Elaine Yeh

Stable Kinetochore-Microtubule Attachment Constrains Centromere Positioning in Metaphase

With a single microtubule attachment, budding-yeast kinetochores provide an excellent system for understanding the coordinated linkage to dynamic microtubule plus ends for chromosome oscillation and positioning. Fluorescent tagging of kinetochore proteins indicates that, on average, all centromeres are clustered, distinctly separated from their sisters, and positioned equidistant from their respective spindle poles during metaphase. However, individual fluorescent chromosome markers near the centromere transiently reassociate with their sisters and oscillate from one spindle half to the other. To reconcile the apparent disparity between the average centromere position and individual centromere proximal markers, we utilized fluorescence recovery after photobleaching to measure stability of the histone-H3 variant Cse4p/CENP-A. Newly synthesized Cse4p replaces old protein during DNA replication. Once assembled, Cse4-GFP is a physically stable component of centromeres during mitosis. This allowed us to follow centromere dynamics within each spindle half. Kinetochores remain stably attached to dynamic microtubules and exhibit a low incidence of switching orientation or position between the spindle halves. Switching of sister chromatid attachment may be contemporaneous with Cse4p exchange and early kinetochore assembly during S phase; this would promote mixing of chromosome attachment to each spindle pole. Once biorientation is attained, centromeres rarely make excursions beyond their proximal half spindle.

Pericentric chromatin is organized into an intramolecular loop in mitosis.

BACKGROUND Cohesin proteins link sister chromatids and provide the basis for tension between bioriented sister chomatids in mitosis. Cohesin is concentrated at the centromere region of the chromosome despite the fact that sister centromeres can be separated by 800 nm in vivo. The function of cohesin at sites of separated DNA is unknown. RESULTS We provide evidence that the kinetochore promotes the organization of pericentric chromatin into a cruciform in mitosis such that centromere-flanking DNA adopts an intramolecular loop, whereas sister-chromatid arms are paired intermolecularly. Visualization of cohesin subunits by fluorescence microscopy revealed a cylindrical structure that encircles the central spindle and spans the distance between sister kinetochores. Kinetochore assembly at the apex of the loop initiates intrastrand loop formation that extends approximately 25 kb (12.5 kb on either side of the centromere). Two centromere loops (one from each sister chromatid) are stretched between the ends of sister-kinetochore microtubules along the spindle axis. At the base of the loop there is a transition to intermolecular sister-chromatid pairing. CONCLUSIONS The C loop conformation reveals the structural basis for sister-kinetochore clustering in budding yeast and for kinetochore biorientation and thus resolves the paradox of maximal interstrand separation in regions of highest cohesin concentration.

Imaging green fluorescent protein fusion proteins in Saccharomyces cerevisiae

Tagging expressed proteins with the green fluorescent protein (GFP) from Aequorea victoria [1] is a highly specific and sensitive technique for studying the intracellular dynamics of proteins and organelles. We have developed, as a probe, a fusion protein of the carboxyl terminus of dynein and GFP (dynein-GFP), which fluorescently labels the astral microtubules of the budding yeast Saccharomyces cerevisiae. This paper describes the modifications to our multimode microscope imaging system [2,3], the acquisition of three-dimensional (3-D) data sets and the computer processing methods we have developed to obtain time-lapse recordings of fluorescent astral microtubule dynamics and nuclear movements over the complete duration of the 90-120 minute yeast cell cycle. This required low excitation light intensity to prevent GFP photobleaching and phototoxicity, efficient light collection by the microscope optics, a cooled charge-coupled device (CCD) camera with high quantum efficiency, and image reconstruction from serial optical sections through the 6 micron-wide yeast cell to see most or all of the astral molecules. Methods are also described for combining fluorescent images of the microtubules labeled with dynein-GFP with high resolution differential interference contrast (DIC) images of nuclear and cellular morphology [4], and fluorescent images of the chromosomes stained with 4,6-diamidino-2-phenylindole (DAPI) [5].

NUCLEOSOME DEPLETION ALTERS THE CHROMATIN STRUCTURE OF SACCHAROMYCES CEREVISIAE CENTROMERES

Saccharomyces cerevisiae centromeric DNA is packaged into a highly nuclease-resistant chromatin core of approximately 200 base pairs of DNA. The structure of the centromere in chromosome III is somewhat larger than a 160-base-pair nucleosomal core and encompasses the conserved centromere DNA elements (CDE I, II, and III). Extensive mutational analysis has revealed the sequence requirements for centromere function. Mutations affecting the segregation properties of centromeres also exhibit altered chromatin structures in vivo. Thus the structure, as delineated by nuclease digestion, correlated with functional centromeres. We have determined the contribution of histone proteins to this unique structural organization. Nucleosome depletion by repression of either histone H2B or H4 rendered the cell incapable of chromosome segregation. Histone repression resulted in increased nuclease sensitivity of centromere DNA, with up to 40% of CEN3 DNA molecules becoming accessible to nucleolytic attack. Nucleosome depletion also resulted in an alteration in the distribution of nuclease cutting sites in the DNA surrounding CEN3. These data provide the first indication that authentic nucleosomal subunits flank the centromere and suggest that nucleosomes may be the central core of the centromere itself.

