ase1Δ cells have disorganized interphase microtubules and fail to organize microtubule overlapping regions. (A) Wild-type (PT47) and ase1Δ (PT311) cells expressing GFP-tub1p. 3D projection images are rotated 90° for cross-sectional view of cells. Wild-type cells have discreet linear bundles of microtubules with medial regions of overlaps aligned parallel to the cell long axis. ase1Δ cells have bundles of microtubules with no apparent medial regions of overlaps and that are not parallel to the cell axis. Bar, 5 μm. (B) Histogram of the number of microtubule bundles per wild-type (green) and ase1Δ cells (red; n = 100). (C) Intensity scans of individual microtubule bundles 1, 2, and 3 taken from A. Comparisons of wild-type microtubule intensity (green) to ase1Δ microtubule intensity (red). ase1Δ cells do not have a medial region of higher fluorescence intensity, consistent with not having medial regions of microtubule overlaps. (D) 3D projection time-lapse images of bilaterally symmetric microtubule organization in wild-type and ase1Δ inter-phase cells. Each 5-min interval produces a complete turnover of microtubule organization. (E) Plot of changes in microtubule symmetry during interphase. Wild-type cells (green) have microtubule bundles that are bilaterally symmetric, leading to a symmetry ratio of 1. ase1Δ cells (red) do not have bilaterally symmetric microtubule bundles, leading to wide fluctuations and deviation from the symmetric ratio of 1.

Microtubule growth and shrinkage dynamics is similar in wild-type and ase1Δ cells. (A) Time-lapse inverted-intensity images of wild-type (PT47) and ase1Δ (PT311) cells expressing GFPtub1p. The wild-type cell shows a microtubule bundle organized in a bilaterally symmetric manner with medial stable region SPB and iMTOC at the cell center and dynamic distal microtubule ends facing the cell tips (dark and light green arrows). Each microtubule end grows and shrinks independently, but still remain connected at the medial region (yellow asterisk). The ase1Δ cell shows two microtubules (red and orange arrows) growing at different time and different orientation. There is no stable medial region that organizes the two microtubules. Bar, 5 μm. (B) Plot of individual microtubule growth and shrinkage velocities. Data were taken from microtubules presented in A. The various color arrows are matched with A. The double-arrows color lines mark duration where the microtubule growing tips make contact with the cell wall. Numbers represent microtubule velocity of each growth and shrinkage phase in μm/min.

ase1Δ cells have defective spindle organization and elongation. (A) 3D projection time-lapse images of wild-type (PT65) and ase1Δ (PT335) mitotic cells expressing GFP-tub1p. Although the wild-type cell forms discrete bundles of single linear astral microtubules during anaphase B (45 min, yellow arrows), the ase1Δ cell forms disorganized, nonlinear astral microtubules that can separate from the SPB (35 and 41 min, red arrows). Bar, 5 μm. (B) High temporal resolution plot of the three phases of spindle elongation. The wild-type cell (green) shows smooth transitions between the different phases. The ase1Δ cell (red) shows abrupt and frequent spindle collapse, particularly during phase II, or metaphase and anaphase A. (C) Zoom image of phase II of the spindles in B, showing abrupt and frequent spindle collapse during phase II or metaphase and anaphase A in the ase1Δ (red) cell. (D) Kymographs of spindle elongation in wild-type and three ase1Δ cells. Ase1Δ cells have relatively shorter spindles. Bar, 10 μm. (E and F) Intensity scans of wild-type and ase1Δ spindles at phase III, or Anaphase B, taken from cells in D. The wild-type cell has a middle region of higher intensity, indicative of the overlapping spindle midzone. In contrast, the ase1Δ cell has no middle region of higher intensity, indicative of no spindle midzone. (G) The spindle structures of wild-type and ase1Δ cells at phase III. Wild-type cell shows stable spindle structure that has extended to the cell tips. Ase1Δ cells show abrupt and premature collapse of the spindle, leading to short spindle structures that have not reached the cell tips. Bar, 5 μm. (H) Histogram of the ratio of spindle length-to-cell length at the instant of spindle breakdown in wild-type and ase1Δ cells (Table 2).

