K-Shrec Analysis of Kinetochore Protein Archetecture at Nm Scale Accuracy

Abstract

Kinetochores link centromeric DNA to the plus ends of spindle microtubules (MTs). They contain more than 70 different proteins that function in chromatin anchorage, microtubule attachment, the spindle assembly checkpoint (SAC), force generation, and correction of MT attachment errors. Protein architecture within the kinetochore is poorly understood because the kinetochore core is only about 100 nm thick and it is has been very difficult to obtain molecular specificity at the resolution of electron microscopy. Based on the single molecule studies by Churchman and Spudich (P.N.A.S., 2005), the light microscopy methods that we developed called “kinetochore-speckle high resolution co-localization” (K-SHREC) allows us to determine the average separation between the centroids of two different color fluorescent probes that label different protein domains within kinetochores of human cells and budding yeast. Accuracy to less than 5 nm was achieved when we applied K-SHREC to metaphase cells where sister kinetochores were aligned in opposite directions. Using K-SHREC we have generated the first nm-scale maps of average protein position and orientation along the kinetochore inner-outer axis for 20 or more key protein complexes, and we have shown a correlation between changes in intrakinetochore separation of the Ndc80 complex from the periphery of the centromere and changes in SAC activity and phosphorylation activity within the kinetochore. (Joglekar et al., Curr. Biol., 2009; Wan, O’Quinn et al., Cell, 2010; Maresca et al., J. Cell Biol., 2009). We have also developed a fluorescence ratio method to measure protein copy number within kinetochores of budding yeast and chicken cells (Joglekar et al., Curr. Biol., 2006; Johnston et al, J. Cell Biol., 2010). Together these studies reveal high conservation in kinetochore protein architecture of the core MT attachment site between yeast and vertebrates. Supported by NIH GM 24364.