The Budding Yeast Point Centromere Associates with Two Cse4 Molecules during Mitosis

Abstract

Summary The centromere is defined by the incorporation of the centro-mere-specific histone H3 variant centromere protein A (CENP-A). Like histone H3, CENP-A can form CENP-A-H4 heterotetramers in vitro [1]. However, the in vivo conformation of CENP-A chromatin has been proposed by different studies as hemisomes, canonical, or heterotypic nucleosomes [2–8]. A clear understanding of the in vivo architecture of CENP-A chromatin is important, because it influences the molecular mechanisms of the assembly and maintenance of the centromere and its function in kinetochore nucleation. Akey determinant of this architecture is the number of CENP-A molecules bound to the centromere. Accurate measurement of this number can limit possible centromere architectures. The genetically defined point centromere in the budding yeast Saccharomyces cerevisiae provides a unique opportunity to define this number accurately, as this 120-bp-long centromere can at the most form one nucleosome or hemisome. Using novel live-cell fluorescence microscopy assays, we demonstrate that the budding yeast centromere recruits two Cse4 (ScCENP-A) molecules. These molecules are deposited during S phase and they remain stably bound through late anaphase. Our studies suggest that the budding yeast centromere incorporates a Cse4-H4 tetramer.

Determining absolute protein numbers by quantitative fluorescence microscopy

“…Therefore, it is possible that Shivaraju et al had very accurately measured the Cse4p content at the centromere (~2 Cse4p per chromosome) while excluding the fluorescence intensity from the extra Cse4p molecules observed by Coffman et al (2011), Lawrimore et al (2011), and Shivaraju et al (2012). The result that the centromere nucleosome contains 2 Cse4p proteins is consistent with TIRF stepwise photobleaching of single nucleosomes in mammalian cells (Padeganeh et al, 2013) and BiFC complementation experiments (Aravamudhan, Felzer-Kim, & Joglekar, 2013). …”

Glucose Starvation Inhibits Autophagy via Vacuolar Hydrolysis and Induces Plasma Membrane Internalization by Down-regulating Recycling

“…For Figs. 3 and 5, images were acquired on a Nikon Ti-E inverted microscope equipped with a 100ϫ, 1.4 numerical aperture oil immersion objective as described previously (22). Fluorescent samples were excited with the Lumencor light engine equipped with individual LEDs for GFP/YFP/mCherry excitation (Lumencor, Inc., Beaverton, OR).…”

How Kinetochore Architecture Shapes the Mechanisms of Its Function

“…What is the axial and circumferential distribution of protein molecules about their average positions? Quantitative answers to these questions obtained from diverse fluorescence microscopy methods and combined with the known structures of kinetochore proteins established a detailed model of the architecture of the KMN network in the budding yeast kinetochore-microtubule attachment [7, 48–50]. Although this architecture invokes certain assumptions, specifically the circular symmetry of kinetochore proteins around the microtubule diameter and the relative positions of the CH-domains and the Dam1 ring, it has enabled powerful predictions regarding the emergent mechanisms of kinetochore function (discussed in the sections below).…”

“…B (left to right = microtubule plus-end to centromere) The conserved, dual pathways (solid arrows – direct interaction, dashed arrow – indirect interaction) that assemble the KMN network, which forms the interface of the kinetochore with the microtubule plus-end. C Reconstruction of the protein architecture of the budding yeast kinetochore using fluorescence microscopy measurements and protein structures [7, 8, 14, 15, 32, 33, 48–50, 73]. Centromere-associated proteins are represented by white, oblong shapes.…”