Tension sensors reveal how the kinetochore shares its load

At metaphase in mitotic cells, pulling forces at the kinetochore-microtubule interface create tension by stretching the centromeric chromatin between oppositely oriented sister kinetochores. This tension is important for stabilizing the end-on kinetochore microtubule attachment required for proper bi-orientation of sister chromosomes as well as for satisfaction of the Spindle Assembly Checkpoint and entry into anaphase. How force is coupled by proteins to kinetochore microtubules and resisted by centromere stretch is becoming better understood as many of the proteins involved have been identified. Recent application of genetically encoded fluorescent tension sensors within the mechanical linkage between the centromere and kinetochore microtubules are beginning to reveal – from live cell assays – protein specific contributions that are functionally important

The Minus End-Directed Motor Kar3 Is Required for Coupling Dynamic Microtubule Plus Ends to the Cortical Shmoo Tip in Budding Yeast

The budding yeast shmoo tip is a model system for analyzing mechanisms coupling force production to microtubule plus-end polymerization/depolymerization. Dynamic plus ends of astral microtubules interact with the shmoo tip in mating yeast cells, positioning nuclei for karyogamy [1]. We have used live-cell imaging of GFP fusions to identify proteins that couple dynamic microtubule plus ends to the shmoo tip. We find that Kar3p, a minus end-directed kinesin motor protein, is required, whereas the other cytoplasmic motors, dynein and the kinesins Kip2p and Kip3p, are not. In the absence of Kar3p, attached microtubule plus ends released from the shmoo tip when they switched to depolymerization. Furthermore, microtubules in cells expressing kar3-1, a mutant that results in rigor binding to microtubules [2], were stabilized specifically at shmoo tips. Imaging of Kar3p-GFP during mating revealed that fluorescence at the shmoo tip increased during periods of microtubule depolymerization. These data are the first to localize the activity of a minus end-directed kinesin at the plus ends of microtubules. We propose a model in which Kar3p couples depolymerizing microtubule plus ends to the cell cortex and the Bim1p-Kar9p protein complex maintains attachment during microtubule polymerization. In support of this model, analysis of Bim1p-GFP at the shmoo tip results in a localization pattern complementary to that of Kar3p-GFP

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

Higher twists in the pion structure function

We calculate the QCD moments of the pion structure function using Drell-Yan data on the quark distributions in the pion and a phenomenological model for the resonance region. The extracted higher twist corrections are found to be larger than those for the nucleon, contributing around 50% of the lowest moment at Q^2=1 GeV^2.Comment: 8 pages, 3 figures, to appear in Phys. Rev.

In Vivo Protein Architecture of the Eukaryotic Kinetochore with Nanometer Scale Accuracy

The kinetochore is a macromolecular protein machine [1] that links centromeric chromatin to the plus-ends of one or more microtubules (MT), and segregates chromosomes during cell division. Its core structure consists of eight multi-component protein complexes, most of which are conserved in all eukaryotes. We use an in vivo two-color fluorescence microscopy technique to determine, for the first time, the location of these proteins along the budding yeast kinetochore axis at nanometer resolution. Together with kinetochore protein counts [2, 3], these localizations predict the 3-D protein architecture of a kinetochore-microtubule attachment, and provide new functional insights. We also find that the kinetochore becomes much shorter in anaphase as metaphase tension is lost. Shortening is mainly due to a decrease in the length of the Ndc80 complex, which may result either from intra-molecular bending of the Ndc80 complex at the kink within the stalk region of the Ndc80/Nuf2 dimer [4, 5] or from a change in its orientation relative to the microtubule axis. Conformational changes within the Ndc80 and Mtw1 complexes may serve as mechanical cues for tension-dependent regulation of MT attachment and the spindle assembly checkpoint. The geometry of the core structure of the budding yeast kinetochore reported here is remarkably similar to that found in mammalian kinetochores, indicating that kinetochore structure is conserved in eukaryotes with either point or regional centromeres

Coordinated Spindle Assembly and Orientation Requires Clb5p-Dependent Kinase in Budding Yeast

