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.