Resultados de investigación

Eukaryotic chromosome segregation relies on the assembly of a bipolar machinery based on microtubules (MTs), named the mitotic spindle. Formation of the mitotic spindle follows a force balance mechanism that ensures the proper capture and separation of sister chromatids. Many proteins have been involved in the establishment of this force balance, although kinesin 5 is well recognized as the major outward pushing force generator, since its inactivation results in monopolar, non-functional spindles. In order to find additional players in the force balance mechanism, we have performed a suppressor screen using a conditional allele of the fission yeast kinesin 5 ortholog Cut7. This screen identified that the lack of the PP6 phosphatase partially suppresses cut7 phenotypes, at least by defective translation of MT regulators, such as the minus end-directed kinesin Klp2, the MT stabilizer Alp7 and the MT bundler Ase1, impacting on the force balance mechanism. Additionally, our data show that the Elongator complex, a target activated by PP6 for efficient tRNA modification, also contributes to the force balance, albeit to a lesser extent. Importantly, this complex has recently been implicated in direct MT polymerization in metazoans, a role not shared by its fission yeast counterpart., Peer reviewed

Eukaryotic chromosome segregation relies on the assembly of a bipolar machinery based on microtubules (MTs), named the mitotic spindle. Formation of the mitotic spindle follows a force balance mechanism that ensures the proper capture and separation of sister chromatids. Many proteins have been involved in the establishment of this force balance, although kinesin 5 is well recognized as the major outward pushing force generator, since its inactivation results in monopolar, non-functional spindles. In order to find additional players in the force balance mechanism, we have performed a suppressor screen using a conditional allele of the fission yeast kinesin 5 ortholog Cut7. This screen identified that the lack of the PP6 phosphatase partially suppresses cut7 phenotypes, at least by defective translation of MT regulators, such as the minus end-directed kinesin Klp2, the MT stabilizer Alp7 and the MT bundler Ase1, impacting on the force balance mechanism. Additionally, our data show that the Elongator complex, a target activated by PP6 for efficient tRNA modification, also contributes to the force balance, albeit to a lesser extent. Importantly, this complex has recently been implicated in direct MT polymerization in metazoans, a role not shared by its fission yeast counterpart., Peer reviewed

A: Serial dilution assay of the wild type, ekc1∆, cut7–24 and cut7–24 ekc1∆ strains incubated for 3 days at the indicated temperatures. B: SDS PAGE analysis of Cut7-3xHA levels on wild type or ppe1∆ cells. The upper panel corresponds to the blot incubated with an anti-HA antibody, while the bottom panel corresponds to the blot incubated with an anti-tubulin antibody. C: Plot showing the total Cut7-3xHA levels relative to the corresponding tubulin levels in wild type and ppe1∆ cells. n for wild type: 3; n for ppe1∆: 3. a.u.: arbitrary units. Statistical significance was determined using a T-test (p = 0.086). D: Upper panel, PhosTag SDS PAGE analysis of Cut7-3xHA in asynchronous and mitotically blocked nda3-KM311 cells in the presence or absence of Ppe1. Bottom panel, the same samples submitted to SDS PAGE analysis using an anti-tubulin antibody. E: SDS PAGE analysis of Pkl1-13xMyc levels on wild type or ppe1∆ cells. The upper panel corresponds to the blot incubated with an anti-Myc antibody, while the bottom panel corresponds to the blot incubated with an anti-tubulin antibody. F: Plot showing the total Pkl1-13xMyc levels relative to the corresponding tubulin levels in wild type and ppe1∆ cells. n for wild type: 3; n for ppe1∆: 3. a.u.: arbitrary units. Statistical significance was determined using a T-test (p = 0.121). G: Upper panel, PhosTag SDS PAGE analysis of Pkl1-13xMyc in asynchronous and mitotically blocked nda3-KM311 cells in the presence or absence of Ppe1. Bottom panel, the same samples submitted to SDS PAGE analysis using an anti-tubulin antibody., Peer reviewed

A: Serial dilution assay of the wild type, elp3∆, cut7–24, cut7–24 elp3∆, cut7–22 and cut7–22 elp3∆ strains incubated for 3 days at the indicated temperatures. B: Selected kymographs showing interphasic MT dynamics in wild type, ppe1∆, elp3∆ and elp3-YY527AA cells expressing GFP-Atb2, showing the inverted green channel in a grey scale. Scale bar: 5 μm. C: Plot showing MT growth rate of the indicated strains expressing GFP-Atb2 (n for wild type: 74; n for ppe1∆: 87; n for elp3∆: 79; n for elp3-YY527AA: 79). Statistical differences were determined using a T-test (wt vs. ppe1∆, p = 0.409; wt vs. elp3∆, p = 0.374; wt vs. elp3-YY527AA, p = 0.001). D: Plot showing MT catastrophe rate of the indicated strains expressing GFP-Atb2 (n for wild type: 95; n for ppe1∆: 73; n for elp3∆: 82; n for elp3-YY527AA: 88). Statistical differences were determined using a T-test (wt vs. ppe1∆, p = 0.054; wt vs. elp3∆, p = 0.003; wt vs. elp3-YY527AA, p = 0.294). E: Plot showing MT dwelling time at the cell end of the indicated strains expressing GFP-Atb2 (n for wild type: 63; n for ppe1∆: 67; n for elp3∆: 67; n for elp3-YY527AA: 65). Statistical differences were determined using a T-test (wt vs. ppe1∆, p = 0.931; wt vs. elp3∆, p = 0.500; wt vs. elp3-YY527AA, p = 0.667). F: Maximum intensity projections from confocal time lapse images of wild type cells expressing mCherry-Atb2 and Elp3 or Elp4 fused to GFP. The upper series shows the magenta (mCherry-Atb2) and green (Elp3-GFP or Elp4-GFP) merged channels. The bottom series shows the inverted green channel in a grey scale. Time 0 corresponds to mitotic entry. The dashed line represents the cell contour. Scale bar: 2 μm. G: Western-blot analysis of SDS PAGE of Elp3-GFP and Elp4-GFP from the strains shown in C., Peer reviewed

A: Serial dilution assay of the wild type, ppe1∆, klp2∆, ppe1∆ klp2∆, cut7–22, cut7–22 ppe1∆, cut7–22 klp2∆ and cut7–22 ppe1∆ klp2∆ strains incubated for 3 days at the indicated temperatures. B: Serial dilution assay of the wild type, ppe1∆, alp7∆, ppe1∆ alp7∆, cut7–22, cut7–22 ppe1∆, cut7–22 alp7∆ and cut7–22 ppe1∆ alp7∆ strains incubated for 3 days at the indicated temperatures. C: Serial dilution assay of the wild type, ppe1∆, ase1∆, ppe1∆ ase1∆, cut7–22, cut7–22 ppe1∆, cut7–22 ase1∆ and cut7–22 ppe1∆ ase1∆ strains incubated for 3 days at the indicated temperatures., Peer reviewed

A: Serial dilution assay of the wild type, ppe1∆ and elp3∆ strains incubated for 3 days in YE5S of YE5S plates supplemented with 5 μg/ml of G418 at 30 °C. B: Serial dilution assay of the wild type, ppe1∆, elp4-S114A and ppe1∆ elp4-S114A strains incubated for 3 days in YE5S of YE5S plates supplemented with 5 μg/ml of G418 at 30 °C. C: Serial dilution assay of the wild type, cut7–22 and cut7–24 strains incubated for 3 days at the indicated temperatures supplemented with 5 μg/ml of G418., Peer reviewed