oscillatory movements could be either the results of equal forces applied on cMTs of the same length but pulling in opposite directions or could be due to the lack of interactions between cMTs and the cell cortex. In contrast, bypassing and oscillations could occur when the forces applied on SPBs are unbalanced either due to strong differences in cMT length or to pulling of several cMTs in one direction. Differences in cMT Dynamics between Budding Yeast and A. gossypii A. gossypii hyphae can grow up to 3 m/min. As shown with Bik1-Cherry, many MTs ends emanating from at least the four to ﬁve most apical SPB and nuclei can reach the tips of the growing hyphae. This suggests that in A. gossypii cMT cytoskeleton has to adapt to these fast-growing cells and need to increase cMT growth speed consequently. Our re- sults show clear differences in cMT dynamics between bud- ding yeast and A. gossypii. Growth rate is indeed increased in A. gossypii and can partially account for differences in cMT length between the two organisms. However, the cMT length differences between yeast and A. gossypii probably not only depend on speed of polymerization or depolymer- ization but also on the length of the polymerization and depolymerization phases (or processivity). Interestingly, the frequency of cMT shrinkage seems to be very low in A. gossypii. These observations especially concern the cMTs longer than 2 m. Because of the high number of cMTs emanating from a SPB (up to 6 cMTs) and the strong ﬂuo- rescence background around it, it is difﬁcult to follow shrinkage or pause of cMTs shorter than 2 m. But why is MT polymerization rate 3 times higher in A. gossypii than in budding yeast and why are catastrophes rare events? Sev- eral recent reviews compiled the current mechanistic models for MT dynamic instability and emphasized its complexity (Hammond et al., 2008; Howard and Hyman, 2009; Slep, 2010). Dynamic instability depends on the capacity of / - tubulins to self-assemble and to hydrolyze GTP at MT tips. In budding yeast, -tubulin is encoded by two genes, TUB1 and TUB3, whereas it is only encoded by one gene in A. gossypii (AgTUB1). ScTub3 represents 10% of -tubulin in the cell and contribute to the intrinsic stability of yeast MTs in vitro by decreasing the rate of depolymerization and the frequency of catastrophe (Bode et al., 2003). On the 39 resi- dues that are different between ScTub1 and ScTub3, 26 res- idues of AgTub1 are identical to ScTub1, eight are identical to ScTub3, and ﬁve are speciﬁc to the A. gossypii protein. These changes could result in conformational changes in the tubulin dimers, modify their GTPase activity, or affect inter- actions with MT-associated proteins and could therefore explain the differences between A. gossypii and budding yeast cMT dynamics. Bik1, the Kinesin Kip2 and cMT Dynamics Previous work based on anti-tubulin staining techniques has proposed that two categories of cMTs can be distinguished in A. gossypii: short cMTs interacting with the cell cortex via their ends ( 5 m long) and 5- m-long cMTs making lateral contact with the cell cortex. These short and long cMTs also were proposed to emanate from SPBs with dif- ferent orientations: perpendicular and tangential to SPB lay- ers respectively (Lang et al., 2010a,b). Our in vivo studies of cMT dynamics using GFP-Tubulin1 and Bik1-Cherry mark- ers showed that distinction between short and long cMTs does not really exists because short cMTs can rapidly grow up to 25 m. The presence of short and long cMTs at one speciﬁc time point of the GFP-Tub1 movies [or anti-tubulin immunostaining or electron microscopy pictures in Lang et Vol. 21, November 1, 2010 Cytoplasmic MTs Behavior in A. gossypii al. (2010a)] results from cMT dynamics. However, cMTs could be distinguished according their ability to accumulate Bik1 at their tips. We observed very short cMTs ( 2 m long) that lack Bik1 at their ends and seem to move more rapidly out of the focal plane than the long cortical cMTs or quickly depolymerize (shrink within a 6-s time frame). Their tips are not necessarily interacting with the cell cortex. Moreover, even though shrinking cMTs ( 2 m long) are rarely observed, they never accumulate Bik1 at their ends. Only the cMTs showing a Bik1-Cherry signal at their tips rapidly grow longer than 5 m and can be followed for up to 2 min. Interestingly, they are in constant growing phase, their tips often grow along the cell cortex and do not show any dynamic instability. This results revealed a clear differ- ence with the budding yeast Bik1 that binds both polymer- izing and depolymerizing cMTs (Carvalho et al., 2004). The mammalian functional homologue of Bik1, CLIP-170 prefer- entially associates with growing rather than depolymerizing MTs. CLIP-170 treadmills along these MT ends to maintain the association with the extending MT tip. Those interac- tions seem to stabilize MTs by connecting them to the cell cortex (Perez et al., 1999; Slep, 2010). Similar to budding yeast, our data strongly support the idea that AgBik1 does not treadmill with the tips but is targeted to cMT ends by the kinesin Kip2. Because Kip2 is essential to bring Bik1 at cMT tips, it will be interesting to characterize Kip2 local- ization in A. gossypii and see whether the tip localization of Kip2 is also reduced or absent on shrinking cMTs. Investi- gating the mechanisms which control Bik1 and Kip2 local- ization at MT ends in budding yeast and in A. gossypii could help in the future to understand the differences in cMT length between the two organisms. CONCLUSIONS The multinucleate growth mode of A. gossypii has resulted in a unique control of cytoplasmic MT dynamics. The near to normal growth of hyphae in the absence of cMTs make A. gossypii an attractive model organism to study biological effects of mutants with altered cMT dynamics. Through our analysis of MT tips behavior and cMT– cell cortex interac- tions, we elucidated the necessity of A. gossypii to produce very long MTs for nuclear migration to compensate the lack of MT capture/shrinkage mechanisms important to position nuclei in other organisms. Our new data demonstrate evo- lutionary diversity of the control of MT dynamics and of the role of MT tips proteins and kinesins between two closely related organisms, S. cerevisiae and A. gossypii. ACKNOWLEDGMENTS We thank Shanon Seger, Eloïse Kremer, and Clara Metz for help in strain construction. We acknowledge the advice and suggestions of Sue Jaspersen and Claudia Lang. We also thank Christine Alberti-Segui for providing un- published data. This work was supported by the Swiss National Science Foundation grant 3100A0-112688 (to P. P.). REFERENCES Adames, N. R., and Cooper, J. A. (2000). Microtubule interactions with the cell cortex causing nuclear movements in Saccharomyces cerevisiae. J. Cell Biol. 149, 863–874. Ahringer, J. (2003). Control of cell polarity and mitotic spindle positioning in animal cells. Curr. Opin. Cell Biol. 15, 73–81. Akhmanova, A., and Steinmetz, M. O. (2008). Tracking the ends: a dynamic protein network controls the fate of microtubule tips. Nat. Rev. Mol. Cell Biol. 9, 309–322. 3691