Cytoplasmic MTs Behavior in A. gossypii Figure 2. cMT polymerization and depolymerization rates in wild-type A. gossypii cells. (A) Quantiﬁcation of cMT polymerization rate using 1Z plane movies as shown in Figure 1B (using Bik1-Cherry GFP-Tub1 cells). The measured cMTs showed a Bik1 signal at their end and a strong green dot (GFP-Tub1 at SPBs) at their end. (B) Quantiﬁcation of cMT shrinkage rate using 1Z plane movies as shown in Figure 1B. The measured cMTs showed no or a very weak Bik1 signal at their end and a strong green dot (GFP-Tub1 at SPBs) at their end. (C) Two Bik1 dots (spots 1 and 2) localizing at the end of cMTs emanating from different SPBs are merging. Their ﬂuorescence intensities are plotted in arbitrary units. Time is in seconds. Bar, 5 m. and the cell cortex. Figure 3A illustrates representative im- ages from three 1Z plane movies made with GFP-Tub1 cells (6-s time intervals). Movie a shows the end of a cMT interacting with the cortex and while growing up to 5 m long (t36), the cMT pushed the SPB against the cortex longi- tudinally (see yellow arrows). In the movie b, the cMT grew against the cortex and pushed the SPB away from it. In contrast to the movie a, the SPB was pushed laterally (see yellow line). In the movie c, one short cMT was rapidly growing to become longer than 5 m. While growing, the cMT slid along the cell cortex and pulled the SPB in the direction of its end (t36–t60, see yellow line and white arrows). Very similar to what has been observed in budding yeast, A. gossypii cMTs are pushed against, and sweep and slide along the cell cortex, generating SPBs and nuclei movements. Capture/Shrinkage Mechanisms Are Not Observed in A. gossypii In yeast, cMTs are directed to the bud neck and the bud tip. After bud emergence, cMTs are growing toward the bud and cMT tips eventually get captured by the bud neck or the bud tip. After being attached to those speciﬁc cortical sites, cMTs are shrinking, therefore pulling nuclei toward the bud tip. In A. gossypii, sites where cMT ends could possibly be captured have not yet been identiﬁed. Therefore, we focused our interest on the interactions between MT ends (Bik1-Cherry signal) and the cell cortex to check whether Bik1 dots eventually stably associate with the cell cortex. As mentioned, this was never the case. While quan- tifying MT polymerization rate (18 cMTs were analyzed for Vol. 21, November 1, 2010 23.1 min in total, with 6-s intervals), we could never observe a Bik1 dot localizing at a same site for two consecutive time frames, except for Bik1 localization at hyphal tips. Bik1 dots always moved along the cortex, suggesting that in contrast to budding yeast, cMT tips were not captured by speciﬁc cortical sites. Even though the ends of cMTs do not stably interact with speciﬁc cortical sites, we often see that cMTs maintain lateral contact along their side while sliding for- ward. Figure 3B shows an example of the sliding forces applied on a SPB by the lateral interaction of a cMT with the cell cortex: the cMT end touches the cell cortex (t36), grows and curves along it (t42) and then is strongly pulled toward the hyphal tip (t42–t54). However, in contrast to budding yeast, the interactions of the cMT with the hyphal tip do not induce its shrinkage (t54–t66). Even if this is probably indi- rectly due to its inherent property of being a small surface at the cell end, the cortex of the hyphal tip (and also the septa; see Supplemental Figure 1) could be distinguished from the rest of the cortex because it received a high number of cMT ends (Figure 1A). Together, by using Bik1 signal as a marker for MT ends, we concluded that ends can localize to any place along the hyphal cortex and then actively move along it for relatively long periods ( 2 min). No speciﬁc cortical MT capture sites seemed to exist in A. gossypii; thus, the capture/ shrinkage mechanism observed in budding yeast to trigger nuclear movements does not exist in A. gossypii. Bik1 Promotes Microtubule Growth in A. gossypii The deletion of BIK1 in budding yeast or of CLIP-170 its mammalian homologue has a drastic effect on MT dynamics. 3683