Localization of Cdc28 with astral MT plus ends is dependent on Clb4. (A) The indicated cell types with CDC28-4GFP SPC42-RFP were analyzed for Cdc28-4GFP bud neck localization (arrows). Cdc28 was strongly enriched inside the nucleus (Maekawa et al. 2003). The cartoon cells on the right show the SPB (red), Cdc28 at astral MT plus ends (green), astral MTs (thin black line), nuclear MTs (thick black line), and the nucleus (blue). (B) In 100% of Δclb4 cells (n > 100) Cdc28-4GFP failed to associate with astral MT plus ends (arrows). Bars, 5 μm.

Cdc28 has to be in an active state for efficient localization to astral microtubule plus ends. (A) CDC28-4GFP and CDC28^K40L-4GFP accumulated to a similar degree. Immunoblots of yeast cell extracts with anti-GFP and anti-Tub2 antibodies. (B-E) Cdc28^K40L bound only slightly reduced to SPBs, but its association with astral MT plus ends was strongly reduced. (B) Colocalization of Cdc28-4GFP and Cdc28^K40L-4GFP with the SPB marker Spc42-RFP (asterisks). In contrast to Cdc28-GFP (arrows), Cdc28^K40L-4GFP fails to bind to the bud neck in the majority of cells. (C) Cdc28-4GFP colocalizes as a dot with astral MT plus ends (CFP-Tub1). Such a localization was observed only rarely with Cdc28^K40L-4GFP. The arrow marks the plus end of astral MTs. (D) Quantification of C. The percentage of preanaphase cells with a clear dot-like Cdc28-4GFP or Cdc28^K40L-4GFP signal at astral MT plus ends directed into the bud is shown. (E) The signal intensity of the Cdc28-4GFP or Cdc28^K40L-4GFP dots of C was quantified. Bars, 5 μm.

Kar9 directs Clb4 to SPBs. (A) Colocalization of Clb4 and Kar9 at astral MTs. Fixed CLB4-GFP KAR9-eqFP611 CFP-TUB1 cells were analyzed by fluorescence microscopy. (B) SPB localization of Clb4 is dependent on Kar9. Localization of Clb4-GFP in KAR9 SPC42-CFP and Δkar9 SPC42-CFP cells. (C) Clb4 fails to associate with astral MT plus ends in Δkar9 cells. The arrow points toward the plus end of an astral MT. (D) Kar9 localization in Δclb4 cells. Live images of preanaphase cells were examined. The three major cell types are shown. In type 1 cells, Kar9 is only associated with the budward-directed old SPB and/or astral MTs organized by this SPB. In type 2 cells, the majority of Kar9-GFP signal (>70%) is associated with the old SPB. A weaker signal is at the new SPB in the mother cell. In type 3 cells, both SPBs and associated astral MTs have an equal Kar9-GFP signal. (E) Quantification of D, n > 100. The cartoon indicates the regions that were considered as budward- and motherward-directed signals. (F) SPB localization of Kar9-GFP in mutants (S288c background) lacking two B-type cyclins. Experiment as in D and E. (G) The distribution of Kar9-GFP at SPBs, along astral MTs and at astral MT plus ends in preanaphase CLB4 and Δclb4 cells (both with KAR9-GFP CFP-TUB1). n > 100. (H) In Δclb4 cells, Kar9-GFP is frequently dispersed along the length of astral MTs. In preanaphase CLB4 KAR9-GFP CFP-TUB1 cells, Kar9 is predominately associated with the plus end of astral MTs. In preanaphase Δclb4 cells, a clear Kar9 signal is seen at astral MT plus ends. However, an additional Kar9 signal is dispersed along the length of the astral MTs. Bars, 5 μm.

Clb4 is transported from the SPB to astral MT plus ends. (A) CLB4-GFP is functional. A growth spot test of the indicated cell types with the URA3-based pRS316-ACT5 plasmid on selective plates (SC-Ura) and 5-FOA plates. 5-FOA selects for the cells that spontaneously lost the pRS316-ACT5 plasmid. The fact that Δclb4 Δact5 cells grew worse than CLB4 Δact5 and CLB4-GFP Δact5 cells indicates that the CLB4-GFP construct is functional. (B) Growth test of the indicated cell types at 23°C, 30°C, and 37°C. (C) Cell cycle-dependent localization of Clb4. Cells with CLB4-GFP SPC42-RFP in G1 (panels a,b), early S (panels c,d), S/G2 (panels e,f), and anaphase (panels g,h) were analyzed for Clb4-GFP localization. Because of the slow folding of the RFP molecule, the pre-existing old SPB is more strongly stained by RFP than the newly formed SPB (Pereira et al. 2001). The arrows point toward Clb4-GFP signals at the bud neck. Asterisks indicate Clb4 associating with the SPB. (D) Clb4 is concentrated at astral MT plus ends. Preanaphase CFP-TUB1 CLB4-GFP cells were analyzed for Clb4-GFP localization. Arrows point toward the plus end of MTs. (E) Preanaphase CLB4-GFP SPC42-CFP cells were fixed and analyzed as indicated for SPB association of Clb4. Quantification of Supplementary Figure 1B. n = 180. (F) Single-plane time-lapse analysis of CLB4-GFP SPC42-RFP cells. The merged Clb4-GFP (in green), Spc42-RFP signals (red), and phase contrast images were taken every 10 sec. Bars, 5 μm.

