Cells expressing Spc105GILK::AAAA (21-GILK-24::AAAA) showed abnormal recruitment of Bub3-Bub1 to bioriented kinetochores, similar to Spc105BPM (Fig. SAC silencing, thereby ensuring accurate chromosome segregation. Introduction During cell division, chromosomes often form syntelic attachments, wherein both sister kinetochores establish end-on attachments with microtubules from the same spindle pole (Fig. 1 A). For accurate chromosome segregation, these erroneous attachments must be corrected before the cell enters anaphase. However, recent studies show that end-on kinetochoreCmicrotubule attachments, whether they are monopolar, syntelic, or bipolar, can silence the spindle assembly checkpoint (SAC; Etemad et al., 2015; Tauchman et al., 2015). To prevent chromosome missegregation, the kinetochore must allow SAC silencing only after bipolar attachments form (Fig. 1 A). How the kinetochore meets this requirement is usually unclear, because the same enzyme, protein phosphatase 1 (PP1), antagonizes both the SAC and the error correction machinery. PP1 silences the SAC by dephosphorylating the kinetochore protein KNL1/Spc105 to enable anaphase onset (London et al., 2012; Meadows et al., 2011; Nijenhuis et al., 2014; Rosenberg et al., 2011). It stabilizes kinetochoreCmicrotubule attachments by dephosphorylating microtubule-binding kinetochore components such as the Ndc80 complex (Liu et al., 2010; Posch et al., 2010). This dual role of PP1 creates the possibility of a harmful cross-talk between SAC silencing and error correction: if PP1 is usually recruited for SAC silencing before chromosome biorientation, it can inadvertently stabilize syntelic attachments and thus cause chromosome missegregation. Therefore, LY 344864 racemate it is important to understand how the LY 344864 racemate kinetochore ensures that the correction of syntelic attachments and chromosome biorientation precedes SAC silencing. Open in a separate window Figure 1. The basic patch near the N-terminus of Spc105 contributes to Glc7 recruitment. (A) Model of how cross-talk between SAC silencing and error correction can interfere with the correction of syntelic attachments and promote chromosome missegregation. (B) Functional domains of Spc105 and the amino acid sequence of its N-terminus. The mutations in Spc105 used in this study are noted at the bottom. (C) Representative micrographs of TetO-TetR-GFP spots. achieves biorientation faster in cells expressing Spc105BPM compared with WT cells (data presented as mean + SEM; P = 0.0041 at 45 min using two-way ANOVA). Sister centromere separation is higher in cells expressing Spc105BPM compared with WT cells, even though the spindle length is not. Scale bar: 3.2 m. The measurements were pooled from three experiments; for WT, = 273 and 342 at 30 and 45 min, respectively; for BPM, = 176 and 281 at 30 and 45 min; **, P < 0.01 for the fraction of cells with bioriented at 45 min; *, P < 0.05 for sister centromere separation at 45 min. (D) Left: V-plots display the normalized distribution of kinetochores along the spindle axis for the indicated strains (> 50 for each time point). LY 344864 racemate Each row of pixels in the plot represents the symmetrized distribution of Spc105222GFP or Spc105BPM,222GFP along the spindle axis in one cell. Rows are ranked according to spindle length (see Materials and methods and Marco et al. ). Scale bar: 1.6 m. Right, top: Average sister kinetochore separation (data presented as mean + SEM; P = 0.0005 [***] and 0.0121 [*] for 45 and 60 min, respectively, using unpaired test). Right, bottom: Distance between two spindle poles LY 344864 racemate TEAD4 remains unchanged (data presented as mean + SEM; P = 0.6523 and 0.1932 for 45 and 60 min, respectively, using unpaired test, from two experiments). (E) Top: Workflow. Middle: Representative micrographs of yeast cells expressing the indicated proteins. Scale bar: 3.2 m. Bottom: Frequency of metaphase cells with visible Bub3 and Mad1 at the kinetochores (pooled from two experiments; for Bub3-mCherry, = 204, LY 344864 racemate 196, and 179, respectively; for Mad1-mCherry, = 101, 94, and 123). In this and subsequent assays yielding two-category (presence or absence of visible recruitment) scoring data for WT and mutant Spc105, we used Fishers exact test for the fractions calculated from the total number of observations. P < 0.0001 for Bub3-mCherry and P < 0.0003 for Mad1-mCherry recruitment. (F) Fusion of an extra basic patch with or without RVSF and their effect on Bub3-Bub1 localization in metaphase cells. Top: Schematics of the fragments fused to the N-terminus of Spc105ABPM. (F, i) Left: Representative micrographs. Scale bar: 3.2 m. Right: Bar graph shows the fraction of cells with visible Bub3-mCherry recruitment (= 90, 70, 96, and 114, respectively, pooled from two experiments; P < 0.0001 using Fishers exact test). (F, ii) Left: Representative micrographs of cells expressing the indicated proteins. Scale bar: 3.2 m. Right: Bar graph shows the fraction of.
- T-cell epitopes are peptides derived from antigens and identified by the T-cell receptor (TCR) when bound to MHC molecules displayed within the cell surface of APCs
- Cloning of gene fragments encoding diagnostic antigens
- Epitopes are present on a single HLA (private epitope) or shared by multiple antigens (public epitope)
- Spleens were harvested in 1 (C) or 2 wpi (B, C) and cells were analyzed by movement cytometry in comparison to na?ve mice
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