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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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Various MAPs are associated with the Q-MT bundle and influence its shape. (A and B) Mto2 relocalizes from the nuclear membrane onto the Q-MT bundle. (A) Cells expressing Mto2-GFP (green) and RFP-Atb2 (red) are shown. Colocalization coefficient between Mto2-GFP and RFP-Atb2 are indicated (see Materials and methods for details). (B) Line-scan analyses (right) indicate that Mto2 localizes at MT extremities within the Q-MT bundle. (C) Numerous MAPs colocalize with the Q-MT bundle. Cells expressing RFP-Atb2 and the indicated MAP fused to GFP were grown 5 d and imaged. Graphs are line scan analyses of the red and green fluorescence. In B and C, data are representative of the fluorescence intensity variations measured out of four experimental repeats. (D–F) The Q-MT bundle shape is influenced by the deletion of specific MAPs. (D) Q-MT bundle was imaged in 7-d-old cells expressing GFP-Atb2 and deleted for the indicated MAP. (E and F) Q-MT bundle length (n > 200; E) and Q-MT bundle intensity (n > 100; F) were measured in the indicated mutants. ***, P < 10−4. Blue and red asterisks shows positive and negative difference, respectively. Bars, 2 µm.
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fig4: Various MAPs are associated with the Q-MT bundle and influence its shape. (A and B) Mto2 relocalizes from the nuclear membrane onto the Q-MT bundle. (A) Cells expressing Mto2-GFP (green) and RFP-Atb2 (red) are shown. Colocalization coefficient between Mto2-GFP and RFP-Atb2 are indicated (see Materials and methods for details). (B) Line-scan analyses (right) indicate that Mto2 localizes at MT extremities within the Q-MT bundle. (C) Numerous MAPs colocalize with the Q-MT bundle. Cells expressing RFP-Atb2 and the indicated MAP fused to GFP were grown 5 d and imaged. Graphs are line scan analyses of the red and green fluorescence. In B and C, data are representative of the fluorescence intensity variations measured out of four experimental repeats. (D–F) The Q-MT bundle shape is influenced by the deletion of specific MAPs. (D) Q-MT bundle was imaged in 7-d-old cells expressing GFP-Atb2 and deleted for the indicated MAP. (E and F) Q-MT bundle length (n > 200; E) and Q-MT bundle intensity (n > 100; F) were measured in the indicated mutants. ***, P < 10−4. Blue and red asterisks shows positive and negative difference, respectively. Bars, 2 µm.

Mentions: To get insights into the mechanism of Q-MT bundle formation, we looked for MAPs colocalizing with this structure. First, we found that astonishingly, in quiescent cells, Mto1 and Mto2 were no longer found as dots around the nuclear envelope but rather localized exclusively onto the Q-MT bundle (Fig. 4 A and Fig. S3 A). Line scan analyses revealed that Mto1/2 localized as dots that most likely mark the MT ends (Fig. 4 B and Fig. S3 A), in agreement with Mto1/2 being a MT minus end binding complex (Sawin et al., 2004; Venkatram et al., 2004, 2005; Samejima et al., 2005; Janson et al., 2007). Second, the regular spacing between MTs within the Q-MT bundle observed by EM (Fig. 1 E) strongly suggested the presence of a MT bundling protein, and, accordingly, we found that Ase1 localized all along the Q-MT bundle (Fig. 4 C). In S. pombe, the CLASP family member Cls1 (Peg1) has been involved in MT stabilization and is known to localize to the overlapping zone of interphase MT bundle (Bratman and Chang, 2007). Similarly, in quiescent cells, we found that Cls1 localized with the denser region of the Q-MT bundle (Fig. 4 C). Finally, the MT plus end tracking proteins Alp7 (the TACC protein orthologue) and Alp14 and Dis1 (two TOG-related proteins) localized as dots that probably correspond to MT extremities within the Q-MT bundle (Fig. 4 C), just like the MT minus end–directed kinesin-14 Klp2, although fewer and fainter Klp2 dots were detected (Fig. 4 C). Of note, Mal3, the EB1 homologue, could not be detected in quiescent cells (unpublished data).


