How the kinetochore couples microtubule force and centromere stretch to move chromosomes

The Ndc80 complex (Ndc80, Nuf2, Spc24, Spc25) is a highly conserved kinetochore protein essential for end-on anchorage to spindle microtubule plus-ends and for force generation coupled to plus-end polymerization and depolymerization. Spc24/Spc25 at one end of the Ndc80 complex binds the kinetochore. The N-terminal tail and CH domains of Ndc80 bind microtubules, and an internal domain binds microtubule-associated proteins (MAPs) such as the Dam1 complex. To determine how the microtubule and MAP binding domains of Ndc80 contribute to force production at the kinetochore in budding yeast, we have inserted a FRET tension sensor into the Ndc80 protein about halfway between its microtubule binding and internal loop domains. The data support a mechanical model of force generation at metaphase where the position of the kinetochore relative to the microtubule plus-end reflects the relative strengths of microtubule depolymerization, centromere stretch and microtubule binding interactions with Ndc80 and Dam1 complexes

Bending the Rules: Widefield Microscopy and the Abbe Limit of Resolution

One of the most fundamental concepts of microscopy is that of resolution–the ability to clearly distinguish two objects as separate. Recent advances such as structured illumination microscopy (SIM) and point localization techniques including photoactivated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM) strive to overcome the inherent limits of resolution of the modern light microscope. These techniques, however, are not always feasible or optimal for live cell imaging. Thus, in this review, we explore three techniques for extracting high resolution data from images acquired on a widefield microscope–deconvolution, model convolution, and Gaussian fitting. Deconvolution is a powerful tool for restoring a blurred image using knowledge of the point spread function (PSF) describing the blurring of light by the microscope, although care must be taken to ensure accuracy of subsequent quantitative analysis. The process of model convolution also requires knowledge of the PSF to blur a simulated image which can then be compared to the experimentally acquired data to reach conclusions regarding its geometry and fluorophore distribution. Gaussian fitting is the basis for point localization microscopy, and can also be applied to tracking spot motion over time or measuring spot shape and size. All together, these three methods serve as powerful tools for high-resolution imaging using widefield microscopy

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

Tension sensing of bi-oriented 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

The microtubule-based motor Kar3 and plus end–binding protein Bim1 provide structural support for the anaphase spindle

In budding yeast, the mitotic spindle is comprised of 32 kinetochore microtubules (kMTs) and ∼8 interpolar MTs (ipMTs). Upon anaphase onset, kMTs shorten to the pole, whereas ipMTs increase in length. Overlapping MTs are responsible for the maintenance of spindle integrity during anaphase. To dissect the requirements for anaphase spindle stability, we introduced a conditionally functional dicentric chromosome into yeast. When centromeres from the same sister chromatid attach to opposite poles, anaphase spindle elongation is delayed and a DNA breakage-fusion-bridge cycle ensues that is dependent on DNA repair proteins. We find that cell survival after dicentric chromosome activation requires the MT-binding proteins Kar3p, Bim1p, and Ase1p. In their absence, anaphase spindles are prone to collapse and buckle in the presence of a dicentric chromosome. Our analysis reveals the importance of Bim1p in maintaining a stable ipMT overlap zone by promoting polymerization of ipMTs during anaphase, whereas Kar3p contributes to spindle stability by cross-linking spindle MTs

The spatial segregation of pericentric cohesin and condensin in the mitotic spindle

The mitotic chromatin spring is organized into a rosette of intramolecular loops of pericentric chromatin by condensin and cohesin. Model convolution reveals that condensin clusters along the spindle axis, while cohesin is dispersed radially along pericentromere loops.In mitosis, the pericentromere is organized into a spring composed of cohesin, condensin, and a rosette of intramolecular chromatin loops. Cohesin and condensin are enriched in the pericentromere, with spatially distinct patterns of localization. Using model convolution of computer simulations, we deduce the mechanistic consequences of their spatial segregation. Condensin lies proximal to the spindle axis, whereas cohesin is radially displaced from condensin and the interpolar microtubules. The histone deacetylase Sir2 is responsible for the axial position of condensin, while the radial displacement of chromatin loops dictates the position of cohesin. The heterogeneity in distribution of condensin is most accurately modeled by clusters along the spindle axis. In contrast, cohesin is evenly distributed (barrel of 500-nm width × 550-nm length). Models of cohesin gradients that decay from the centromere or sister cohesin axis, as previously suggested, do not match experimental images. The fine structures of cohesin and condensin deduced with subpixel localization accuracy reveal critical features of how these complexes mold pericentric chromatin into a functional spring

Pat1 protects centromere-specific histone H3 variant Cse4 from Psh1-mediated ubiquitination

