Two XMAP215/TOG Microtubule Polymerases, Alp14 and Dis1, Play Non-Exchangeable, Distinct Roles in Microtubule Organisation in Fission Yeast
Abstract
:1. Introduction
2. Results
2.1. Creation of Temperature-Sensitive alp14 Mutants in the Absence of Dis1
2.2. Alp14 and Dis1 Collaborate to Regulate Proper Mitotic Progression and Promote Spindle Elongation
2.3. Alp14, But Not Dis1, Promotes Microtubule Nucleation
2.4. Microtubule Intensities Are Reduced by Simultaneous Inactivation of Alp14 and Dis1
2.5. Short Spindles Often Collapse in alp14-26dis1∆ Cells, Leading to a Lethal “Cut” Phenotype
2.6. Alp14 and Dis1 Are Not Functionally Exchangeable
3. Discussion
3.1. Alp14 and Dis1 Have Evolved to Execute Functionally Non-Exchangeable Roles
3.2. Dis1 May Be a Specialised Regulator of Microtubule Dynamics at the Kinetochore-Microtubule Interface
4. Materials and Methods
4.1. Strains, Media, and Genetic Methods
4.2. Preparation and Manipulation of Nucleic Acids
4.3. Strain Construction, Gene Disruption and N-Terminal and C-Terminal Epitope Tagging
4.4. Isolation of alp14 Temperature-Sensitive Mutants in the dis1∆ Background
4.5. Fluorescence Microscopy and Time-Lapse Live Cell Imaging
4.6. Quantification of Fluorescent Signal Intensities
4.7. Statistical Data Analysis
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
GBP | GFP-binding protein |
MAPs | Microtubule-Associated Proteins |
MT | Microtubule |
NLS | Nuclear Localisation Signal |
SPB | Spindle Pole Body |
ts | temperature sensitive |
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Strains | Genotypes | Figures Used | Derivations |
---|---|---|---|
MS345 | h− alp14::ura4+ leu1 ura4 | 1A | Our lab stock |
MA003 | h+ alp14-GFP-kanR leu1ura4 his2 | 1A | Our lab stock |
MY2097 | h− alp14-21-GFP-kanR leu1ura4 | 1A | This study |
TK469 | h− alp14-26-GFP-kanR leu1ura4 | 1A | This study |
MY2099 | h− alp14-27-GFP-kanR leu1ura4 | 1A | This study |
TK475 | h− alp14-31-GFP-kanR leu1ura4 | 1A | This study |
TK484 | h− alp14-32-GFP-kanR leu1ura4 | 1A | This study |
MY2101 | h− alp14-33-GFP-kanR leu1ura4 | 1A | This study |
TK358 | h− dis1::hphR alp14-GFP-kanR leu1 ura4 | 1A | This study |
TK400 | h− alp14-21-GFP-kanR dis1::hphR leu1 ura4 | 1A–B | This study |
TK405 | h− alp14-26-GFP-kanR dis1::hphR leu1 ura4 | 1A–B | This study |
TK406 | h− alp14-27-GFP-kanR dis1::hphR leu1 ura4 | 1A–B | This study |
TK457 | h− alp14-31-GFP-kanR dis1::hphR leu1 ura4 | 1A–B | This study |
TK458 | h− alp14-32-GFP-kanR dis1::hphR leu1 ura4 | 1A–B | This study |
TK459 | h− alp14-33-GFP-kanR dis1::hphR leu1 ura4 | 1A–B | This study |
TK551 | h+ plo1-GFP-HA-kanR aur1R-Pnda3-mCherry-atb2 leu1 ura4 his2 | 2A–E, 4E | This study |
TK583 | h− dis1::ura4+ plo1-GFP-HA-kanR aur1R-Pnda3-mCherry-atb2 leu1 ura4 | 2A–E, 4E | This study |
