S. Grava and P. Philippsen Figure 6. KIP2 deletion affects Bik1 localization at cMT tips. (A) Bik1 localization in WT (GFP-TUB1) and kip2 mutant. In the mutant, Bik1 accumulation is reduced or abolished at cMT tip (*) and increased at SPB (arrowheads). (B) 1Z plane movie of Bik1-Cherry GFP-Tub1 in kip2 background. Numbers indicate time in seconds. Bars, 5 m. two tubulin dimers (Varga et al., 2006; Varga et al., 2009). Here, we tested whether AgKip3 (47% identity to ScKip3) has also a role in cMT dynamics and nuclear movements in A. gossypii. We deleted the KIP3 gene in hyphae with H4- GFP–labeled nuclei and in hyphae expressing Bik1-Cherry plus GFP-Tub1. The kip3 strains grew as well as control strains on full medium and did not show sensitivity or resistance to benomyl (33–132 M; Figure 7A). However the distances between neighboring nuclei increased clearly in kip3 compared with WT cells. The category of N-N dis- tances “over 9 m” was 6 times more represented in kip3 than in WT (Supplemental Figure 3A). If AgKip3 localizes to the mitotic spindle like its budding yeast homologue, then the increase in N-N distances is probably due to a decrease of the mitotic index in kip3 (Miller et al., 1998; Gupta et al., 2006). Overall, this result suggests that the nuclear distance can be increased to a certain extend without affecting the growth rate. 3688 Figure 7. Kinesin Kip3 regulates cMT length and its absence enhances nuclear dynamics in A. gossypii. (A) kip3 mutant grows like WT. Plates were incubated for 5 d at 30°C on full medium (AFM) containing DMSO (negative control), benomyl at 33 M, or both. (B) Quan- tiﬁcation of cMT length in WT and kip3 cells by using anti-Tub1 immunostaining pictures as shown in C. Bar, 5 m. (D) Quantiﬁcation of nuclei dynamics in kip3 hyphae. Distances between the center of each nucleus and a ﬁxed point were measured for each time point. (E) Quantiﬁcation of the number of steps the 35 nuclei from Table 2 could consecutively move backward or forward during the 30-min obser- vations. (F) MT polymerization rate in kip3 mutant strain. Measurements were made on 6-s interval movies with cells expressing GFP- Tub1 and Bik1-cherry fusions (only 1Z plane was observed). cMTs were measured from their end (SPB observed with GFP-Tub1) un- til their tip (Bik1-cherry signal). Due to in- creased cMT length in kip3 strain, SPBs and tips were often in different focal planes. As- terisk (*) corresponds to the actual length of the cMTs (SPB to tip), whereas the other MTs have an underestimated size (visible part of cMT to tip). 1 time frame corresponds to 6 s. The average WT cMT polymerization rate is shown as reference (black line). Molecular Biology of the Cell