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)

Various MAPs are associated with the Q-MT bundle and influence its shape. (A and B) Mto2 relocalizes from the nuclear membrane onto the Q-MT bundle. (A) Cells expressing Mto2-GFP (green) and RFP-Atb2 (red) are shown. Colocalization coefficient between Mto2-GFP and RFP-Atb2 are indicated (see Materials and methods for details). (B) Line-scan analyses (right) indicate that Mto2 localizes at MT extremities within the Q-MT bundle. (C) Numerous MAPs colocalize with the Q-MT bundle. Cells expressing RFP-Atb2 and the indicated MAP fused to GFP were grown 5 d and imaged. Graphs are line scan analyses of the red and green fluorescence. In B and C, data are representative of the fluorescence intensity variations measured out of four experimental repeats. (D–F) The Q-MT bundle shape is influenced by the deletion of specific MAPs. (D) Q-MT bundle was imaged in 7-d-old cells expressing GFP-Atb2 and deleted for the indicated MAP. (E and F) Q-MT bundle length (n > 200; E) and Q-MT bundle intensity (n > 100; F) were measured in the indicated mutants. ***, P < 10−4. Blue and red asterisks shows positive and negative difference, respectively. Bars, 2 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4494004&req=5

fig4: Various MAPs are associated with the Q-MT bundle and influence its shape. (A and B) Mto2 relocalizes from the nuclear membrane onto the Q-MT bundle. (A) Cells expressing Mto2-GFP (green) and RFP-Atb2 (red) are shown. Colocalization coefficient between Mto2-GFP and RFP-Atb2 are indicated (see Materials and methods for details). (B) Line-scan analyses (right) indicate that Mto2 localizes at MT extremities within the Q-MT bundle. (C) Numerous MAPs colocalize with the Q-MT bundle. Cells expressing RFP-Atb2 and the indicated MAP fused to GFP were grown 5 d and imaged. Graphs are line scan analyses of the red and green fluorescence. In B and C, data are representative of the fluorescence intensity variations measured out of four experimental repeats. (D–F) The Q-MT bundle shape is influenced by the deletion of specific MAPs. (D) Q-MT bundle was imaged in 7-d-old cells expressing GFP-Atb2 and deleted for the indicated MAP. (E and F) Q-MT bundle length (n > 200; E) and Q-MT bundle intensity (n > 100; F) were measured in the indicated mutants. ***, P < 10−4. Blue and red asterisks shows positive and negative difference, respectively. Bars, 2 µm.
Mentions: To get insights into the mechanism of Q-MT bundle formation, we looked for MAPs colocalizing with this structure. First, we found that astonishingly, in quiescent cells, Mto1 and Mto2 were no longer found as dots around the nuclear envelope but rather localized exclusively onto the Q-MT bundle (Fig. 4 A and Fig. S3 A). Line scan analyses revealed that Mto1/2 localized as dots that most likely mark the MT ends (Fig. 4 B and Fig. S3 A), in agreement with Mto1/2 being a MT minus end binding complex (Sawin et al., 2004; Venkatram et al., 2004, 2005; Samejima et al., 2005; Janson et al., 2007). Second, the regular spacing between MTs within the Q-MT bundle observed by EM (Fig. 1 E) strongly suggested the presence of a MT bundling protein, and, accordingly, we found that Ase1 localized all along the Q-MT bundle (Fig. 4 C). In S. pombe, the CLASP family member Cls1 (Peg1) has been involved in MT stabilization and is known to localize to the overlapping zone of interphase MT bundle (Bratman and Chang, 2007). Similarly, in quiescent cells, we found that Cls1 localized with the denser region of the Q-MT bundle (Fig. 4 C). Finally, the MT plus end tracking proteins Alp7 (the TACC protein orthologue) and Alp14 and Dis1 (two TOG-related proteins) localized as dots that probably correspond to MT extremities within the Q-MT bundle (Fig. 4 C), just like the MT minus end–directed kinesin-14 Klp2, although fewer and fainter Klp2 dots were detected (Fig. 4 C). Of note, Mal3, the EB1 homologue, could not be detected in quiescent cells (unpublished data).

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