(A) Time-lapsed images of cell expressing mCherry-atb2 (tubulin) and klp9p-GFP. During interphase, prophase, metaphase and anaphase A, klp9p-GFP is located in the nucleoplasm. At the onset of anaphase B (yellow arrow heads), klp9p-GFP goes to the spindle and the spindle midzone. At the end of anaphase B, the spindle breaks down, and klp9p-GFP is transiently at the site of the presumptive contractile ring (yellow dashed circles).
(B) Kymographs of klp9p dynamics throughout the cell cycle. Mitosis in fission yeast lasts ~30 min. Anaphase B (yellow arrow heads) marks a relatively fast spindle elongation rate of ~0.7 μm/min and lasts ~12 min before spindle breakdown. Klp9p-GFP localizes to the spindle at anaphase B onset, and focuses to the spindle midzone throughout anaphase B (yellow dashed circle). Dashed white lines mark the position of the nucleus and divided nuclei. Bar, 5 μm.
(C) EGS cross-linking of recombinant His-klp9p revealed dimers and tetramers.
(D) Comparative plot of spindle length vs. time and box plot of anaphase B spindle elongation velocities of wildtype cells (wt, blue), klp9Δ cells expressing exogenous wildtype klp9 (klp9Δ+klp9^wt, green), klp9Δ cells (klp9Δ, red), and klp9Δ cells expressing exogenous klp9 motor dead mutant (klp9^md, yellow).

(A) Time-lapsed images of cells expressing either cdc13p-GFP (cyclin B, binding partner of cdc2p) or clp1p-GFP during mitosis. Prior to anaphase B, cdc13p-GFP localizes to the spindle. At anaphase B onset (yellow arrow head), cdc13p-GFP delocalizes from the spindle (yellow dashed oval). In contrast, prior to anaphase B, clp1p-GFP localizes to the spindle poles and nucleolus. At anaphase B onset (yellow arrow head), clp1p-GFP localizes to the spindle and subsequently the spindle midzone (yellow dashed oval). Bar, 5 μm.
(B) Klp9p and ase1p are phosphorylated by cdc2p in vitro. Recombinant His-klp9p and His-klp9p^{phosphoinhibit} (His-klp9p^S>A) and His-ase1p and His-ase1p^{phosphoinhibit} (His-ase1p^S>A) were incubated with cdc2p.
(C) Klp9p and ase1p are dephosphorylated by clp1p in vitro. Recombinant GST-klp9p and GST-ase1p was first phosphorylated by cdc2p. The products were next incubated with recombinant MBP-clp1p. (Asterisk * corresponds to MBP-clp1p)
(D) Ase1-phosphoinhibit version ase1p^S>A cannot enter the nucleus during closed mitosis. Shown are cells expressing mCherry-atb2 (tubulin) and GFP-tagged ase1^wt, ase1^{phosphoinhibit} (ase1^S>A), or ase1^phosphomimic (ase1^S>D). At mitosis ase1^wt and ase1^S>D localize to the spindle midzone. In contrast, ase1^S>A is not strongly visible at the spindle midzone (yellow dashed circle). Bar, 5 μm.
(E) Dual GFP- and NLS-tagged versions of ase1^wt, ase1^{phosphoinhibit} (ase1^S>A), or ase1^phosphomimic (ase1^S>D). Prominent is the strong signal of ase1^phosphomimic (ase1^S>D), which now can enter the nucleus. Arrow heads mark the spindle poles. Bar, 5 μm.

(A) Coarse kymograph of fluorescence recovery of cells expressing GFP-klp9p under the wildtype, ase1Δ, or clp1^off cell background. Note that cells also express alp4p-GFP spindle pole marker to show precise spindle length and the timing of anaphase B. Yellow boxes, bleached region. Yellow arrows, FRAP start. Bar, 5 μm.
(B) Coarse kymograph of fluorescence recovery of cells expressing ase1p-GFP under the wildtype, klp9Δ, or clp1^off cell background.
(C) Comparative plots of fluorescence recovery after bleaching. Shown are normalized fluorescent signals with pre-bleached at 100% (mean ± sd). Left panel, GFP-klp9p in wildtype (green), ase1Δ (red), and clp1^off (blue). Right panel, ase1p-GFP in wildtype (green), ase1Δ (red), and clp1^off (blue). Note the relatively higher mobility of klp9p and ase1p in the mutant backgrounds compared to wildtype.

(A) Time-lapsed images of cell expressing mCherry-atb2p (tubulin) and ase1p-GFP through different phases of mitosis. Ase1p stabilizes the spindle midzone throughout anaphase B (Loiodice et al., 2005; Yamashita et al., 2005). Bar, 5 μm.
(B) Typical wildtype spindle length vs. time plot. Anaphase B is marked by a dramatic increase in spindle length and spindle elongation velocity.
(C) Temperature shift experiment of cut7-24 mutant cells expressing GFP-atb2p. Within two minutes of shifting to the restrictive temperature of 35°C, the metaphase cell (bottom cell) immediately exhibited spindle collapse, which became a mono-polar spindle (see enlargement box) (Hagan and Yanagida, 1990; Hagan and Yanagida, 1992). In contrast, the anaphase cell (top cell) continued through anaphase B spindle elongation (yellow arrow). Bar, 5 μm.
(D) Time-lapsed images of cell expressing mCherry-atb2p (tubulin) and cut7p-3GFP. No cut7p-3GFP was observed at the anaphase B spindle midzone. Bar, 5 μm.
(E) Box plot comparison of anaphase B spindle elongation velocities in wildtype cells and motor mutant cells. Only klp9Δ shows a significant decrease in velocity. At the higher temperature required for cut7-24, spindle elongation velocity increased for both wildtype and klp9Δ correspondingly.

