The kinases Hsl1 and Gin4 are important for proper microtubule dynamics at the bud neck. (A,B) Analysis of microtubule organization in cells lacking Hsl1 and Gin4 as in Figure 3B and C. (C) Length distribution of microtubules attached at the bud neck in wild-type and in hsl1 gin4 double-mutant cells (n = 56 in wild type and 64 in the mutant).

Nuclear-positioning defect in the absence of septin function is not owing to actin or morphological defects. (A) Actin polarity in wild-type and septin-defective cells. Cells grown at the restrictive temperature for 2.5 h were fixed with formaldehyde and stained with rhodamine-phalloidin. (B) Measurement of the bud neck width in wild-type cells and the cdc12-1 mutant grown at 33°C for 3 h (n = 78 for wild type and 89 for cdc12-1).

The septin cytoskeleton is involved in the control of nuclear positioning. (A) Micrographs of wild-type (WT) and cdc12-1 mutant cells arrested at the G₂/M transition with hydroxyurea for 3 h and stained with DAPI to visualize nuclei. Bar, 2 μm. (B) Quantification of nuclear-positioning defects in strains mutated for components of the cleavage apparatus. kar9 and dhc1 mutants are added for comparison. Cells were arrested prior to anaphase, and the frequency of mislocalized nuclei (≳1 μm away from the bud neck) was determined (n ≥ 500 for each strain, in at least two independent counts of 200–300 cells each). (C) Nuclear migration in small- and medium-budded cells of unsynchronized wild-type and cdc12-1 mutant strains. The percentage of cells with a nucleus in the vicinity of the bud neck (within 1 μm from the bud neck) is shown. The cells were shifted for 2 h to the restrictive temperature.

Role of the septins in coordination of cytoplasmic and nuclear events in cell division. (A) Role of the septins in spindle alignment. Unlike kar9 and bni1 mutants, cells lacking the septin ring are fully competent for spindle alignment. Deletion of the formin BNR1 has no effect. Results of at least two counts with a minimum of 80 cells per count. (B) Model: Astral microtubule interaction with the septin-dependent cytoskeleton participates in bringing the nucleus to the bud neck. The dynamics of microtubules at the bud neck is regulated by SDK Hsl1 and Gin4. Spindle alignment occurs through microtubule interaction with the bud cortex. Both processes are Kar9-dependent. (C) Model: The septins are involved in at least three independent processes: the recruitment of Bnr1 and Myo1, which are required for cytokinesis; the recruitment and activation of Hsl1 and Gin4; and the Kar9-dependent capture of microtubules. In turn, Hsl1 and Gin4 regulate both entry into mitosis (via repression of Swe1) and microtubule dynamics at the bud neck. Microtubule capture and shrinkage are required to position the spindle relative to the cleavage apparatus. After proper anaphase (chromosome segregation), the mitotic exit network (MEN) induces cytokinesis.

Role of septins in Kar9 function and localization. (A) Genetic interactions between cdc12-1 and mutations in genes participating in either the Dhc1 (dhc1, bik1, num1) or the Kar9 pathways (kar9, kip3) for nuclear migration. Strains of indicated genotypes were streaked out on the same plates and were incubated at either 24°C or 30°C for 3 d. See Materials and Methods for the construction of the strains. (B) Micrographs of wild-type cells expressing Kar9–GFP under the endogenous promoter. The two upper cells show Kar9 in the mother cell and at the cortex of the bud. The two lower cells have one dot each at the bud neck. Bar, 1 μm. (C) Quantification of Kar9 distribution to the mother cell, bud neck, and bud cortex in wild-type cells, cdc12-1 mutants, and gin4 hsl1 double mutants. Quantification was made in cells stained for both tubulin and Kar9. Only the Kar9 dots associated with microtubule distal tips and the cortex were counted (n = 140 for WT, 268 for cdc12-1, 110 for hsl1 gin4; average of at least two independent counts).

Function of microtubule attachment to the bud neck in nuclear migration. (A) Microtubule capture at the bud neck is associated with shrinkage of the microtubule and pulling of the spindle toward the bud neck. A wild-type cell expressing Kar9–GFP and GFP–Tub1 at endogenous levels is shown. Each frame was generated by projecting images of six focal planes taken through the cell. Frames are taken at 6-sec intervals. The line drawn through all frames marks the position of the spindle at the beginning of the contact event. An astral microtubule grows toward the bud neck (frames 1–6) and remains attached there (defined by the presence of a clear Kar9–GFP dot at the end of the microtubule) during 30 sec (until frame 10). At frames 10–11 it detaches and shrinks back to the spindle pole (frame 11–12). During the second half of the attachment period (frames 6–10), the microtubule shrinks and pulls the spindle toward the bud neck. Bar, 1 μm. (B) Dynamics of microtubules contacting either the mother or the bud cortex. The fraction of time spent in either growth or shrinkage is shown depending on the cortical domain contacted (mother cell or bud neck). (C) Effect of microtubule attachments on spindle movement, depending on the attachment site. The position of the spindle relative to the bud neck was compared at the beginning and at the end of capture events (n = 89 at bud neck, 40 at bud cortex, 43 at mother cortex).

Astral microtubules establish septin-dependent interactions with the bud neck. (A) Kar9 dots localize at the plus-end of microtubules. Cells expressing Tub1–GFP, Tub1–GFP Kar9–GFP, and Tub1–CFP Kar9–YFP are shown. Both tagged proteins were expressed at endogenous levels. No dot can be seen at the plus-end of microtubules when Kar9 is not tagged with GFP. Double staining shows that these dots correspond to Kar9 dots. Thus, staining of Tub1 and Kar9 both with GFP permits us to visualize microtubule interaction with the cortex. (B) Micrographs of wild-type and mutant cells (genotypes are indicated) expressing GFP–Tub1 and Kar9–GFP at endogenous levels. The spindle is prominent, and astral microtubules are fainter and emanate from the spindle poles (see drawing in the case of wild type). A Kar9 dot (marked with a red arrow) can be seen at the tip of many astral microtubules. Bar, 1 μm. (C) Quantification of microtubule organization in cells with a small spindle. The cells were sorted within three categories (see drawing): (1) no astral microtubule touching either the bud neck or the bud cortex, (2) cells with astral microtubules touching the bud neck cortex, (3) cells with astral microtubules touching the bud cortex. Cells that have astral microtubules touching both the bud neck and the bud cortex were rare (<3%) and are excluded from the graph. Results of at least five independent counts per strain, with at least 100 cells per count. (D) Analysis of astral microtubule organization (as in B) in wild-type and cdc12-1 mutant cells that express only GFP–Tub1. The cdc12-1 and wild-type control cells were shifted to 33°C for 3 h.