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.