Cytoplasmic MTs Behavior in A. gossypii Figure 7. Continued. Using anti-tubulin staining, we also noticed that cMTs were longer in kip3 , suggesting that AgKip3 has a role in shortening or destabilizing cMTs (Figure 7, B and C, and Table 1; average MT length in WT, 5.46 m, 32.8% of cMTs 6 m; in kip3 , 6.90 m, 50.4% of cMTs 6 m). As described previously for WT and the kip2 mutant, we quantiﬁed the position of SPBs relative to the hyphal cortex in the kip3 mutant and found that 58.7% of the SPBs of kip3 cells were situated near the cell cortex. This is similar to WT (54.2% of SPBs near the cortex; Figure 4D) and con- sistent with our previous conclusion that long cMTs are required to maintain SPBs in proximity to the cortex. The most striking phenotype was the increase in nuclear bypass- ing frequencies and oscillation amplitudes in kip3 hyphae (Figure 7, D and E, and Table 2). As described above for the bik1 mutant, we quantiﬁed the nuclear movements of 35 nuclei for 30 min and observed 3 times more bypassing events in the kip3 mutant than in WT. Forward and back- ward events were, respectively, increased 1.5 and 2 times compared with WT, and the rate of consecutive forward and backward movements also increased (consecutive forward steps 1 in WT, 21.7%; in kip3 , 42.4%; consecutive back- ward steps 1 in WT, 15.8%; in kip3 , 21.7%). In addition, the distance between the ﬁrst nucleus and the hyphal tip in kip3 cells compared with WT is not signiﬁcantly changed, similarly as in bik1 and kip2 hyphae which form cMTs shorter than WT (Supplemental Figure 3B). Thus, the ob- served size differences in cMTs in these three mutants still allow to keep contact of the leading nucleus with the grow- ing tip. Vol. 21, November 1, 2010 Together, these results strongly suggest that AgKip3 is a MT depolymerase like its budding yeast homologue and that the frequency and amplitude of nuclear movements in A. gossypii are cMT dependent. Oscillation and Bypassing Events Are Mainly Due to Pulling Forces Applied on cMTs and Not Pushing Forces To test how cMT dynamics was changed in kip3 , we mea- sured cMT polymerization rate in the mutant labeled with Bik1-Cherry and GFP-Tub1. Interestingly, we observed that the cMT polymerization rate in kip3 was slightly reduced compared with WT (kip3 , 0.077 0.016 m/s; WT, 0.108 0.035 m/s; Figure 7F). Because the maximal oscillation amplitude measured for kip3 cells was 8.92 m/min, we could conclude that this event was not caused by the push of a fast-growing cMT against the cell cortex (average cMT growth rate of kip3 , 4.62 m/min; minimum, 2.90 and maximum, 6.54 m/min). Then, these fast moving nuclei in kip3 cells were most probably resulting from increased pulling forces applied on cMTs. We propose therefore a model in which nuclei bypassing and oscillatory movements are mainly dependent on a MT motor exerting pulling forces on SPBs via their cMTs. This motor (probably dynein) would be associated to the cell cortex because cMT sliding along the cortex are often visible in A. gossypii. In this model, the loading of the dynein motor on cMTs could be proportional to the length of the cMTs. The higher dynein concentration is on cMTs the faster nuclei can be pulled and oscillate. Alternatively, increasing the length of cMTs also could in- crease their chance of interacting with the cell cortex and 3689