TK556 | h− alp14-26-myc-hphR plo1-GFP-HA-kanR aur1R-Pnda3-mCherry-atb2 leu1 ura4 | 2A–E, 4E | This study |
TK585 | h− alp14-26-myc-hphR dis1::ura4+ plo1-GFP-HA-kanR aur1R-Pnda3-mCherry-atb2 leu1 ura4 | 2A–E, 4D-E | This study |
TK586 | h− alp14::ura4+ plo1-GFP-HA-kanR aur1R-Pnda3-mCherry-atb2 leu1 ura4 | 2A–E, 4E | This study |
MO100 | h− cut12-GFP-ura4+ aur1R-Pnda3-mCherry-atb2 leu1 ura4 | 3A–C, 4A-B | Our lab stock |
TK547 | h+ dis1::natR cut12-GFP-ura4+ aur1R-Pnda3-mCherry-atb2 leu1 ura4 his2 | 3A–C | This study |
TK580 | h+ alp14-26-myc-hphR cut12-GFP-ura4+ aur1R-Pnda3-mCherry-atb2 leu1 ura4 | 3A–C | This study |
TK572 | h− alp14-26-myc-hphR dis1::natR cut12-GFP-ura4+ aur1R-Pnda3-mCherry-atb2 leu1 ura4 | 3A–C, 4A–C | This study |
TK582 | h+ alp14::hphR cut12-GFP-ura4+ aur1R-Pnda3-mCherry-atb2 leu1 ura4 his2 | 3A–C | This study |
TK576 | h− dis1-GFP-kanR hphR-GBP-mCherry-alp4 leu1 ura4 | 5B–C | This study |
TK591 | h− alp14::ura4+ dis1-GFP-kanR hphR-GBP-mCherry-alp4 leu1 ura4 | 5B–C | This study |
TK590 | h+ alp7::ura4+ dis1-GFP-kanR hphR-GBP-mCherry-alp4 leu1 ura4 his2 | 5B–C | This study |
TK578 | h− dis1-GFP-kanR leu1 ura4 | 5C | This study |
TK602 | h+ alp14::ura4+ dis1-GFP-kanR leu1 ura4 his2 | 5C | This study |
MS187 | h− alp7::ura4+ dis1-GFP-kanR leu1 ura4 | 5C | Our lab stock |
MY2021 | h− alp14-NLS-GFP-kanR mis12-GBP-6HIS-mCherry-natR leu1 ura4 | 6B–C | This study |
MY2041 | h− dis1::hphR alp14-NLS-GFP-kanR mis12-GBP-6HIS-mCherry-natR leu1 ura4 | 6B–C | This study |
MY2027 | h− alp7::ura4+ alp14-NLS-GFP-kanR mis12-GBP-6HIS-mCherry-natR leu1 ura4 | 6B–C | This study |
MS836 | h+ alp14-NLS-GFP-kanR leu1 ura4 his2 | 6C | Our lab stock |
TK360 | h− dis1::hphR alp14-NLS-GFP-kanR leu1 ura4 | 6C | This study |
MS838 | h− alp7::ura4+ alp14-NLS-GFP-kanR leu1 ura4 | 6C | Our lab stock |
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Yukawa, M.; Kawakami, T.; Pinder, C.; Toda, T. Two XMAP215/TOG Microtubule Polymerases, Alp14 and Dis1, Play Non-Exchangeable, Distinct Roles in Microtubule Organisation in Fission Yeast. Int. J. Mol. Sci. 2019, 20, 5108. https://doi.org/10.3390/ijms20205108
Yukawa M, Kawakami T, Pinder C, Toda T. Two XMAP215/TOG Microtubule Polymerases, Alp14 and Dis1, Play Non-Exchangeable, Distinct Roles in Microtubule Organisation in Fission Yeast. International Journal of Molecular Sciences. 2019; 20(20):5108. https://doi.org/10.3390/ijms20205108
Chicago/Turabian StyleYukawa, Masashi, Tomoki Kawakami, Corinne Pinder, and Takashi Toda. 2019. "Two XMAP215/TOG Microtubule Polymerases, Alp14 and Dis1, Play Non-Exchangeable, Distinct Roles in Microtubule Organisation in Fission Yeast" International Journal of Molecular Sciences 20, no. 20: 5108. https://doi.org/10.3390/ijms20205108