S. Grava and P. Philippsen Figure 8. Amplitudes of nuclear oscillation and bypassing events depend on cMT length. Long cMTs in Agkip3 have longer lateral interactions with the cell cortex compared with WT and are sub- jected to more pulling forces (most probably dynein dependent forces, red dots). The shorter cMTs in Agbik1 (and also in Agkip2 ; not shown) rarely have lateral interactions with the cor- tex. For reasons of clarity, numbers of nuclei, cMTs, and hyphal tip-accumulated cMT ends are underrepresented in the depicted hyphae. with some speciﬁc proteins required to stimulate the pulling activity of the dynein motor (Figure 8). The clustering of nuclei in hyphal tips, reported previously for an Agdyn1 strain seems to contradict the model of this ﬁgure (Alberti- Segui et al., 2001). It is very likely that the nuclear clustering which is already observed in tips of A. gossypii germlings is caused by the extremely long cMTs, which form in the absence of dynein, thus pushing the nuclei toward the tips as documented in Alberti-Segui et al. (2001). DISCUSSION The two major goals of this work were to understand how cMT dynamics participates to nuclear movements and to investigate how cMT dynamics is controlled in the multinu- cleated A. gossypii cells. We have characterized the dynamic intracellular distribution of Bik1. Our results revealed a Kip2-dependent mechanism for targeting Bik1 to cMT ends and showed that in contrast to its budding yeast homologue, AgBik1 dissociates from shrinking cMTs. We showed that the kinesin Kip3 contributes to cMT depoly- merization and that cMT shrinkage/catastrophe are seldom events in A. gossypii. Finally, our ﬁndings showed that the overall length of cMTs is a more important factor for the control of nuclear movements than cMT dynamicity. Nuclear Movements in A. gossypii Are Not Controlled by cMT Dynamic Instability MT dynamics is determined by four parameters: the speed of MT growth, the speed of MT shrinkage, the frequency of catastrophe (transitions from growth to shrinkage phase), and the frequency of rescues (transitions from shrinkage to growth phase). MTs can also be in a pause state (Desai and Mitchison, 1997; Akhmanova and Steinmetz, 2008; van der Vaart et al., 2009). In A. gossypii, we ﬁrst hypothesized that like in other organisms, dynamic instability would occur to allow cMTs to probe the cell for speciﬁc cortical sites. In budding yeast, cMTs ensure that the mitotic spindle is prop- erly positioned relative to the cleavage apparatus. The sep- tins are required to form a cMT-capture site at the bud neck cortex, and septin-dependent kinases are suggested to in- duce cMT catastrophe and pulling forces on the spindle, which then move toward the bud neck (Kusch et al., 2002). The incipient bud site is another important site for cMTs 3690 capture followed by shrinkage (Adames and Cooper, 2000; Lee et al., 2000). The Bim1, Bud6, Bni1, and Kar9 proteins involved in the capture/shrinkage mechanism in budding yeast are all present in A. gossypii. However, our preliminary experiments have shown that the absence of Kar9 and Bim1 proteins does not signiﬁcantly affect nuclear distribution (Grava, unpublished data). This supports our interpreta- tions that in A. gossypii, cMT ends are not captured by speciﬁc cortical sites. However we cannot exclude rare events similar to capture/shrinkage observed in S. cerevisiae. Here, we also show that cMTs do not show alternated phases of growth and shrinkage. cMT ends are therefore not inducing nuclear movements via depolymerization. In A. gossypii, it is possible that the mechanism of speciﬁc cMT capture and positioning of nuclei has not been developed or has been lost during evolution because cell separation does not occur and hyphal compartments can share eight to nine nuclei. The strong nuclei and SPBs oscillations observed in hyphae are therefore probably due to MT motor-dependent sliding of cMTs along the cell cortex. Nuclear Movements in A. gossypii Require Prolonged MT Growth Phases Our time lapse microscopy of GFP-labeled MTs has pro- vided key insight into the regulation of nuclear movements in A. gossypii. On one hand, when a short cMT grows against the cell cortex, it pushes the SPB and nucleus away from the cortex and induces micromovements of SPB and nuclei. On the other hand, when a cMT grows for a longer period, prolonged lateral interaction of this cMT with the cell cortex induces its sliding along the cell cortex. These sliding events produce much more efﬁcient nuclear movements (toward the site of cortical attachment) than pushing events. The idea that cMT length is an essential factor in the control of nuclear movements is also supported by 1) the loss of oscillation and bypassing events in the bik1 and kip2 mutants, in which cMTs are much shorter than in wild-type cells; and 2) the higher frequency and amplitude of nuclear movements in the kip3 mutant, in which cMTs are longer than in WT. In budding yeast, sliding of cMTs along the cell cortex depends on a dynein-driven pulling mechanism. The “off-loading model” proposes that dynein ﬁrst accumulates at cMT tips, then gets anchored to the cortex, and ﬁnally its end stepping behavior moves the MT past the ﬁxed motor (Lee et al., 2003; Sheeman et al., 2003). If dynein is responsible for cMT pulling in A. gossypii, the nuclear oscillation differences between kip2 /bik1 and kip3 mutants could be explained in the following way: the pulling force applied on long cMTs is increased due to a higher concentration of dynein on MTs (or speciﬁcally at their ends). It is not known exactly how dynein gets loaded on budding yeast MTs. However, it has been shown that the accumulation of dynein at ends requires Bik1 and Kip2. If dynein binds at a random position on MTs and then moves toward the end via Kip2/Bik1, the longer a cMT is, the higher the concentration of Kip2/Bik1 and/or dynein could be on cMTs. Alternatively, the pulling force applied on longer cMTs could be increased due to longer lateral interactions of cMTs with the cell cortex. If the MT motor responsible for cMT sliding in A. gossypii is associated with the plasma membrane, increasing cMT length will increase the probability a cMT can interact with such a MT motor and will also increase the number of motors interacting with the cMT (Figure 8). Considering that an SPB can nucleate up to six cMTs, another concern for the future will be to understand how each SPB can coordinate the forces applied on cMTs. Non- Molecular Biology of the Cell