S. Grava and P. Philippsen Figure 3. cMT interactions with the cell cortex generate SPB movements in A. gossypii cells. (A) Examples of 1Z plane time-lapse movies with GFP-Tub1 cells. a, cMT grows and sweeps the cortex. The SPB does not signiﬁcantly move laterally but is pushed longitudinally. Yellow arrows represent the position of the SPB at t0. b, cMT grows and pushes the SPB against the cortex. c, cMT grows and slides along the cortex resulting in a longitudinal SPB movement. a– c, dashed lines represent the uppermost positions of SPBs in the image sequences. The cMT ends are marked with white arrows. (B) A cMT grows and then slides along the cortex in a GFP-Tub1 Bik1-Cherry hypha (sliding occurs at t36–t54). Asterisk (*) shows a Bik1 dot moving along the cMT. Numbers indicate time in seconds. Bars, 5 m. CLIP-170 promotes a high MT rescue frequency and avoids persistent depolymerization when MTs reach the cell pe- riphery (Komarova et al., 2002). Deletion of BIK1 in budding yeast leads to very short cMTs (Berlin et al., 1990). ScBik1 stimulates MT dynamics by increasing the rates of growth and shrinkage and by reducing the amount of time cMTs spend in the pause state (Wolyniak et al., 2006). In ﬁlamen- tous fungi, deletions of CLIP-170 homologues have milder effects on cMTs. Aspergilus nidulans hyphae lacking the CLIP-170 homologue CLIPA still show cMTs extending to hyphal tips even though cMT dynamics is slightly deregu- lated (Eﬁmov et al., 2006). We deleted the entire open reading frame of the AgBIK1 gene in wild-type and GFP-TUB1 cells. Anti- -tubulin im- munostaining revealed that short cMTs were still present but the long cMTs observed in wild type were no longer visible in bik1 cells (Figure 4A and Table 1). The average cMT length in bik1 was 1.67 m (n 489; maximum length, 9.5 m) whereas in wild type, average cMT length was 5.64 m (n 335; maximum length, 26.5 m). To understand how AgBik1 inﬂuences cMT dynamics, we per- formed time-lapse microscopy on bik1 GFP-TUB1 hyphae. As mentioned, cMTs were difﬁcult to distinguish in GFP- Tub1 hyphae, but short and long cMTs could be occasionally 3684 tracked for 30 s. As shown in Figure 4B, in GFP-TUB1 hyphae, cMTs (white arrows) grew rapidly to reach the cortex, with which they associated. In bik1 GFP-TUB1 cells, we could only rarely follow cMTs and only for short periods. Symbols in Figure 4B mark cMTs that either depolymerized (arrowhead) or paused (asterisk) after a short growth phase (arrows). We calculated a cMT polymerization rate of 0.086 0.025 m/s (mean SD; n 17) in bik1 hyphae, which is similar to the polymerization rate of 0.108 0.035 m/s in WT hyphae (Figure 4C). Together, these data suggest that in contrast to its homo- logues in other ﬁlamentous fungi, Bik1 is crucial for the control of cMT dynamics in A. gossypii. Rather than enhanc- ing the speed of cMT polymerization, AgBik1 seems to promote the processivity of cMT polymerization and stabi- lize cMTs by avoiding too long pausing time or complete depolymerization. Long cMTs Enhance the Frequency of MT Sliding Events and Maintain SPBs and Nuclei Close to the Cell Cortex While measuring cMT dynamics, we noticed that SPBs in bik1 mutants were more frequently placed in the center of the hyphae compared with SPBs in wild-type cells. To quan- tify this, we longitudinally divided hyphae into three equal Molecular Biology of the Cell