A High-Resolution Multimode Digital Microscope System

In this chapter we describe the development of a high-resolution, multimode digital imaging system based on a wide-field epifluorescent and transmitted light microscope and a cooled charge-coupled device (CCD) camera. Taylor and colleagues (Farkas et al., 1993; Taylor et al., 1992) have reviewed the advantages of using multiple optical modes to obtain quantitative information about cellular processes and described instrumentation they have developed for multimode digital imaging. The instrument described here is somewhat specialized for our microtubule and mitosis studies, but it is also applicable to a variety of problems in cellular imaging including tracking proteins fused to the green fluorescent protein (GFP) in live cells (Cubitt et al., 1995; Heim and Tsien, 1996; Olson et al., 1995). For example, the instrument has been valuable for correlating the assembly dynamics of individual cytoplasmic microtubules (labeled by conjugating X-rhodamine to tubulin) with the dynamics of membranes of the endoplasmic reticulum (ER, labeled with DiOC6) and the dynamics of the cell cortex [by differential interference contrast (DIC)] in migrating vertebrate epithelial cells (Waterman-Storer and Salmon, 1997). The instrument has also been important in the analysis of mitotic mutants in the powerful yeast genetic system Saccharo-myces cerevisiae. Yeast cells are a major challenge for high-resolution imaging of nuclear or microtubule dynamics because the preanaphase nucleus is only about 2 μm wide in a cell about 6 μm wide. We have developed methods for visualizing nuclear and spindle dynamics during the cell cycle using high-resolution digitally enhanced DIC (DE-DIC) imaging (Yang et al., 1997; Yeh et al., 1995). Using genetic and molecular techniques. Bloom and coworkers (Shaw et al., 1997a,b) have been able to label the cytoplasmic astral microtubules in dividing yeast cells by expression of cytoplasmic dynein fused to GFP. Overlays of GFP and DIC images of dividing cells have provided the opportunity to see for the first time the dynamics of cytoplasmic microtubules in live yeast cells and how these dynamics and microtubule interactions with the cell cortex change with mitotic cell cycle events in wild-type and in mutant strains (Shaw et al., 1997a,b). Our high-resolution multimode digital imaging system is shown in Fig. 1 and diagrammed in Fig. 2. The legend to Fig. 2 provides model numbers and sources of the key components of the instrument. There are three main parts to the system: a Nikon FXA microscope, a Hamamatsu C4880 cooled CCD camera, and a MetaMorph digital imaging system. First we will consider our design criteria for the instrument, then consider separately the major features of the microscope components, the cooled CCD camera, and the MetaMorph digital imaging system. The reader is referred to other sources for general aspects of microscope optics (Inoue and Oldenbourg, 1993; Keller, 1995; Spencer, 1982); DIC microscopy (Salmon, 1998); fluorescence microscopy (Taylor and Salmon, 1989); video cameras, slow-scan cameras, and video microscopy (Aikens et al., 1989; CLMIB staff, 1995; Inoue, 1986; Inoue and Spring, 1997; Shotten, 1995) and 3D imaging methods (Carrington et al., 1995; Hiroka et al., 1991; Pawley, 1995). Fig. 1 Photograph of the multimode digital imaging system including the Nikon FXA upright microscope sitting on an air table (Newport Corp., Irvine, CA, VW-3660 with 4-inch-high tabletop). Images are captured with a Hamamatsu C4880 cooled CCD camera. Image acquisition, ... Fig. 2 Component parts are L1, 100-watt quartz halogen lamp; S1, Uniblitz shutter (No. 225L2A1Z523398, Vincent Associates, Rochester, NY); G, ground glass diffuser: FBI, manual filter changers including KG4 heat cut and green interference filters; I1, field ... II. Design Criteria A. Fluorescence Considerations When we began building our instrument 4 years ago, our primary objective was to obtain quantitative time-lapse records of spindle microtubule dynamics and chromosome movements for mitosis in live tissue culture cells and in in vitro assembled spindles in Xenopus egg extracts. Fluorescence optical components were chosen, in part, based on the fluorophores that were available for labeling microtubules, chromosomes, and other microtubule- or spindle-associated components. Microtubules could be fluorescently labeled along their lengths by incorporating X-rhodamine-labeled tubulin into the cytoplasmic tubulin pool (Fig. 3; Murray et al., 1996; Salmon et al., 1994). In addition, we needed to use fluorescence photoactivation methods to produce local marks on spindle microtubules to study the dynamics of their assembly (Mitchison and Salmon, 1993; Waters et al., 1996). This is accomplished by the addition of a cagedfluorescein-labeled tubulin to the cytoplasmic pool and fluorescence photoactivation with a 360-nm microbeam as described by Mitchison and coworkers (Mitchison, 1989; Sawin and Mitchison, 1991; Sawin et al., 1992). In extracts and some living cells, chromosomes can be vitally stained with the DNA intercalating dyes DAPI or Hoescht 33342 (Murray et al., 1996; Sawin and Mitchison, 1991). Thus, in fluorescence modes, we needed to be able to obtain images in a “red channel” for X-rhodamine microtubules, a “green channel” for photoactivated fluorescein marks, and a “blue channel” for chromosomes. Fig. 3 Views of a living, dividing yeast, Saccharomyces cerevisiae, by (A) fluorescence of GFP protein bound to nuclear histones and (B) DIC.* (C) Images in DIC of the 0.24-μm spacing between rows of surface pores of the diatom Amphipleura illuminated ... B. Live Cell Considerations Photobleaching and photodamage are a major concern during time-lapse imaging of fluorescently tagged molecules in live cells or in in vitro extracts. Minimizing these problems requires that the specimen be illuminated by light shuttered between camera exposures, that the imaging optics have high transmission efficiencies, that the camera detectors have high quantum efficiency at the imaging wavelengths, and that light can be integrated on the detector to reduce illumination intensity and allow longer imaging sessions without photo-damage. This later point is true not only for epifluorescence but also for transmitted light (phase-contrast or DIC) imaging. In our studies, the detector also needed a high dynamic range (12 bits = 4096 gray levels), as we wanted to be able to quantitate the fluorescence of a single microtubule or the whole mitotic spindle, which could have 1000 or more microtubules. In addition, the red, green, and blue images needed to be in focus at the same position on the detector so that the fluorophores within the specimen could be accurately correlated.

