Figures and data
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The formation of the Q-nMT bundle is a three-step process.
(A) Nuclear MT length in WT cells expressing mTQZ-Tub1, before (grey) or after a 15 min Noc treatment (blue) upon entry into quiescence.
(B) MT fluorescence intensity was used as a proxy of Q-nMT bundle width in WT cells expressing mTQZ-Tub1; thin line: intensity of an individual cell; bold line: mean intensity; n>60 for each phase. Mean intensity measurement for half pre-anaphase mitotic spindles (purple) phase I (green), phase II (orange) or phase III (red) Q-nMT bundle. To help comparison, in each graph, the dash line indicate the mean intensity in half pre-anaphase mitotic spindle. Representative cells are shown (images in pseudo-colors).
(C) Morphometric Q-nMT bundle property distribution (length and width) in each phase before and after a 15 min Noc treatment in WT cells expressing mTQZ-Tub1.
(D) Single WT cells expressing mTQZ-Tub1 (red) and Nuf2-GFP (green) in phase II (23h) or phase III (50h) were deposited on an agarose pad containing Noc and imaged. Blue arrowheads: SPB; white arrows: Nuf2-GFP clusters; time is in min after pad deposition.
(E) Tub4-mTQZ fluorescence intensity measured at the SPB upon entry into quiescence. Representative cells are shown (images in pseudo-colors). Bar is 1µm
(F) WT cells expressing mTQZ-Tub1 (red) under the TUB1 promoter and mRuby-Tub1 (green) under the ADH2 promoter. Representative cells at the indicated time after glucose exhaustion and the associated line-scans are show. The graph shows the percentage of cells harboring both mTQZ and mRuby fluorescence along the Q-nMT bundle. Each circle is the percentage obtained for an independent cell culture, n>200).
(G) Schematic of the Q-nMT bundle formation. During phase I, stable MTs (green) elongate from the SPB (in grey). During phase II, new MTs (orange) elongated from the SPB and are stabilized along the phase I MTs, but stay dynamic when longer than phase I MTs. In the meantime, Tub4 (blue) increases at the SPB (grey). After phase III, all MTs are stable (red).
(H) Upon glucose exhaustion, WT cells expressing mTQZ-Tub1 (green) and Nuf2-GFP (red) were pulsed treated with Noc (blue) or DMSO (grey) for 24 h. Noc or DMSO were then chased using carbon exhausted medium and cells were imaged. Representative images of cells 2 d after the chase are shown. Right panel: same experiment done in WT prototroph cells and representative images of cells 4 d after the chase are shown. Tubulin (green) was detected by immunofluorescence, actin (red) by phalloidin and DNA (blue) with DAPI. The mean Q-nMT bundle length (±SD) in the population is indicated.
In A, C, E an H, each circle corresponds to a single cell. In A, E, F and H mean and SD are shown, and unpaired T-test p-values are indicated; ***: p-value<10-5. In A, D, F and H bar is 2µm.
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Q-nMT bundle formation is influenced by the alpha-tubulin amount and isoform
(A) WT cell (4 d) expressing either Tub3-3GFP (green) and mTQZ-Tub1 (red, top panel) or Tub3-RFP (red) and mWasabi-Tub1 (green, bottom panel). Blue arrowheads point to SPB.
(B) Nuclear MT length in WT and tub3Δ cells expressing mTQZ-Tub1, 36 h (phase II, yellow) and 90 h (phase III, red) after glucose exhaustion, treated 15 min (blue) or not (grey) with Noc. Representative images are shown on the right.
(C) Nuclear MT length in WT and Tub1-only cells expressing mTQZ-Tub1, 36 h (phase II, yellow) and 90 h (phase III, red) after glucose exhaustion, treated 15 min or not with Noc. Representative images are shown on top.
(D) Fluorescence intensity along the Q-nMT bundle in WT (grey) and Tub1-only (blue) cells expressing mTQZ-Tub1 grown for 10 h (phase I), 36 h (phase II), and 90 h (phase III). Thin lines indicate individual cells fluorescence intensity and bold lines, the mean intensity. Dashed lines indicate the maximal mean fluorescence intensity measured in phase I.
In B and C, each circle corresponds to a single cell. MT mean length, SD, and unpaired T-test p-value are indicated. In all panels, bar is 2µm.
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Kinetochore-kinetochore interactions are required for Q-nMT bundle formation.
(A) Nuclear MT length distribution in WT (grey) and ndc80-1 (violet) cells expressing mTQZ-Tub1 (green) and Nuf2-GFP (red), shifted to 37 °C upon glucose exhaustion for the indicated time and imaged after a 20 min Noc treatment. Representative cells shifted for 12 h at 37°C are shown.
(B) WT (grey) and ipl1-1 cells (pink) expressing mTQZ-Tub1 were shifted upon glucose exhaustion to 37 °C for 48 h, and imaged after a 20 min Noc treatment. Representative cells are shown.
(C) WT cells (2 d) expressing mTQZ-Tub1 (red) and Bir1-GFP or Sli15-GFP (green) were imaged. Graphs show Bir1-GFP or Sli15-GFP fluorescence intensity along normalized Q-nMT bundles (plain and dash lines: mean and SD respectively).
(D) Nuclear MT length distribution in cells of the indicated genotype (4 d) expressing mTQZ-Tub1 treated or not with Noc. Representative cells are shown.
(E) WT cells expressing mTQZ-Tub1 (red) and Slk19-GFP (green) 4 h after glucose exhaustion. Blue arrowhead: SPB.
(F) Nuclear MT length distribution in cells of the indicated genotype (4 d) expressing mTQZ-Tub1 (green) and Nuf2-GFP (red) were imaged before or after Noc treatment. Representative cells are shown. White arrowheads point to Nuf2 dots.
