Fig. 2: Bridgin assembles onto the KMN network platform.
From: Bridgin connects the outer kinetochore to centromeric chromatin
a Left, enrichment of Bgi1 at M-phase kinetochores in SHR870 (BGI1::BGI1-V5-GFP) cells by cross-linked chromatin immunoprecipitation (ChIP) followed by quantitative polymerase chain reaction (qPCR). The data represent the mean ± standard deviation (S.D.) of three independent experiments. Right, schematic representing the location of qPCR primers used. Centromeric sequences are defined by grey boxes. b Steps followed to determine localization interdependency at the kinetochore among its subcomplexes. c–e Left, a representative image of Bgi1-V5-GFP in c SHR906 (DAD2::GAL7p-mCherry-DAD2), d SHR907 (MIS12MTW1::GAL7p-mCherry-MIS12MTW1) and e SHR945 (KNL1SPC105::GAL7p-mCherry-KNL1SPC105) in permissive (+) or nonpermissive (−) conditions of the GAL7p. Scale bar, 3 μm; 1 μm for magnified inset. Right, Bgi1-V5-GFP signals and c mCherry-Dad2, d mCherry-Mis12Mtw1, or e mCherry-Knl1Spc105 in cells with or without Dad2, Mis12Mtw1, or Knl1Spc105 expression, respectively, were quantified and normalized to their respective mean signals in the permissive condition of the GAL7p. Signals were measured after 18, 12, and 18 h in nonpermissive conditions for Dad2, Mis12Mtw1, and Knl1Spc105, respectively. For d, the strong influence of Mis12Mtw1 on kinetochore localization of bridgin resulted in signals that were below detectable levels (B.D.L.). For e, bridgin signals in 24 out of 300 cells were below detectable levels. f Left, representative images of Bgi1-V5-GFP in wild-type H99 (SOS7) or in sos7 null mutant SHR908 (SOS7::sos7Δ) cells. Scale bar, 2 μm. Right, Bgi1-V5-GFP signal intensities were quantified and normalized to the mean wild-type signal. c–f The results of three independent experiments with 100 cells each are represented. The red dot represents the mean of one experiment; mean ± S.D. is shown. For statistical comparison of differences between the samples, Mann–Whitney two-tailed analysis was applied, p values show significant differences. g Schematic describes the observed localization interdependency of inner (blue) and outer (green) kinetochore protein complexes at the C. neoformans kinetochore. Purple arrows highlight the dependency of bridgin on the KMN network. Source data are available as a Source Data file.