A novel Pat1-dependent mechanism is identified for the protection of kinetochore-associated Cse4 from ubiquitination in order to ensure faithful chromosome segregation and genomic stability.Evolutionarily conserved histone H3 variant Cse4 and its homologues are essential components of specialized centromere (CEN)-specific nucleosomes and serve as an epigenetic mark for CEN identity and propagation. Cse4 is a critical determinant for the structure and function of the kinetochore and is required to ensure faithful chromosome segregation. The kinetochore protein Pat1 regulates the levels and spatial distribution of Cse4 at centromeres. Deletion of PAT1 results in altered structure of CEN chromatin and chromosome segregation errors. In this study, we show that Pat1 protects CEN-associated Cse4 from ubiquitination in order to maintain proper structure and function of the kinetochore in budding yeast. PAT1-deletion strains exhibit increased ubiquitination of Cse4 and faster turnover of Cse4 at kinetochores. Psh1, a Cse4-specific E3-ubiquitin ligase, interacts with Pat1 in vivo and contributes to the increased ubiquitination of Cse4 in pat1∆ strains. Consistent with a role of Psh1 in ubiquitination of Cse4, transient induction of PSH1 in a wild-type strain resulted in phenotypes similar to a pat1∆ strain, including a reduction in CEN-associated Cse4, increased Cse4 ubiquitination, defects in spatial distribution of Cse4 at kinetochores, and altered structure of CEN chromatin. Pat1 interacts with Scm3 and is required for its maintenance at kinetochores. In conclusion, our studies provide novel insights into mechanisms by which Pat1 affects the structure of CEN chromatin and protects Cse4 from Psh1-mediated ubiquitination for faithful chromosome segregation

Individual pericentromeres display coordinated motion and stretching in the yeast spindle

During mitosis, cohesin and condensin cross-link pericentromeres of different chromosomes to coordinate centromere attachment sites.The mitotic segregation apparatus composed of microtubules and chromatin functions to faithfully partition a duplicated genome into two daughter cells. Microtubules exert extensional pulling force on sister chromatids toward opposite poles, whereas pericentric chromatin resists with contractile springlike properties. Tension generated from these opposing forces silences the spindle checkpoint to ensure accurate chromosome segregation. It is unknown how the cell senses tension across multiple microtubule attachment sites, considering the stochastic dynamics of microtubule growth and shortening. In budding yeast, there is one microtubule attachment site per chromosome. By labeling several chromosomes, we find that pericentromeres display coordinated motion and stretching in metaphase. The pericentromeres of different chromosomes exhibit physical linkage dependent on centromere function and structural maintenance of chromosomes complexes. Coordinated motion is dependent on condensin and the kinesin motor Cin8, whereas coordinated stretching is dependent on pericentric cohesin and Cin8. Linking of pericentric chromatin through cohesin, condensin, and kinetochore microtubules functions to coordinate dynamics across multiple attachment sites

Free Zinc as a Predictive Marker for COVID-19 Mortality Risk

Free zinc is considered to be the exchangeable and biological active form of zinc in serum, and is discussed to be a suitable biomarker for alterations in body zinc homeostasis and related diseases. Given that coronavirus disease 2019 (COVID-19) is characterized by a marked decrease in total serum zinc, and clinical data indicate that zinc status impacts the susceptibility and severity of the infection, we hypothesized that free zinc in serum might be altered in response to SARS-CoV-2 infection and may reflect disease severity. To test this hypothesis, free zinc concentrations in serum samples of survivors and nonsurvivors of COVID-19 were analyzed by fluorometric microassay. Similar to the reported total serum zinc deficit measured by total reflection X-ray fluorescence, free serum zinc in COVID-19 patients was considerably lower than that in control subjects, and surviving patients displayed significantly higher levels of free zinc than those of nonsurvivors (mean ± SD; 0.4 ± 0.2 nM vs. 0.2 ± 0.1 nM; p = 0.0004). In contrast to recovering total zinc concentrations (r = 0.706, p < 0.001) or the declining copper–zinc ratio (r = −0.646; p < 0.001), free zinc concentrations remained unaltered with time in COVID-19 nonsurvivors. Free serum zinc concentrations were particularly low in male as compared to female patients (mean ± SD; 0.4 ± 0.2 nM vs. 0.2 ± 0.1 nM; p = 0.0003). This is of particular interest, as the male sex is described as a risk factor for severe COVID-19. Overall, results indicate that depressed free serum zinc levels are associated with increased risk of death in COVID-19, suggesting that free zinc may serve as a novel prognostic marker for the severity and course of COVID-19.DFG, 316442145, FOR 2558: Interaktionen von essenziellen Spurenelementen in gesunden und erkrankten älteren Menschen(TraceAge)DFG, 424957847, TRR 296: Lokale Kontrolle der Schilddrüsenhormonwirkung (LocoTact

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

The budding yeast kinetochore is ~68nm in length with a diameter slightly larger than a 25nm microtubule [1]. 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 [2]. If there were 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