast orthologue Bik1 are also crucial for cMT interactions with the cell cortex and for targeting of dynein at cMT ends (Coquelle et al., 2002; Sheeman et al., 2003; Carvalho et al., 2004).The tip localization of Bik1 seems to depend on the kinesin Kip2 (Carvalho et al., 2004). In budding yeast, nuclear movements have been de- scribed to be random or directed (DeZwaan et al., 1997; Shaw et al., 1997; Lee et al., 1999; Adames et al., 2000). In G1 phase, nuclei are randomly pushed in all directions by the growth of cMTs against the cortex. Then, after spindle pole body (SPB) duplication and bud emergence, nuclear move- ments become directed. cMTs that have been captured by the bud neck or the bud tip shrink to move the nucleus close to the bud neck (capture/shrinkage step). This requires a high dynamicity of cMTs (DeZwaan et al., 1997; Shaw et al., 1997; Lee et al., 1999; Kusch et al., 2002). In early anaphase, the dynein pathway triggers the sliding of cMTs along the bud cortex to pull the nucleus through the narrow bud neck (Adames and Cooper, 2000). The ﬁlamentous fungus Ashbya gossypii is an interesting model to study the evolution of the cMT cytoskeleton and nuclear migration because its genome is closely related to the budding yeast genome (Dietrich et al., 2004), but their cMT cytoskeleton and nuclear mobility are markedly differ- ent. In contrast to budding yeast, A. gossypii grows as very