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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.

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.

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Q-MT bundle formation does not depend on GTP depletion but rather involves the SPB. (A) GTP concentration decreases upon quiescence entry. (B) In proliferating cells, a MPA treatment (100 µg/ml) causes a massive GTP concentration drop. Proliferating cells were incubated 30 min or 2 h with either MPA, MPA + guanine (0.3 mM) to reverse the MPA effect, or guanine alone as a control. In A and B, N = 4 experiments with two samples per experiment. (C) MPA-induced GTP droop does not affect MT dynamics in proliferating cells. MT growth and shrinkage speed were determined in proliferating WT cells expressing GFP-Atb2 treated (2 h) or not with MPA. (D and E) MT stabilization involves the SPB. (D) At various times after carbon exhaustion (left, red dashed line), cell expressing GFP-Atb2 (green) and Sfi1-CFP (red) were treated for 10 min or 8 h with benomyl and then imaged. Numbers indicate the mean number of MT bundle per cell (N = 2 experiments and n > 200 cells for each time point). (E) WT cells were grown 6 h after carbon starvation and incubated with benomyl for the indicated times (left). A cartoon (middle) representing either a MT bundle random stabilization model or model biased toward the stabilization of the SPB-associated MT bundle. Red, SPB; blue, nuclear membrane; green, MT bundle. Cells displaying multiple MT bundles (red bars) or only a single MT bundle associated with the SPB (green bars) or not (gray bars) were scored for cells incubated with benomyl for the indicated times. N = 3 experiments and n > 200 cells per time point. Error bars are SD. The percentage of cells displaying a unique MT bundle associated with the SPB calculated theoretically using a random stabilization prediction (black asterisks) or SPB-biased stabilization (blue askterisks) are indicated (see Materials and methods for details). For all graphs, means and SD are indicated. Bars, 2 µm.
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fig3: Q-MT bundle formation does not depend on GTP depletion but rather involves the SPB. (A) GTP concentration decreases upon quiescence entry. (B) In proliferating cells, a MPA treatment (100 µg/ml) causes a massive GTP concentration drop. Proliferating cells were incubated 30 min or 2 h with either MPA, MPA + guanine (0.3 mM) to reverse the MPA effect, or guanine alone as a control. In A and B, N = 4 experiments with two samples per experiment. (C) MPA-induced GTP droop does not affect MT dynamics in proliferating cells. MT growth and shrinkage speed were determined in proliferating WT cells expressing GFP-Atb2 treated (2 h) or not with MPA. (D and E) MT stabilization involves the SPB. (D) At various times after carbon exhaustion (left, red dashed line), cell expressing GFP-Atb2 (green) and Sfi1-CFP (red) were treated for 10 min or 8 h with benomyl and then imaged. Numbers indicate the mean number of MT bundle per cell (N = 2 experiments and n > 200 cells for each time point). (E) WT cells were grown 6 h after carbon starvation and incubated with benomyl for the indicated times (left). A cartoon (middle) representing either a MT bundle random stabilization model or model biased toward the stabilization of the SPB-associated MT bundle. Red, SPB; blue, nuclear membrane; green, MT bundle. Cells displaying multiple MT bundles (red bars) or only a single MT bundle associated with the SPB (green bars) or not (gray bars) were scored for cells incubated with benomyl for the indicated times. N = 3 experiments and n > 200 cells per time point. Error bars are SD. The percentage of cells displaying a unique MT bundle associated with the SPB calculated theoretically using a random stabilization prediction (black asterisks) or SPB-biased stabilization (blue askterisks) are indicated (see Materials and methods for details). For all graphs, means and SD are indicated. Bars, 2 µm.

Mentions: Tubulin binds GTP and can hydrolyze this nucleotide. The influence of the tubulin nucleotide status on MT dynamics in vivo is still a mater of debate (Kueh and Mitchison, 2009). We have analyzed the GTP concentration variation upon entry into quiescence after carbon exhaustion using high pressure ionic chromatography and observed a drastic decrease of the intracellular pool of this nucleotide (Fig. 3 A). Mycophenolic acid (MPA) is a drug that specifically inhibits inosine-5′-monophosphate dehydrogenase, the enzyme that catalyzes the conversion of inosine-5′-monophosphate into guanosine-5′-monophosphate (Allison and Eugui, 2000), the GTP precursor. As in other organisms (Qiu et al., 2000), MPA treatment of proliferating S. pombe cells caused GTP depletion (Fig. 3 B). The GTP drop was reversed by addition of guanine in the medium, demonstrating the specificity of MPA treatment (Escobar-Henriques et al., 2001). Interestingly, MPA treatment did not significantly affect MT growth or shrinkage rates (Fig. 3 C) nor the MT catastrophe and rescue frequencies (not depicted), excluding the possibility that MT stabilization would be an immediate consequence of GTP depletion upon quiescence entry.


