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A stable microtubule array drives fission yeast polarity reestablishment upon quiescence exit.

Laporte D, Courtout F, Pinson B, Dompierre J, Salin B, Brocard L, Sagot I - J. Cell Biol. (2015)

Bottom Line: Astonishingly, MTs are also stabilized and rearranged into a novel antiparallel bundle associated with the spindle pole body, named Q-MT bundle.Finally and importantly, we reveal that Q-MT bundle elongation is involved in polarity reestablishment upon quiescence exit and thereby the efficient return to the proliferative state.Our work demonstrates that quiescent S. pombe cells assemble specific cytoskeleton structures that improve the swiftness of the transition back to proliferation.

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Affiliation: Université de Bordeaux, Institut de Biochimie et Génétique Cellulaires, 33000 Bordeaux, France Centre National de la Recherche Scientifique, UMR5095 Bordeaux, 33077 Bordeaux, France.

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In quiescence, S. pombe assemble a unique MT bundle associated with the SPB. (A) Cells expressing GFP-Atb2 (green) and Sfi1-CFP (red) are shown (top). The variation of the number of MT bundles per cell is presented as a function of time (bottom; glucose exhaustion is marked by a red dashed line; n > 200 cells per time point). (B) The number of MTs per bundle increases upon quiescence entry (p-values are indicated; n > 100 cells per time point). (C) The Q-MT bundle is associated with the SPB (N = 2 experiments and n > 100 cells). Examples of quiescent cells (5 d) expressing GFP-Atb2 (green), Sfi1-CFP (red), and Cut11-RFP (blue). Bars, 2 µm. (D and E) The Q-MT bundle visualized longitudinally (D) and transversally (E) by freeze substitution EM in WT cells (7 d). Green arrows point at MTs, red arrowheads point at the nuclear membrane, and a blue arrow indicates the SPB. (F) Distance distribution between two MTs measured using images in E. (G) Using serial section electron tomograms, 3D models of Q-MT bundles were designed. MTs are in green, the nuclear membrane is in red, and the SPB is in blue. Two cells are shown using different view angles.
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fig1: In quiescence, S. pombe assemble a unique MT bundle associated with the SPB. (A) Cells expressing GFP-Atb2 (green) and Sfi1-CFP (red) are shown (top). The variation of the number of MT bundles per cell is presented as a function of time (bottom; glucose exhaustion is marked by a red dashed line; n > 200 cells per time point). (B) The number of MTs per bundle increases upon quiescence entry (p-values are indicated; n > 100 cells per time point). (C) The Q-MT bundle is associated with the SPB (N = 2 experiments and n > 100 cells). Examples of quiescent cells (5 d) expressing GFP-Atb2 (green), Sfi1-CFP (red), and Cut11-RFP (blue). Bars, 2 µm. (D and E) The Q-MT bundle visualized longitudinally (D) and transversally (E) by freeze substitution EM in WT cells (7 d). Green arrows point at MTs, red arrowheads point at the nuclear membrane, and a blue arrow indicates the SPB. (F) Distance distribution between two MTs measured using images in E. (G) Using serial section electron tomograms, 3D models of Q-MT bundles were designed. MTs are in green, the nuclear membrane is in red, and the SPB is in blue. Two cells are shown using different view angles.

Mentions: Upon carbon source exhaustion, fission yeast cells leave the cell cycle and enter quiescence from interphase (Bostock, 1970; Costello et al., 1986). In these conditions, we have analyzed MT organization in wild-type (WT) cells expressing the α-tubulin 2 (Atb2) fused to GFP. As expected, proliferating interphase cells displayed two to five long cytoplasmic bundles composed of 4 ± 1 MTs (Fig. 1, A and B; Höög et al., 2007). Strikingly, we observed that after carbon exhaustion, the number of MT bundles progressively decreased (Fig. 1 A). Four days after carbon exhaustion, the majority of the cells displayed a single MT bundle (Fig. 1 A and Fig. S1 A) that we named Q-MT bundle, standing for quiescent cell MT bundle. This unique MT bundle was composed of more than 15 MTs (Fig. 1 B) that were not necessarily of the same length, as exemplified by the arrow shape of the bundle extremities (Fig. S1 B). In fact, in ∼15% of the cells, the Q-MT bundle could display internal thickness variations (Fig. S1 C). Importantly, by imaging cells coexpressing GFP-Atb2 with the SPB-associated protein Sfi1 fused to CFP and the nuclear membrane protein Cut11 fused to RFP, we observed that the Q-MT bundle was generally associated with the SPB (>70% of the cells; Fig. 1, C, D, and G), even when quiescence was prolonged (Fig. S1 D). Yet, MT bundles not associated with the SPB displayed the same length and intensity than the SBP-associated ones (Fig. S1, E and F). EM analysis of quiescent WT cells showed that within the Q-MT bundle MTs were regularly spaced (Fig. 1, D–G; and Fig. S1 G). Finally, the Q-MT bundle organization and localization within the cell were confirmed by 3D models constructed using serial section electron tomograms (Fig. 1 G).


