The formation of the Q-nMT bundle is a three-step process

(A) Nuclear MT length in WT cells expressing mTQZ-Tub1, before (-) or after (+) a 15 min Noc treatment (30 µg/ml) upon entry into quiescence. Each circle corresponds to the length of an individual MT structure. The results for 3 independent experiments are shown (pale blue, cyan and dark blue, n > 160 for each point in each experiment). The mean and SD are shown. Student test or ANOVA (sample >2) were used to compare inter-replicates. #: p-value > 0.05, $: p-value < 0.05. A student test (t-test with two independent samples) was used to compare the results obtained with or without Noc, the indicated p-values being the highest measured among experiments. ***: p-value < 1.10−5. Images of representative cells are shown. Bar is 2 µm.

(B) MT fluorescence intensity as a proxy of MT structure width in WT cells expressing mTQZ-Tub1. Mean intensity measurement for half pre-anaphase mitotic spindles (purple), phase I (green), phase II (orange) or phase III (red) Q-nMT bundle. A line scan along the MT structure for an individual cell is shown as thin line, the mean as a bold line (n > 60 / phase), all the lines being aligned at 0,5 µm before the fluorescence intensity increase onset on the SPB side. The blue lines are results obtained after a 15 min Noc treatment (30 µg/ml). In each graph, horizontal dashed line indicates the mean intensity. Images in pseudo-colors of a representative cell for each phase are shown. Bar is 2 µm.

(C) MT bundle length as a function of MT bundle width for individual cells in each phase before and after a 15 min Noc treatment (30 µg/ml) in WT cells expressing mTQZ-Tub1. Each circle represents an individual MT structure.

(D) WT cells expressing mTQZ-Tub1 (red) and Nuf2-GFP (green) in phase II (23 h) or phase III (50 h) were deposited on an agarose pad containing 30 µg/ml Noc and imaged. Blue arrowheads: SPB; white arrowheads: Nuf2-GFP clusters. Time is in min after deposition on the pad. Bar is 1 µm.

(E) Tub4-mTQZ fluorescence intensity measured at the SPB upon entry into quiescence. Each circle represents an individual cell. The mean and SD are shown; t-tests were used to compare independent samples (N = 3, n > 150), *: 0.05 > p-value > 1.10−3, ***: p-value < 1.10−5. Images in pseudo-colors of representative cells are shown. Bar is 2 µm.

(F) WT cells expressing mTQZ-Tub1 under the TUB1 promoter and mRuby-Tub1 under the ADH2 promoter. The percentage of cells harboring both mTQZ and mRuby fluorescence along the Q-nMT bundle is shown; each circle being the percentage for an independent experiment, with n > 200 counted for each experiment. The mean and SD are shown. Images of representative cells at the indicated time after glucose exhaustion are shown. Bar is 2 µm.

(G) Schematic of the Q-nMT bundle formation. During phase I, stable MTs (phase I MT - green) elongate from the SPB (grey). During phase II, the amount of Tub4 (cyan) increases at the SPB. In the meantime, new MTs (phase II MT - orange) elongated from the SPB and are stabilized along the phase I MTs, yet their +ends remain dynamic (dashed lines). After phase III, all MTs are stabilized (red). Nuf2 is schematized as dark blue squares.

(H) Upon glucose exhaustion, WT cells expressing mTQZ-Tub1 (green) and Nuf2-GFP (red) were pulsed treated with 30 µg/ml Noc (blue) or DMSO (grey) for 24 h. Noc or DMSO were then chased using carbon exhausted medium and cells were imaged. Each circle corresponds to MT structure length in an individual cell. The mean and SD are shown (N = 3, n > 100). Images of representative cells 2 d after the chase and representative cells 4 days 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.

Q-nMT bundle formation is influenced by the alpha-tubulin amount and isotype

(A) WT cells (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, bar is 2 µm.

