maged using a 60 1.35 NA oil immersion objective lens on a microscope system equipped with filter sets for DAPI, EGFP/Alexa Fluor 488, Alexa Fluor 555, and Alexa Fluor 647; a CoolSNAP HQ2 camera; and MetaMorph 7.5 imaging software. Image z stacks comprising 12 images 0.2 m apart were collected and maximum projected to give a single image for each color channel. The different color channels were then combined in MetaMorph to give a 24-bit RGB image. These were cropped in Photoshop CS3 and imported into Illustrator CS3 for figure production. Quantitations of FD&C Green No. 3 kinetochore intensities were performed using Volocity or ImageJ software. Background-corrected kinetochore intensities determined by placing a 7 7-pixel circular region of interest over individual kinetochores were normalized to CREST or CenpA signal as indicated. Analysis of kinetochore intensities was performed in Excel. All immunofluorescence experiments shown are representatives of at least three independent experiments. Spindle checkpoint silencing assay For SAC silencing assays, HeLa cells were seeded into 6-well dishes with coverslips and treated with siRNA to phosphatase subunits for 48 h. See Submitted: 25 June 2014 Accepted: 21 August 2014 Accurate chromosome segregation during mitosis depends on the kinetochore, which supports chromosome movements by attaching to microtubules and also acts as a signaling hub to control the spindle assembly checkpoint. The kinetochore is thought to assemble on centromeric chromatin according to a temporal and spatial hierarchy of functionally distinct complexes. In addition, the kinetochore changes its size and composition in response to microtubule attachment status. The molecular basis of this adaptive response is not clear. Based on classic electron microscopy images of a trilaminar structure, the kinetochore has been divided into inner and outer regions. 
Recent studies using super-resolution fluorescence microscopy techniques and immunoelectron microscopy, combined with epistasis analyses has led to a hierarchical view of kinetochore assembly upon centromeric chromatin marked by the centromere-specific histone H3 variant CENP-A. The inner kinetochore includes the constitutive centromereassociated network proteins, which Correspondence to Hironori Funabiki: [email protected]; or David J. Wynne: [email protected] Abbreviations used in this paper: CCAN, constitutive centromereassociated network; CPC, chromosomal passenger complex; CSF, cytostatic factor; DB, dilution buffer; FL, full length; OMX, optical microscope experimental; OTF, optical transfer function; PP1, protein phosphatase 1; RPE-hTERT, human telomerase-immortalized retinal pigmented epithelial; SAC, spindle assembly checkpoint; SIM, structured illumination PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19833994 microscopy. recognize CENP-A and are present on centromeres throughout the cell cycle. During mitosis, two CCAN proteins, CENP-C and CENP-T, recruit outer kinetochore protein complexes via distinct mechanisms. CENP-T directly recruits the Ndc80 complex, whereas CENP-C recruits the KMN network, composed of the KNL1 complex, the Mis12 complex, and the Ndc80C through direct binding to the Mis12C. Thus, both CENP-C and CENP-T contribute to the recruitment of the Ndc80C, which generates the stable attachments to microtubule ends essential for anaphase chromosome movement. In contrast, KNL1 and its binding partner Zwint primarily act as a scaffold for the transient SAC proteins complex, and Spindly; Varma and Salmon, 201