ase1p-GFP has different mobility at interphase and mitosis. (A) Interphase and mitotic wild-type (PT306) cells expressing ase1p-GFP. A bar of ase1p-GFP is present in the interphase cell, and ase1p-GFP is present at the spindle midzone and the SPBs in the mitotic cell. The yellow dotted box shows the region of subsequent FRAP. (B) Time-lapse images of the FRAP experiments. During the time of FRAP (0 s, FRAP), almost 100% of the GFP signals are photobleached. Within 8 s of recovery, both the interphase ase1pGFP bar and the mitotic SPB-organized ase1p-GFP dot appear as robust signals. At 50 s of recovery, both the interphase ase1p-GFP bar and the SPB-organized ase1p-GFP dot have almost fully recovered their fluorescence. In contrast, the spindle midzone does not recover as effectively during these times. Bar, 5 μm. (C) Intensity plots of the FRAP experiment from B. (D) Western blot shows two forms of ase1p during mitosis (M) compared with interphase (G₂ and G₁/S), as assayed with the G₂/M cell cycle arrest mutant cdc25-22 expressing ase1p-GFP (PT403).

ase1Δ cells grow slower, are longer, and are sensitive to microtubule depolymerization drug. (A) Differential interference contrast (DIC) image of wild-type (PT286) and ase1Δ (PT309) cells. ase1Δ cells are generally longer, have nonlinear cell shape, and have septum-positioning defects compared with wild-type (yellow bars). Bar, 5 μm. (B) Doubling time of wild-type (PT286) and ase1Δ (PT309) cells under a range of temperature. Student's t statistics showed that ase1Δ cells takes longer to double than wild-type (p < 0.05). (C) Histograms of cell lengths at time of mitosis. Wild-type (PT286) cells are 13.41 ± 2.12 μm (n = 100), and ase1Δ (PT309) cells are 15.40 ± 2.18 μm (n = 100) [mean ± SD; p < 0.05]. (D) Carbendazim (MBC) sensitivity assay for wild-type (PT286) and ase1Δ (PT309) cells grown on YE5S plates with varying concentrations of MBC.

ase1p is required to stabilize and anchor microtubule overlapping regions to the nuclear membrane from MBC-induced microtubule depolymerization. (A) Wild-type (PT47) and ase1Δ (PT311) cells expressing GFP-tub1p. A flow chamber was constructed for efficient perfusion of 50 μg/ml MBC and subsequent washout with fresh media. Before MBC treatment, both wild-type and ase1Δ cells have long microtubule bundles (–1 min). Immediately after MBC treatment (0 through 5 min), microtubules initiate catastrophe and depolymerize from the distal cell tips toward the cell center (1 min). After 5 min of MBC treatment, stable remnants of microtubules are still localized to the cell center in wild-type cells (5 min). Similar treatment and duration for ase1Δ cells yielded fewer stable microtubule remnants around the cell center. On washout (6 min), microtubule repolymerized from the stable remnants at the cell center in wild-type cells. In ase1Δ cells, microtubule repolymerization can be seen in the cytoplasm far from the cell center (6 min). Bar, 5 μm. (B) Histogram of the number of stable microtubule remnants remaining per cell after 5 min of MBC treatment (n = 100). (C) High temporal resolution of microtubule repolymerization after wash-out of MBC in wild-type and ase1Δ cells expressing GFPtub1p. Immediately after MBC wash-out (0 s) the wild-type cell repolymerized its microtubules from the stable microtubule remnants constricted at the cell nucleus in the middle of the cell. In contrast, microtubule repolymerization in the ase1Δ cell occurred at the nuclear region as well as the cell cytoplasm away from the nuclear region (yellow arrows). Bar, 5 μm.