(B) Nuclear MT length in WT and tub3Δ cells expressing mTQZ-Tub1, 36 h (phase II, orange) and 90 h (phase III, red) after glucose exhaustion, treated 15 min (blue) or not (grey) with 30 µg/ml Noc. Each circle corresponds to the length of an individual MT structure, mean and SD are shown. ANOVA was used to compare inter-replicates (n > 200, N = 5); #: p-value > 0.05, $: p-value < 0.05. A student test (t-test with two independent samples) was used to compare (+) or (-) Noc data. The indicated p-values are the highest calculated among the 5 experiments; ***: p-value < 1.10−5. Images of representative cells are shown, bar is 2 µm.

(C) Nuclear MT length in WT and Tub1-only cells expressing mTQZ-Tub1, 36 h (phase II, orange) and 90 h (phase III, red) after glucose exhaustion, treated 15 min or not with 30 µg/ml Noc. Statistical representations are as in (B). Images of representative cells are shown, bar is 2 µm.

(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). Individual fluorescent intensity is shown as thin line, the mean as a bold line (n > 60 / phase), all the lines being aligned at 0,5 µm before the fluorescence intensity increase onset on the SPB side. Dashed lines indicate the maximal mean fluorescence intensity. Images in pseudo-color of representative cells are shown, bar is 2 µm.

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), transferred to 37 °C upon glucose exhaustion and maintained at 37 °C for the indicated time. Cells were imaged after a 20 min Noc treatment (30 µg/ml). Each circle corresponds to the length of an individual MT structure, mean and SD are shown. A student test (t-test with two independent samples) was used to compare (+) or (-) Noc samples from the same experiment (n > 250, N = 2). The indicated p-values are the highest p-values calculated among the 2 repeated experiments. ***: p-value < 1.10−5. Images of representative cells incubated 12 h at 37 °C are shown, bar is 2 µm.

(B) WT (grey) and ipl1-5as (pink) cells expressing mTQZ-Tub1 were treated for 60 h with 50 µM NA-PP1 after glucose exhaustion, and imaged after a 15 min Noc treatment (30 µg/ml). Same statistical representation as in (A); N = 2, n > 250. Images of representative cells are shown, bar is 2 µm.

(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 (see material and method section, plain and dash lines: mean and SD respectively). Images of representative cells are shown, bar is 2 µm.

(D) Nuclear MT length distribution in cells of the indicated genotype (4 d) expressing mTQZ-Tub1 treated or not with Noc (30 µg/ml). Same statistical representation as in (A); N = 3, n > 200. Images of representative cells are shown, bar is 2 µm.

(E) WT cells expressing mTQZ-Tub1 (red) and Slk19-GFP (green) 4 h after glucose exhaustion. Blue arrowhead: SPB, bar is 2 µm.

(F) Nuclear MT length distribution in cells of the indicated genotype (4 d) expressing mTQZ-Tub1 (green) and Nuf2-GFP (red) imaged before or after Noc treatment (30 µg/ml). Same statistical representation as in (A); N = 2, n > 200. Images of representative cells are shown, white arrowheads point to Nuf2-GFP dots, bar is 2 µm.

(G) Cells of the indicated genotype (4 d) expressing mTQZ-Tub1 (green) and Nup2-RFP (red); bar is 1 µm.

(H) Nuclear MT length distribution in cells of the indicated genotype (4 d) expressing mTQZ-Tub1 treated or not with Noc (30 µg/ml). Same statistical representation as in (A); N ≥ 2, n > 200. Images of representative cells are shown, bar is 2 µm.

(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. Images of representative cells are shown, blue arrowhead: SPB, white arrowhead: cMT, bar is 2 µm. Graph indicates the variation of length in released fragments (n > 120) and histograms show the % of released or SBP attached fragments that are either stable, or that shorten or grow within a 30 min period after the laser-induced breakage. Bar is 1 µm

Each phase of Q-nMT formation requires a specific kinesin

(A) Images of representative WT cells (2 d) expressing Kar3-3GFP (green) and mTQZ-Tub1 (red) and corresponding fluorescence intensity along normalized Q-nMT bundles (see material and method section), bar is 2 µm.

(B) Morphometric Q-nMT bundle properties distribution in 4 d WT (grey), kar3Δ (red), vik1Δ (blue), and cik1Δ cells (green) expressing mTQZ-Tub1 after Noc treatment (30 µg/ml) – see Sup. Fig. 4A for the control without Noc. Each circle corresponds to an individual Q-nMT bundle. Black crosses are mean and SD. Images of representative cells are shown, bar is 2 µm.