A stable microtubule array drives fission yeast polarity reestablishment upon quiescence exit.

Laporte D, Courtout F, Pinson B, Dompierre J, Salin B, Brocard L, Sagot I - J. Cell Biol. (2015)

In quiescence, S. pombe assemble a unique MT bundle associated with the SPB. (A) Cells expressing GFP-Atb2 (green) and Sfi1-CFP (red) are shown (top). The variation of the number of MT bundles per cell is presented as a function of time (bottom; glucose exhaustion is marked by a red dashed line; n > 200 cells per time point). (B) The number of MTs per bundle increases upon quiescence entry (p-values are indicated; n > 100 cells per time point). (C) The Q-MT bundle is associated with the SPB (N = 2 experiments and n > 100 cells). Examples of quiescent cells (5 d) expressing GFP-Atb2 (green), Sfi1-CFP (red), and Cut11-RFP (blue). Bars, 2 µm. (D and E) The Q-MT bundle visualized longitudinally (D) and transversally (E) by freeze substitution EM in WT cells (7 d). Green arrows point at MTs, red arrowheads point at the nuclear membrane, and a blue arrow indicates the SPB. (F) Distance distribution between two MTs measured using images in E. (G) Using serial section electron tomograms, 3D models of Q-MT bundles were designed. MTs are in green, the nuclear membrane is in red, and the SPB is in blue. Two cells are shown using different view angles.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
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fig1: In quiescence, S. pombe assemble a unique MT bundle associated with the SPB. (A) Cells expressing GFP-Atb2 (green) and Sfi1-CFP (red) are shown (top). The variation of the number of MT bundles per cell is presented as a function of time (bottom; glucose exhaustion is marked by a red dashed line; n > 200 cells per time point). (B) The number of MTs per bundle increases upon quiescence entry (p-values are indicated; n > 100 cells per time point). (C) The Q-MT bundle is associated with the SPB (N = 2 experiments and n > 100 cells). Examples of quiescent cells (5 d) expressing GFP-Atb2 (green), Sfi1-CFP (red), and Cut11-RFP (blue). Bars, 2 µm. (D and E) The Q-MT bundle visualized longitudinally (D) and transversally (E) by freeze substitution EM in WT cells (7 d). Green arrows point at MTs, red arrowheads point at the nuclear membrane, and a blue arrow indicates the SPB. (F) Distance distribution between two MTs measured using images in E. (G) Using serial section electron tomograms, 3D models of Q-MT bundles were designed. MTs are in green, the nuclear membrane is in red, and the SPB is in blue. Two cells are shown using different view angles.
Mentions: Upon carbon source exhaustion, fission yeast cells leave the cell cycle and enter quiescence from interphase (Bostock, 1970; Costello et al., 1986). In these conditions, we have analyzed MT organization in wild-type (WT) cells expressing the α-tubulin 2 (Atb2) fused to GFP. As expected, proliferating interphase cells displayed two to five long cytoplasmic bundles composed of 4 ± 1 MTs (Fig. 1, A and B; Höög et al., 2007). Strikingly, we observed that after carbon exhaustion, the number of MT bundles progressively decreased (Fig. 1 A). Four days after carbon exhaustion, the majority of the cells displayed a single MT bundle (Fig. 1 A and Fig. S1 A) that we named Q-MT bundle, standing for quiescent cell MT bundle. This unique MT bundle was composed of more than 15 MTs (Fig. 1 B) that were not necessarily of the same length, as exemplified by the arrow shape of the bundle extremities (Fig. S1 B). In fact, in ∼15% of the cells, the Q-MT bundle could display internal thickness variations (Fig. S1 C). Importantly, by imaging cells coexpressing GFP-Atb2 with the SPB-associated protein Sfi1 fused to CFP and the nuclear membrane protein Cut11 fused to RFP, we observed that the Q-MT bundle was generally associated with the SPB (>70% of the cells; Fig. 1, C, D, and G), even when quiescence was prolonged (Fig. S1 D). Yet, MT bundles not associated with the SPB displayed the same length and intensity than the SBP-associated ones (Fig. S1, E and F). EM analysis of quiescent WT cells showed that within the Q-MT bundle MTs were regularly spaced (Fig. 1, D–G; and Fig. S1 G). Finally, the Q-MT bundle organization and localization within the cell were confirmed by 3D models constructed using serial section electron tomograms (Fig. 1 G).

Bottom Line: Astonishingly, MTs are also stabilized and rearranged into a novel antiparallel bundle associated with the spindle pole body, named Q-MT bundle.Finally and importantly, we reveal that Q-MT bundle elongation is involved in polarity reestablishment upon quiescence exit and thereby the efficient return to the proliferative state.Our work demonstrates that quiescent S. pombe cells assemble specific cytoskeleton structures that improve the swiftness of the transition back to proliferation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Université de Bordeaux, Institut de Biochimie et Génétique Cellulaires, 33000 Bordeaux, France Centre National de la Recherche Scientifique, UMR5095 Bordeaux, 33077 Bordeaux, France.

Show MeSH
Related in: MedlinePlus