Nuclear positioning is defective in ase1Δ cells. (A) Time-lapse images of wild-type (PT104) and ase1Δ (PT337) cells expressing the SPB, iMTOC, and nuclear membrane marker GFP-sad1p. In the wild-type cell, the SPB (dark green arrow) and one iMTOC (light green arrow) are present in this optical section. They undergo microtubule-dependent oscillation within the cell. In contrast, the ase1Δ cell shows only the single SPB (red arrow), which remains relatively static, with no oscillation. Bar, 5 μm. (B) Plot of SPB and iMTOC oscillations in wild-type and ase1Δ cells. Data were taken from SPB and iMTOC presented in A. (C) DIC and merged image of wild-type and ase1Δ cells expressing GFPsad1p. The wild-type cells have linear rod-shaped cell morphology, with the nuclei located at the geometrical center of the cells. In contrast, the ase1Δ cells appear long, nonlinear, with the nucleus misplaced from the cell center (yellow arrow). Bar, 5 μm. (D) Comparison of nuclei positions in wild-type and ase1Δ cells. The septum divides the cell into two halves. Ratio of short-to-long cell halves provides a measure of nuclear and septum deviation from the geometrical center of the cell (n = 100).

ase1p colocalizes with sites of microtubule overlaps and are highly dynamic during interphase. (A) 3D projection images of wild-type (PT338) cells coexpressing CFP-tub1p (red) and ase1p-GFP (green). The merged images show that ase1p-GFP localizes to bright sites of microtubule overlaps throughout the cell cycle, i.e., microtubule overlaps associated with the iMTOCs (white arrows) during interphase, the SPBs (red arrows) and the eMTOCs (blue arrow) and with the spindle midzone (yellow arrow) during mitosis. Bar, 5 μm. (B) Histogram of the number of microtubule bundles and ase1p-GFP bars per cell during inter-phase (n = 100). (C) 3D projection time-lapse images of a wild-type (PT306) cell expressing ase1p-GFP. During interphase, ase1p-GFP forms multiple bars that exhibit assembly and disassembly dynamics. Yellow arrows highlight a newly assembled ase1p-GFP dot, which increases in length to form an ase1p-GFP bar and then subsequently disassembled (3–7 and 11–12 min). During mitosis, the interphase ase1p-GFP bars disassembled (see the transition from 19 to 25 min), and new ase1p-GFP is recruited to the spindle midzone (41 min, red arrow) and the SPBs (red asterisks). During late mitosis, ase1p-GFP bars are recruited to the site of the septum, presumably to organize the PAA microtubules (52 and 59 min, blue arrows). Bar, 5 μm. (D) High temporal resolution of interphase ase1p-GFP dynamics. The single optical section time-lapse images show a dynamic central bar (0 s, red asterisk) of ase1p-GFP. A single dot of ase1p-GFP appears (5 s, green arrows) and moves toward and incorporates into the central bar. At a later time (1060 s), the ase1p-GFP bar has moved to a new position, and a new dot of ase1p-GFP appears and moves toward the bar (1060 s, green arrow). The direction of movements of the ase1pGFP dots are from the cell tips toward the cell center or from the microtubule plus end toward the microtubule minus end. Bar, 5 μm.

A model for ase1p function in fission yeast cell cycle. (A) During interphase, the mto1p complex of proteins, which may be anchored to the nuclear membrane by sad1p, recruits γ-TuRC to the nuclear membrane. The γ-TuRC nucleates cytoplasmic microtubules. Nonphosphorylated ase1p, coupled to a minus end motor, is delivered to the microtubule minus ends, where ase1p can bundle two microtubules in minus end to minus end antiparallel manner, forming the iMTOC structures. (B) During mitosis, ase1p is phosphorylated by Cdk, and enters the nucleus where it is coupled to a plus end motor to move toward the microtubule plus ends, where it bundles microtubules in plus end to plus end antiparallel manner, forming the spindle midzone structure.