Cytoplasmic MTs Behavior in A. gossypii Table 2. Quantiﬁcation of numbers and amplitude of oscillation events in WT and mutant A. gossypii strains WT bik1 kip2 kip3 Forward eventsa 164 76 75 245 Backward eventsa 98 46 32 194 Bypassing eventsa 9 0 0 28 Max. forward amplitudeb 3.75 2.6 4.14 8.92 Max. backward amplitudeb 3.06 2.6 2.63 7.17 Avg. growth speedc 0.75 0.74 0.8 0.77 a Total number of events observed during 30 min for 35 nuclei. The ﬁve front nuclei of seven different hyphae were followed for 30 min. See examples of movies in Figure 5C). Time interval was 1 min. Deﬁnitions for forward and backward are given in the Results. b Amplitudes (in micrometers per minute) indicate the maximal distance covered by a nucleus within a 1-min time interval. c This speed is an average of the growth speed of the seven hyphae used for the quantiﬁcation. the general spacing of nuclei in wild-type and mutant cells, we classiﬁed the distances between two neighboring nuclei (N-N distances) into four categories (Figure 5B). The pro- portion of 3- to 6- m N-N distances was twice lower in bik1 mutant compared with wild type and almost 20% of the N-N distances were longer than 9 m in the mutant (4% in WT). Because Bik1 also localized to the central spindle (Supplemental Figure 1), the increased N-N distances in bik1 also could result from a lower mitotic index. If an- aphases get delayed due to spindle defects, less mitotic events would occur and therefore nuclear density would decrease. Together, our results suggest that the absence of long MTs may only slightly affect the control of nuclear spacing. Bik1 Is Required for Nuclear Oscillation and Bypassing Events We have shown above that long cMTs and sliding events are abolished in bik1 mutants. Nuclear movements defects in bik1 was quantiﬁed using 1-min time interval movies with bik1 H4-GFP cells. The ﬁve nuclei closest to the hyphal tips were tracked for 30 min in seven different wild-type and bik1 hyphae (with comparable growth speeds; Table 2 and Figure 5, B and C). Because the overall nuclear migration follows the hyphal growth direction (and the cytoplasmic stream) and because the hyphal growth speed varies for each hypha, we ﬁrst had to deﬁne forward and backward events in wild type to be able to compare it with our mu- tants. During 1-min time interval, we considered as a for- ward event a step toward the hyphal tip with twice the speed of the hyphal growth speed. A backward step was stated as a step opposite to the hyphal tip with half the speed of the hyphal growth speed. The 35 wild-type nuclei performed 164 forward and 98 backward steps in 30 min. In the bik1 mutant, only 76 forward and 46 backward events were observed. As illus- trated in Figure 5, B and C (also see Table 2), not only the frequency but also the amplitude of bik1 nuclear oscilla- tions was strongly reduced compared with wild type. This lack of oscillation was not due to growth speed differences between mutant and wild type (hyphal growth speed for wild type, 0.92 m/min and for bik1 , 0.98 m/min for examples in Figure 5B; also see Table 2). In addition, no bypassing events were observed for the 35 bik1 nuclei during these 30-min movies (9 events in wild type; Table 2). In conclusion, our results conﬁrmed that long cMTs are required to generate nuclear movements such as bypassing and oscillation. However, it is still unclear whether long cMTs are directly involved in nuclear dynamics by ran- domly pushing nuclei away from the cell cortex or away Vol. 21, November 1, 2010 from each others, or whether MT motors require long cMTs for effective pulling and directed nuclear movements. Kinesin Kip2 Is Involved in the Control of cMT Length and Bik1 Localization at cMT Tips In budding yeast, Bik1 is targeted to the MT end by a kinesin-dependent transport mechanism. Bik1 forms a com- plex with the kinesin Kip2 and both comigrate along indi- vidual MTs (Carvalho et al., 2004). To determine the mech- anism of Bik1 targeting at end, we tested whether the absence of AgKip2 (42% identity with ScKip2) affected Bik1 localization in A. gossypii. Similar to the AgBIK1 deletion, the deletion of AgKIP2 did not affect the hyphal growth rate but led to a severe decrease in cMT length (Supplemental Figure 2 and Table 1). In kip2 hyphae, Bik1 accumulation at cMT ends is strongly reduced or abolished. In contrast, the Bik1-Cherry signal is strongly increased at the SPBs (Figure 6). Therefore, like in budding yeast, AgKip2 has a role in transporting AgBik1 to the end and probably also in main- taining Bik1 at cMT ends. In conclusion, the A. gossypii kinesin Kip2 is also involved in the control of MT dynamics by delivering Bik1 at MT tips. Because Kip2 has to bring Bik1 at cMT end, its velocity along cMTs has to be higher than the cMT polymerization rate (6.5 m/min). In budding yeast, the Bik1/Kip2 complex moves along the cMTs with an average speed of 6.6 m/min (Carvalho et al., 2004). Finally, consistent with its role in localizing Bik1 at the cMT ends, we showed that Kip2 is also required for nuclear dynamics. As in bik1 mutant, forward and backward events are strongly decreased in kip2 cells and bypassing events are abolished (Supplemental Figure 2 and Table 2). Deletion of the Kinesin Kip3 Increases cMT Length and Frequencies of Oscillation and Bypassing Recent work has shown that the budding yeast kinesin Kip3 has unique properties. It is both a end-directed motor and a end-speciﬁc depolymerase that is necessary to regulate the length of MTs attached to the cell cortex and to a lesser extend interpolar MTs (Gardner et al., 2008). ScKip3 is ob- served as speckles that travel toward, and accumulate at, the ends of growing MTs. However, its accumulation at MT ends is reduced or absent from depolymerizing ends (Gupta et al., 2006). Varga et al. (2006, 2009) proposed a model (the antenna model) in which Kip3 binds randomly along the length of the MT and its high processivity allows it to efﬁciently target MT ends. The longer a MT is, the higher the concentration of Kip3 will be at the end, and the higher the depolymerization rate will be. The dissociation of Kip3 from the ends is greatly accelerated by incoming Kip3 molecules and is accompanied by removal of one or 3687