(C) Nuclear MT length distribution in WT and cin8Δ cells expressing mTQZ-Tub1 treated (+) or not (-) with 15 min Noc (30 µg/ml). Each circle corresponds to the length of an individual MT structure, mean and SD are shown. A student test (t-test with two independent samples) was used to compare (+) or (-) Noc samples from the same experiment (n > 250, N = 2). The indicated p-values are the highest p-values calculated among the 2 repeated experiments. ***: p-value < 1.10−5.

(D) Fluorescence intensity along Q-nMT bundles in WT and cin8Δ cells expressing mTQZ-Tub1 7 h and 24 h after glucose exhaustion. Thin line: intensity from an individual cell; bold line: mean intensity (n > 60 / phase). Images of representative cells are shown, bar is 2 µm.

(E) WT and cin8Δ cells expressing mEOS3.2-Tub1 were imaged using PALM. Images in pseudo-colors of representative cells are shown. Full width at half maximum (FWHM) was measured at the indicated distance from the SPB. Each line in the bottom graphs corresponds to a single cell; p-value between WT and cin8Δ are indicated (unpaired t-test). Bar is 2 µm.

(F) Images of representative 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). Bar is 2 µm, blue arrowhead: SPB.

(G) Representative images of WT, kip1Δ and kip3Δ cells expressing mTQZ-Tub1 treated (+) or not (-) with 15 min Noc (30 µg/ml), Bar is 2 µm.

(H) Nuclear MT length distribution in WT, kip1Δ and kip3Δ cells expressing mTQZ-Tub1 treated (+) or not (-) 15 min with Noc (30µg/ml). Legend is the same as in (C); n > 250; *: p-value < 1.10−3; ***: p-value < 1.10−6. MT mean length and SD are indicated.

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 DMSO (top panel, Nuf2-GFP, n=33) or with CHX (bottom panel, Stu2-GFP, n=49). Each line corresponds to an individual cell. In the upper panel, time was set to zero at the onset of MT bundle depolymerisation. In the lower panel, time was set to zero when cells were deposited on the agarose pad, bar is 2 µm.

(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 shrinking Q-nMT bundle is shown on the left, bar is 1 µm.

(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 (30 µg/ml), to remove all dynamic cMTs. Each circle corresponds to a single cell. MT mean length and SD are indicated. A student test (t-test with two independent samples) was used to compare samples from the same experiment (n > 250, N = 4). The indicated p-values are the highest p-value calculated among the 4 repeated experiments. ***: p-value < 1.10−5. MT mean length and SD are indicated. Images of representative 4-day-old cells of the indicated genotype expressing mTQZ-Tub1 before and 60 min after quiescence exit are shown, bar is 2 µm.

(D) Representative images of a kip3Δ cell expressing Nuf2-GFP upon quiescence exit. Blue and white arrowheads: SPBs and Q-nMT bundle extremities respectively, bar is 2 µm.

(E) Percentage of 6-day-old cells expressing Spc42-mRFP1 with separated SPB as a function of time upon quiescence exit (n > 200).

(F) 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 SBP with or without Q-nMT bundle or with duplicated SPBs were scored (N = 4, n > 200), SD are indicated. Images of representative cells are shown, bar is 2 µm. Right bottom panel: actin (phalloidin staining, red) in kip3Δ she1Δ cells (6 d) expressing mTQZ-Tub1 (green) before and 1 h after quiescence exit.

Model for Q-nMT bundle assembly

(a) In G1, the nucleus is in a Rabl-like configuration. (b) Upon quiescence establishment, chromosomes get condensed. MT-kinetochore interaction and Ilp1 are required for the onset of phase I. (c) Kar3 and its regulator Cik1 are essential to initiate Q-nMT bundle elongation. Although deletion of BIM1 has no effect, it becomes critical for phase I if kinetochore-MT interactions are 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. (d) 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-dependent manner. Phase I and phase II MT +ends (>1 µm) remain dynamic until the full-length Q-nMT bundle stabilization is reached via the action of Kip1, about 2 days after glucose exhaustion (e).