The orientation of the mitotic spindle along a polarity axis is critical in asymmetric cell divisions. In the budding yeast, Saccharomyces cerevisiae, loss of the S-phase B-type cyclin Clb5p under conditions of limited cyclin-dependent kinase activity (cdc28-4 clb5Δ cells) causes a spindle positioning defect that results in an undivided nucleus entering the bud. Based on time-lapse digital imaging microscopy of microtubules labeled with green fluorescent protein fusions to either tubulin or dynein, we observed that the asymmetric behavior of the spindle pole bodies during spindle assembly was lost in the cdc28-4 clb5Δ cells. As soon as a spindle formed, both poles were equally likely to interact with the bud cell cortex. Persistent dynamic interactions with the bud ultimately led to spindle translocation across the bud neck. Thus, the mutant failed to assign one spindle pole body the task of organizing astral microtubules towards the mother cell. Our data suggest that Clb5p-associated kinase is required to confer mother-bound behavior to one pole in order to establish correct spindle polarity. In contrast, B-type cyclins, Clb3p and Clb4p, though partially redundant with Clb5p for an early role in spindle morphogenesis, preferentially promote spindle assembly

Asymptotic Freedom for Non-Relativistic Confinement

Some aspects of asymptotic freedom are discussed in the context of a simple two-particle non-relativisitic confining potential model. In this model asymptotic freedom follows from the similarity of the free-particle and bound state radial wave functions at small distances and for the same angular momentum and the same large energy. This similarity, which can be understood using simple quantum mechanical arguments, can be used to show that the exact response function approaches that obtained when final state interactions are ignored. A method of calculating corrections to this limit is given and explicit examples are given for the case of the harmonic oscillator.Comment: 16 pages, 5 figures, RevTex

Subprocess Size in Hard Exclusive Scattering

The interaction region of hard exclusive hadron scattering can have a large transverse size due to endpoint contributions, where one parton carries most of the hadron momentum. The endpoint region is enhanced and can dominate in processes involving multiple scattering and quark helicity flip. The endpoint Fock states have perturbatively short lifetimes and scatter softly in the target. We give plausible arguments that endpoint contributions can explain the apparent absence of color transparency in fixed angle exclusive scattering and the dimensional scaling of transverse rho photoproduction at high momentum transfer, which requires quark helicity flip. We also present a quantitative estimate of Sudakov effects.Comment: 16 pages, 4 figures, JHEP style; v2: quantitative estimate of Sudakov effects and more detailed discussion of endpoint behaviour of meson distribution amplitude added, few other clarifications, version to appear in Phys. Rev.

Extraction of the D13(1520) photon-decay couplings from pion- and eta-photoproduction data

We compare results for the D13(1520) photon-decay amplitudes determined in analyses of eta- and pion-photoproduction data. The ratio of helicity amplitudes (A_3/2 / A_1/2), determined from eta-photoproduction data, is quite different from that determined in previous analyses of pion-photoproduction data. We consider how strongly the existing pion-photoproduction data constrain both this ratio and the individual photon-decay amplitudes.Comment: 7 pages, 2 figure

Molecular architecture of the kinetochore-microtubule attachment site is conserved between point and regional centromeres

Point and regional centromeres specify a unique site on each chromosome for kinetochore assembly. The point centromere in budding yeast is a unique 150-bp DNA sequence, which supports a kinetochore with only one microtubule attachment. In contrast, regional centromeres are complex in architecture, can be up to 5 Mb in length, and typically support many kinetochore-microtubule attachments. We used quantitative fluorescence microscopy to count the number of core structural kinetochore protein complexes at the regional centromeres in fission yeast and Candida albicans. We find that the number of CENP-A nucleosomes at these centromeres reflects the number of kinetochore-microtubule attachments instead of their length. The numbers of kinetochore protein complexes per microtubule attachment are nearly identical to the numbers in a budding yeast kinetochore. These findings reveal that kinetochores with multiple microtubule attachments are mainly built by repeating a conserved structural subunit that is equivalent to a single microtubule attachment site