Resultados de investigación

Additional file 2. Macroscopic lesions, histopathology and parasite DNA detection in lymph nodes draining the small intestine., Ministerio de Ciencia e Innovación; Comunidad de Madrid; H2020 European Institute of Innovation and Technology, Peer reviewed

Additional file 1. Histopathology and parasite DNA detection and quantification in the small intestine., Ministerio de Ciencia e Innovación; Comunidad de Madrid; H2020 European Institute of Innovation and Technology, Peer reviewed

Additional file 3. Histological lesions in the placentomes., Ministerio de Ciencia e Innovación; Comunidad de Madrid; H2020 European Institute of Innovation and Technology, Peer reviewed

Additional file 4. βAPP immunolabelling in foetal brains. For each foetal brain, 4 different areas were studied., Ministerio de Ciencia e Innovación; Comunidad de Madrid; H2020 European Institute of Innovation and Technology, Peer reviewed

Additional file 5. βAPP staining of foetal brains. Representative images of the two different labelling patterns observed: i) staining of areas of variable size in the white matter (A, foetus from sheep 3.1) and ii) sparse areas where the cytoplasm of different cell types in foetal brains from infected sheep was labelled (B, D and F, corresponding to foetuses from sheep 3.4, 1.2 and 1.1, respectively), as opposed to those from the uninfected controls (C, E and G, corresponding to foetuses from sheep 5.2, 4.3 and 5.3, respectively). Neurons (B–C), microglia (D–E), and Purkinje cells in the cerebellum (F–G). Magnification: 40x (A) and 400x (B–G)., Ministerio de Ciencia e Innovación; Comunidad de Madrid; H2020 European Institute of Innovation and Technology, 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

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