(G) Cells of the indicated genotype (4 d) expressing mTQZ-Tub1 (green) and Nup2-RFP (red).
(H) Nuclear MT length distribution in cells of the indicated genotype (4 d) expressing mTQZ-Tub1 treated or not with Noc. Representative cells are shown.
(I) Length variation of nuclear MT bundle fragments after laser ablation (pink dash line) in cells expressing mRuby-TUB1 (red) and Dad2-GFP (green). Time is in min. Blue arrowhead: SPB, white arrowhead: cMT.
In A, B, D, F, and H, each circle corresponds to a single cell. Mean, SD, and unpaired T-test p-values are indicated (*: p-value <0.05, and ***: p-value <10-6). Bar is 2 µm except in (I) where it is 1 µm.
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Each phase of Q-nMT formation requires a specific kinesin.
(A) Images and corresponding line scans of WT cells (2 d) expressing Kar3-3GFP (green) and mTQZ-Tub1 (red).
(B) Morphometric Q-nMT bundle properties distribution in 4 d WT (grey), kar3Δ (red), vik1Δ (blue), cik1Δ cells (green) expressing mTQZ-Tub1 after Noc treatment. Blue crosses are SD. Each circle corresponds to an individual Q-nMT bundle. Representative cells are shown.
(C) Nuclear MT length distribution in WT and cin8Δ cells expressing mTQZ-Tub1 treated (dashed boxes) or not (plain boxes) with Noc.
(D) Fluorescence intensity along Q-nMT bundle in WT and cin8Δ cells expressing mTQZ-Tub1 7 h and 24 h after glucose exhaustion. Representative cells are shown.
(E) WT and cin8Δ cells expressing mEOS3.2-Tub1 were imaged using PALM (images are in pseudo-colors). Full width at half maximum (FWHM) was measured at the indicated distance from the SPB. Each line in the bottom graph corresponds to a single cell. P-value between WT and cin8Δ are indicated (unpaired T-test).
(F) WT cells (3 d) expressing Kip1-GFP (green) and mTQZ-Tub1 (red). Graphs show fluorescence intensity along normalized Q-nMT bundles (plain and dash lines: mean and SD respectively).
(G-H) Representative images and nuclear MT length distribution in WT, kip1Δ and kip3Δ cells expressing mTQZ-Tub1 treated (dashed boxes) or not (plain boxes) with Noc.
In C and H, *: p-value <0.05, and ***: p-value <10-6 (unpaired T-test). Means and SD are indicated. In A, B, D, E, F and G, bar is 2 µm.
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Q-nMT bundle disassembly always occurs before SPB separation upon quiescence exit.
(A) WT cells expressing Spc42-RFP (red) and Nuf2-GFP (green) (5 d) were re-fed on an YPDA microscope pad. Individual Q-nMT bundles were measured in cells treated with CHX (blue, Stu2-GFP) or with DMSO alone (grey, Nuf2-GFP). Each line corresponds to an individual cell. For each cell, time was set to zero at the onset of MT bundle depolymerization (black dashed line).
(B) Q-nMT bundle length (green) and fluorescence intensity at full width half maximum (FWHM - orange) were measured upon quiescence exit in WT cells (5 d) expressing mTQZ-Tub1. Representative example of shirking Q-nMT bundle is shown on the left.
(C) Cells of the indicated genotype expressing mTQZ-Tub1 were grown for 4 d, and re-fed. Q-nMT bundle length was measured at the indicated time points, 15 min after a Noc treatment.
(D) Western blot (GFP antibodies) on total protein extracts from WT cells expressing Kip3-GFP grown for the indicated time. Sac6 was used as a loading control (Sac6 antibodies).
(E) WT and kip3Δ cells (5 d) expressing Nuf2-GFP were re-fed on an YPDA microscope pad. Each line corresponds to an individual cell. For each cell, time was set to zero at the onset of SPB separation.
(F) Representative images of kip3Δ cells expressing Nuf2-GFP upon quiescence exit. White arrowheads: Q-nMT bundle extremities.
(G) Percentage of cells expressing Spc42-mRFP1 with separated SPB as a function of time upon quiescence exit.
(H) WT and kip3Δshe1Δ cells (6 d) expressing Spc42-mRFP1 (red) and mTQZ-Tub1 (green) were re-fed on a YPDA microscope pad. Percentage of cells with a single or a duplicated SPB in the presence or absence of Q-nMT bundle were scored. Representative cells are shown. Right bottom panel: actin (phalloidin staining, red) in kip3Δ she1Δ cells (6 d) expressing mTQZ-Tub1 (green) before and 1 h after quiescence exit.
In F and H, blue arrowheads: SPB. In all panels, bar is 2 µm.
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Model for Q-nMT bundle assembly
MT-kinetochore interaction and Ilp1 (Aurora B) are required for the onset of phase I. Kar3 (kinesin-14) and its regulator Cik1 are essential to initiate Q-nMT bundle elongation. Although deletion of BIM1 (EB1) has no effect, it becomes critical for phase I if kinetochore-MT interactions are already destabilized by the absence of Chromosome Passenger Complex components. Kinetochore clustering by the monopolin complex and Slk19 is needed to maintain MT bundling while phase I MTs elongate. During phase I, Tub4 (ɣ-Tubulin) accumulates at the SPB. In phase II, a second wave of MT nucleation and elongation occur. Phase II MTs are concurrently stabilized along pre-existing phase I MTs, in a Cin8 (kinesin-5)-dependent manner. Phase I and phase II MTs + end (>1 µm) remain dynamic until the full length Q-nMT bundle stabilization is reached via the action of Kip1 (kinesin-5), about 2 days after glucose exhaustion.