Kar9 anchors astral MTs to the bud cortex in the absence of Cdc28-Clb activity. (A) Cdc28-4GFP localization in SIC1^S arrested cells. Cells were released from α-factor block, and cell cycle progression was arrested by the overexpression of SIC1^{T5V,T33V,S76A}. (B) Synchronized KAR9-GFP SPC42-RFP cells were arrested in late G1 through overexpression of Gal1-SIC1-ΔNT. Kar9-GFP associated dot-like with the SPB (Spc42-RFP, asterisks), astral MT plus ends, and along astral MTs (arrows). (C) Kar9 localization of cells of B was determined by fluorescence microscopy. n > 100. (D) In pMet25-SIC1^{T5V,T33V,S76A}-HA cells, astral MT association with the bud tip is dependent on KAR9 but not BUD6. G1 cells of indicated cell types with GFP-Tub1 arrested by α-factor were released into medium lacking methionine, allowing the expression of SIC1^{T5V,T33V,S76A}. (E) SPBs were frequently pulled into the bud in SIC1^{T5V,T33V,S76A} cells. The same experiment as D. Δact5 cells were analyzed in parallel to the corresponding wild type in a separate experiment. Numbers refer to minutes after release from the α-factor block. The position of the SPB (the intersection of astral and nuclear MTs) was determined for each time point in ∼100 cells. (F) Experiment of E but the astral MT organization was scored as belonging to one of four categories (see cartoon cells). (1) Cells lacking astral MTs touching either the bud neck or the bud cortex; (2) cells with astral MTs contacting the bud neck; (3) cells with astral MT contacting the bud cortex (excluding the bud tip region); (4) cells with astral MTs contacting the bud tip. n > 100.

Clb4 regulates the binding of astral MTs with cortical domains of the bud. (A) MT organization of α-factor synchronized CLB4 and Δclb4 cells (both with GFP-TUB1). The yellow arrows in preanaphase CLB4 cells point toward astral MTs touching the bud neck. In contrast, in most Δclb4 cells, the astral MTs were directed toward the bud tip (red arrows). Shown are Z-series projections. (B) Quantification of astral MT bud cortex interactions of cells in A. The thick bar in the cartoon cells indicates the monopolar (late G1 or early S phase) or short bipolar nuclear spindle (S/G2 phase cells). The astral MTs are shown as thin lines. Astral MT bud cortex interactions were scored as in Figure 5F. An additional category was cells with both astral MT bundles directed into the bud. n > 100. (C) Analysis of astral MT interactions with the cell cortex by three-dimensional time-lapse video microscopy using an Ultraview spinning-disk confocal microscope. The position of astral MT plus ends were as outlined in the cartoon shown on the bottom of the subfigure: (a) astral MT end at or near SPB; (b) astral MT end in mother cell body; (c) astral MT end contacting the bud neck; (d) astral MT end contacting the bud cortex; (e) astral MT end contacting the bud tip. Fourteen wild-type and Δclb4 cells were analyzed >12 min each. Four representative examples are shown for each cell type. (D) Budward-directed astral MTs interact in Δclb4 cells longer with the cortex than in CLB4 wild-type cells. Duration of bud cortex interaction of cells imaged in C was determined and grouped as outlined in the figure. CLB4 wild-type cells, n = 44; Δclb4 cells, n = 35. (E) Synchronized CLB4 and Δclb4 cells expressing GFP-TUB1 were analyzed for the position of the preanaphase spindle as indicated. (F) Time-lapse analysis of Δclb4 GFP-TUB1 cells. The movie shows that the short spindle was virtually entirely pulled into the bud after 110 sec. The short spindle then reversed back through the mother-bud neck (130-150 sec). The length of the spindle remained relatively constant at ∼2 μm throughout the sequence. Supplementary Movie 3 shows another example. (G) Astral MT interactions with the bud tip in Δclb4 cells are dependent on MYO2. The indicated cell types (with GFP-TUB1) were shifted for 5 min from 23°C (t = 0 min) to 36°C (t = 5 min). (H) Logarithmically growing GFP-TUB1 cells were treated with Lat-A or DMSO for 10 min. Astral MT cell cortex interactions of fixed cells were determined by fluorescent microscopy. (I) Length of astral MTs in wild-type and Δclb4 cells after Lat-A treatment. Both cell types were treated as in H. The length of budward-directed astral MTs was determined in DMSO-treated cells (n > 60). In Lat-A-treated cells, the length of astral MTs originating from the budward-directed SPB but not contacting the bud cortex was determined (see cartoon on the left; n > 60). Bars, 5 μm.