Function and Assembly of DNA Looping, Clustering, and Microtubule Attachment Complexes within a Eukaryotic Kinetochore

The kinetochore is a complex protein-DNA assembly that provides the mechanical linkage between microtubules and the centromere DNA of each chromosome. Centromere DNA in all eukaryotes is wrapped around a unique nucleosome that contains the histone H3 variant CENP-A (Cse4p in Saccharomyces cerevisiae). Here, we report that the inner kinetochore complex (CBF3) is required for pericentric DNA looping at the Cse4p-containing nucleosome. DNA within the pericentric loop occupies a spatially confined area that is radially displaced from the interpolar central spindle. Microtubule-binding kinetochore complexes are not involved in pericentric DNA looping but are required for the geometric organization of DNA loops around the spindle microtubules in metaphase. Thus, the mitotic segregation apparatus is a composite structure composed of kinetochore and interpolar microtubules, the kinetochore, and organized pericentric DNA loops. The linkage of microtubule-binding to centromere DNA-looping complexes positions the pericentric chromatin loops and stabilizes the dynamic properties of individual kinetochore complexes in mitosis.

Bub1 Kinase and Sgo1 Modulate Pericentric Chromatin in Response to Altered Microtubule Dynamics

BACKGROUND Tension sensing of bioriented chromosomes is essential for the fidelity of chromosome segregation. The spindle assembly checkpoint (SAC) conveys lack of tension or attachment to the anaphase promoting complex. Components of the SAC (Bub1) phosphorylate histone H2A (S121) and recruit the protector of cohesin, Shugoshin (Sgo1), to the inner centromere. How the chromatin structural modifications of the inner centromere are integrated into the tension sensing mechanisms and the checkpoint are not known. RESULTS We have identified a Bub1/Sgo1-dependent structural change in the geometry and dynamics of kinetochores and the pericentric chromatin upon reduction of microtubule dynamics. The cluster of inner kinetochores contract, whereas the pericentric chromatin and cohesin that encircle spindle microtubules undergo a radial expansion. Despite its increased spatial distribution, the pericentric chromatin is less dynamic. The change in dynamics is due to histone H2A phosphorylation and Sgo1 recruitment to the pericentric chromatin, rather than microtubule dynamics. CONCLUSIONS Bub1 and Sgo1 act as a rheostat to regulate the chromatin spring and maintain force balance. Through histone H2A S121 phosphorylation and recruitment of Sgo1, Bub1 kinase softens the chromatin spring in response to changes in microtubule dynamics. The geometric alteration of all 16 kinetochores and pericentric chromatin reflect global changes in the pericentromeric region and provide mechanisms for mechanically amplifying damage at a single kinetochore microtubule.