(A) In vivo colocalization of klp9p and ase1p at the spindle midzone during anaphase B. Time-lapsed images of cell expressing mRFP-ase1p and klp9p-GFP. Dashed yellow lines show positions of the spindle poles. The microtubule antiparallel bundler mRFP-ase1p localizes early on to the spindle (red arrow head). Subsequently, at the onset of anaphase B (yellow arrow heads), klp9p-GFP colocalizes with mRFP-ase1p at the spindle and spindle midzone. Bar, 5 μm.
(B) Kymographs of wildtype and ase1Δ cells expressing klp9p-GFP. (Anaphase B onset, yellow arrows). The wildtype cell shows a clear focusing of klp9p-GFP at the spindle midzone as anaphase B progressed. In contrast, the ase1Δ cell fails to focus klp9p-GFP at the midzone region.
(C) Kymographs of wildtype and klp9Δ cells expressing ase1p-GFP. (Anaphase B onset, yellow arrows). The wildtype cell shows a clear focusing of ase1p-GFP at the spindle midzone as anaphase B progressed. In contrast, the klp9Δ cell fails to focus ase1p-GFP at the midzone region. Bar, 5 μm.
(D) Klp9p binds to ase1p in vitro. Pull-down assays of full-length recombinant GST-klp9p with different truncated versions of recombinant His-ase1p. Schematics of klp9p and ase1p, showing multiple cyclin-dependent kinase phosphorylation sites at their carboxyl termini (red circled P). Dashed red boxes (lanes 3 and 6) indicate no physical interaction. Dashed blue boxes (lane 5) indicate physical interaction.

(A) Pull-down assays of recombinant GST-klp9p and GST-klp9p^phosphomimic (GST-klp9p^S>D) with recombinant His-ase1p and His-ase1p^phosphomimic (His-ase1p^S>D). The strongest binding is observed with GST-klp9p and His-ase1p. Bindings are also observed with either GST-klp9p or His-ase1p phosphomimic versions. No binding is observed between GST-klp9p^S>D and His-ase1p^S>D (red dashed circle).
(B) Kymographs of mutant clp1^off cells and double mutant klp9^phosphomimicase1^phosphomimic (klp9^S>Dase1^S>D) cells expressing klp9p-GFP during mitosis. Mutant clp1^off cells, which have no phosphastase activity, and double mutant klp9^S>Dase1^S>D cells show GFP-klp9p and ase1p-GFP dispersed to wider regions of the spindle. Bar, 5 μm.
(C) Quantification of klp9p and ase1p focusing at the spindle midzone. Kymograph of anaphase B shows two regions of interest: the area A₁ covered by the spindle (red boundary), and the area A₂ covered by klp9p-GFP or ase1p-GFP at the midzone (yellow boundary). We defined the ratio of the A₂/A₁ as the index of focusing. Box plot shows that klp9p-GFP and ase1p-GFP are highly focused at the spindle midzone in wildtype cells. In contrast, phospho-mutants of klp9 and ase1 and clp1^off show klp9p-GFP and ase1p-GFP spread out on the spindle.
(D) Comparative plot of spindle length vs. time and box plot of anaphase B spindle elongation velocity of wildtype cells (wt, blue), mutant clp1^off cells (clp1^off, red), and double mutant klp9 ^phosphomimicase1^phosphomimic cells (klp9^S>Dase1^S>D, yellow).

(A) Cytosim model assumption: klp9p is either parallel or antiparallel by itself, but is antiparallel when bound to ase1p. Bar, 1 μm.
(B) Parameters used in simulations (see text). Parameters of klp9p are assumed to be similar to Eg5, a similar mitotic kinesin.
(C) Comparative plot of spindle length vs. time and box plot of anaphase B spindle elongation velocity of simulated wildtype cells (wt, blue) and mutant clp1^off cells (clp1^off, red). Simulation reproduces the experimental results (compare with Fig. 4C).
(D) Comparative box plot of simulated (s) and experimental (e) spindle velocities for wildtype cells (wt, blue), clp1^off cells (clp1^off, red), and ase1Δ cells (ase1Δ, orange). Spindle velocities of ase1Δ cells are intermediate between wt and clp1^off.
(E) Comparative kymographs of simulated klp9p-GFP focusing at the middle of the spindle in wildtype and ase1Δ cells. Dashed yellow lines show positions of the spindle poles. In the absence of ase1p, klp9p is less focused at the spindle midzone, similar to experimental findings (compare with Fig. 3B and 5C). Bar, 1 μm.
(F) Model of how kinase cdc2p (Cdk1) and phosphatase clp1p (Cdc14) regulate the function of klp9p (motor) and ase1p (MAP) for proper anaphase B.