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)

Q-MT bundle formation does not depend on GTP depletion but rather involves the SPB. (A) GTP concentration decreases upon quiescence entry. (B) In proliferating cells, a MPA treatment (100 µg/ml) causes a massive GTP concentration drop. Proliferating cells were incubated 30 min or 2 h with either MPA, MPA + guanine (0.3 mM) to reverse the MPA effect, or guanine alone as a control. In A and B, N = 4 experiments with two samples per experiment. (C) MPA-induced GTP droop does not affect MT dynamics in proliferating cells. MT growth and shrinkage speed were determined in proliferating WT cells expressing GFP-Atb2 treated (2 h) or not with MPA. (D and E) MT stabilization involves the SPB. (D) At various times after carbon exhaustion (left, red dashed line), cell expressing GFP-Atb2 (green) and Sfi1-CFP (red) were treated for 10 min or 8 h with benomyl and then imaged. Numbers indicate the mean number of MT bundle per cell (N = 2 experiments and n > 200 cells for each time point). (E) WT cells were grown 6 h after carbon starvation and incubated with benomyl for the indicated times (left). A cartoon (middle) representing either a MT bundle random stabilization model or model biased toward the stabilization of the SPB-associated MT bundle. Red, SPB; blue, nuclear membrane; green, MT bundle. Cells displaying multiple MT bundles (red bars) or only a single MT bundle associated with the SPB (green bars) or not (gray bars) were scored for cells incubated with benomyl for the indicated times. N = 3 experiments and n > 200 cells per time point. Error bars are SD. The percentage of cells displaying a unique MT bundle associated with the SPB calculated theoretically using a random stabilization prediction (black asterisks) or SPB-biased stabilization (blue askterisks) are indicated (see Materials and methods for details). For all graphs, means and SD are indicated. Bars, 2 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4494004&req=5

fig3: Q-MT bundle formation does not depend on GTP depletion but rather involves the SPB. (A) GTP concentration decreases upon quiescence entry. (B) In proliferating cells, a MPA treatment (100 µg/ml) causes a massive GTP concentration drop. Proliferating cells were incubated 30 min or 2 h with either MPA, MPA + guanine (0.3 mM) to reverse the MPA effect, or guanine alone as a control. In A and B, N = 4 experiments with two samples per experiment. (C) MPA-induced GTP droop does not affect MT dynamics in proliferating cells. MT growth and shrinkage speed were determined in proliferating WT cells expressing GFP-Atb2 treated (2 h) or not with MPA. (D and E) MT stabilization involves the SPB. (D) At various times after carbon exhaustion (left, red dashed line), cell expressing GFP-Atb2 (green) and Sfi1-CFP (red) were treated for 10 min or 8 h with benomyl and then imaged. Numbers indicate the mean number of MT bundle per cell (N = 2 experiments and n > 200 cells for each time point). (E) WT cells were grown 6 h after carbon starvation and incubated with benomyl for the indicated times (left). A cartoon (middle) representing either a MT bundle random stabilization model or model biased toward the stabilization of the SPB-associated MT bundle. Red, SPB; blue, nuclear membrane; green, MT bundle. Cells displaying multiple MT bundles (red bars) or only a single MT bundle associated with the SPB (green bars) or not (gray bars) were scored for cells incubated with benomyl for the indicated times. N = 3 experiments and n > 200 cells per time point. Error bars are SD. The percentage of cells displaying a unique MT bundle associated with the SPB calculated theoretically using a random stabilization prediction (black asterisks) or SPB-biased stabilization (blue askterisks) are indicated (see Materials and methods for details). For all graphs, means and SD are indicated. Bars, 2 µm.
Mentions: Tubulin binds GTP and can hydrolyze this nucleotide. The influence of the tubulin nucleotide status on MT dynamics in vivo is still a mater of debate (Kueh and Mitchison, 2009). We have analyzed the GTP concentration variation upon entry into quiescence after carbon exhaustion using high pressure ionic chromatography and observed a drastic decrease of the intracellular pool of this nucleotide (Fig. 3 A). Mycophenolic acid (MPA) is a drug that specifically inhibits inosine-5′-monophosphate dehydrogenase, the enzyme that catalyzes the conversion of inosine-5′-monophosphate into guanosine-5′-monophosphate (Allison and Eugui, 2000), the GTP precursor. As in other organisms (Qiu et al., 2000), MPA treatment of proliferating S. pombe cells caused GTP depletion (Fig. 3 B). The GTP drop was reversed by addition of guanine in the medium, demonstrating the specificity of MPA treatment (Escobar-Henriques et al., 2001). Interestingly, MPA treatment did not significantly affect MT growth or shrinkage rates (Fig. 3 C) nor the MT catastrophe and rescue frequencies (not depicted), excluding the possibility that MT stabilization would be an immediate consequence of GTP depletion upon quiescence entry.

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