Chapter 10 A High-Resolution Multimode Digital Microscope System

This chapter describes the development of a high-resolution, multimode digital imaging system based on a wide-field epifluorescent and transmitted light microscope, and a cooled charge-coupled device (CCD) camera. The three main parts of this imaging system are Nikon FXA microscope, Hamamatsu C4880 cooled CCD camera, and MetaMorph digital imaging system. This chapter presents various design criteria for the instrument and describes the major features of the microscope components—the cooled CCD camera and the MetaMorph digital imaging system. The Nikon FXA upright microscope can produce high resolution images for both epifluorescent and transmitted light illumination without switching the objective or moving the specimen. The functional aspects of the microscope set-up can be considered in terms of the imaging optics, the epi-illumination optics, the transillumination optics, the focus control, and the vibration isolation table. This instrument is somewhat specialized for microtubule and mitosis studies, and it is also applicable to a variety of problems in cellular imaging, including tracking proteins fused to the green fluorescent protein in live cells. The instrument is also valuable for correlating the assembly dynamics of individual cytoplasmic microtubules (labeled by conjugating X-rhodamine to tubulin) with the dynamics of membranes of the endoplasmic reticulum (labeled with DiOC6) and the dynamics of the cell cortex (by differential interference contrast) in migrating vertebrate epithelial cells. This imaging system also plays an important role in the analysis of mitotic mutants in the powerful yeast genetic system Saccharomyces cerevisiae.

A 3D Map of the Yeast Kinetochore Reveals the Presence of Core and Accessory Centromere-Specific Histone

The budding yeast kinetochore is ~68 nm in length with a diameter slightly larger than a 25 nm microtubule. The kinetochores from the 16 chromosomes are organized in a stereotypic cluster encircling central spindle microtubules. Quantitative analysis of the inner kinetochore cluster (Cse4, COMA) reveals structural features not apparent in singly attached kinetochores. The cluster of Cse4-containing kinetochores is physically larger perpendicular to the spindle axis relative to the cluster of Ndc80 molecules. If there was a single Cse4 (molecule or nucleosome) at the kinetochore attached to each microtubule plus end, the cluster of Cse4 would appear geometrically identical to Ndc80. Thus, the structure of the inner kinetochore at the surface of the chromosomes remains unsolved. We have used point fluorescence microscopy and statistical probability maps to deduce the two-dimensional mean position of representative components of the yeast kinetochore relative to the mitotic spindle in metaphase. Comparison of the experimental images to three-dimensional architectures from convolution of mathematical models reveals a pool of Cse4 radially displaced from Cse4 at the kinetochore and kinetochore microtubule plus ends. The pool of displaced Cse4 can be experimentally depleted in mRNA processing pat1Δ or xrn1Δ mutants. The peripheral Cse4 molecules do not template outer kinetochore components. This study suggests an inner kinetochore plate at the centromere-microtubule interface in budding yeast and yields information on the number of Ndc80 molecules at the microtubule attachment site.

Nuclear oscillations and nuclear filament formation accompany single-strand annealing repair of a dicentric chromosome in Saccharomyces cerevisiae

Dicentric chromosomes undergo breakage during mitosis as a result of the attachment of two centromeres on one sister chromatid to opposite spindle poles. Studies utilizing a conditional dicentric chromosome III in Saccharomyces cerevisiae have shown that dicentric chromosome repair occurs primarily by deletion of one centromere via a RAD52-dependent recombination pathway. We report that dicentric chromosome resolution requires RAD1, a gene involved in the single-strand annealing DNA repair pathway. We additionally show that single-strand annealing repair of a dicentric chromosome can occur in the absence of RAD52. RAD52-independent repair requires the adaptation-defective cdc5-ad allele of the yeast polo kinase and the DNA damage checkpoint gene RAD9. Dicentric chromosome breakage in cdc5-ad rad52 mutant cells is associated with a prolonged mitotic arrest, during which nuclei undergo microtubule-dependent oscillations, accompanied by dynamic changes in nuclear morphology. We further demonstrate that the frequency of spontaneous direct repeat recombination is suppressed in yeast cells treated with benomyl, a drug that perturbs microtubules. Our findings indicate that microtubule-dependent processes facilitate recombination.