Yimin Dong

CCAN Makes Multiple Contacts with Centromeric DNA to Provide Distinct Pathways to the Outer Kinetochore

Kinetochore specification and assembly requires the targeted deposition of specialized nucleosomes containing the histone H3 variant CENP-A at centromeres. However, CENP-A is not sufficient to drive full-kinetochore assembly, and it is not clear how centromeric chromatin is established. Here, we identify CENP-W as a component of the DNA-proximal constitutive centromere-associated network (CCAN) of proteins. We demonstrate that CENP-W forms a DNA-binding complex together with the CCAN component CENP-T. This complex directly associates with nucleosomal DNA and with canonical histone H3, but not with CENP-A, in centromeric regions. CENP-T/CENP-W functions upstream of other CCAN components with the exception of CENP-C, an additional putative DNA-binding protein. Our analysis indicates that CENP-T/CENP-W and CENP-C provide distinct pathways to connect the centromere with outer kinetochore assembly. In total, our results suggest that the CENP-T/CENP-W complex is directly involved in establishment of centromere chromatin structure coordinately with CENP-A.

A super-resolution map of the vertebrate kinetochore

A longstanding question in centromere biology has been the organization of CENP-A–containing chromatin and its implications for kinetochore assembly. Here, we have combined genetic manipulations with deconvolution and super-resolution fluorescence microscopy for a detailed structural analysis of chicken kinetochores. Using fluorescence microscopy with subdiffraction spatial resolution and single molecule sensitivity to map protein localization in kinetochore chromatin unfolded by exposure to a low salt buffer, we observed robust amounts of H3K9me3, but only low levels of H3K4me2, between CENP-A subdomains in unfolded interphase prekinetochores. Constitutive centromere-associated network proteins CENP-C and CENP-H localize within CENP-A–rich subdomains (presumably on H3-containing nucleosomes) whereas CENP-T localizes in interspersed H3-rich blocks. Although interphase prekinetochores are relatively more resistant to unfolding than sur-rounding pericentromeric heterochromatin, mitotic kinetochores are significantly more stable, reflecting mitotic kinetochore maturation. Loss of CENP-H, CENP-N, or CENP-W had little or no effect on the unfolding of mitotic kinetochores. However, loss of CENP-C caused mitotic kinetochores to unfold to the same extent as their interphase counterparts. Based on our results we propose a new model for inner centromeric chromatin architecture in which chromatin is folded as a layered boustrophedon, with planar sinusoids containing interspersed CENP-A–rich and H3-rich subdomains oriented toward the outer kinetochore. In mitosis, a CENP-C–dependent mechanism crosslinks CENP-A blocks of different layers together, conferring extra stability to the kinetochore.

Condensin Regulates the Stiffness of Vertebrate Centromeres

When chromosomes are aligned and bioriented at metaphase, the elastic stretch of centromeric chromatin opposes pulling forces exerted on sister kinetochores by the mitotic spindle. Here we show that condensin ATPase activity is an important regulator of centromere stiffness and function. Condensin depletion decreases the stiffness of centromeric chromatin by 50% when pulling forces are applied to kinetochores. However, condensin is dispensable for the normal level of compaction (rest length) of centromeres, which probably depends on other factors that control higher-order chromatin folding. Kinetochores also do not require condensin for their structure or motility. Loss of stiffness caused by condensin-depletion produces abnormal uncoordinated sister kinetochore movements, leads to an increase in Mad2(+) kinetochores near the metaphase plate and delays anaphase onset.

Extracellular Fibrils of Pathogenic Yeast Cryptococcus gattii Are Important for Ecological Niche, Murine Virulence and Human Neutrophil Interactions

Cryptococcus gattii, an emerging fungal pathogen of humans and animals, is found on a variety of trees in tropical and temperate regions. The ecological niche and virulence of this yeast remain poorly defined. We used Arabidopsis thaliana plants and plant-derived substrates to model C. gattii in its natural habitat. Yeast cells readily colonized scratch-wounded plant leaves and formed distinctive extracellular fibrils (40–100 nm diameter ×500–3000 nm length). Extracellular fibrils were observed on live plants and plant-derived substrates by scanning electron microscopy (SEM) and by high voltage- EM (HVEM). Only encapsulated yeast cells formed extracellular fibrils as a capsule-deficient C. gattii mutant completely lacked fibrils. Cells deficient in environmental sensing only formed disorganized extracellular fibrils as apparent from experiments with a C. gattii STE12α mutant. C. gattii cells with extracellular fibrils were more virulent in murine model of pulmonary and systemic cryptococcosis than cells lacking fibrils. C. gattii cells with extracellular fibrils were also significantly more resistant to killing by human polymorphonuclear neutrophils (PMN) in vitro even though these PMN produced elaborate neutrophil extracellular traps (NETs). These observations suggest that extracellular fibril formation could be a structural adaptation of C. gattii for cell-to-cell, cell-to-substrate and/or cell-to- phagocyte communications. Such ecological adaptation of C. gattii could play roles in enhanced virulence in mammalian hosts at least initially via inhibition of host PMN– mediated killing.

Contrasting models for kinetochore microtubule attachment in mammalian cells

Kinetochore function is mediated through its interaction with microtubule plus ends embedded in the kinetochore outer plate. Here, we compare and evaluate current models for kinetochore microtubule attachment, beginning with a brief review of the molecular, biochemical, cellular, and structural studies upon which these models are based. The majority of these studies strongly support a model in which the kinetochore outer plate is a network of fibers that form multiple weak attachments to each microtubule, chiefly through the Ndc80 complex. Multiple weak attachments enable kinetochores to remain attached to microtubule plus ends that are continually growing and shrinking. It is unlikely that rings or “kinetochore fibrils” have a significant role in kinetochore microtubule attachment, but such entities could have a role in stabilizing attachment, modifying microtubule dynamics, and harnessing the energy released from microtubule disassembly. It is currently unclear whether kinetochores control and coordinate the dynamics of individual kinetochore microtubules.

Releasing the spindle assembly checkpoint without tension

Eukaryotic cells have evolved a spindle assembly checkpoint (SAC) that facilitates accurate genomic segregation during mitosis by delaying anaphase onset in response to errors in kinetochore microtubule attachment. In contrast to the well-studied molecular mechanism by which the SAC blocks anaphase onset, the events triggering SAC release are poorly understood. Papers in this issue by Uchida et al. (Uchida, K.S.K., K. Takagaki, K. Kumada, Y. Hirayama, T. Noda, and T. Hirota. 2009. J. Cell Biol. 184:383–390) and Maresca and Salmon (Maresca, T.J., and E.D. Salmon. 2009. J. Cell Biol. 184:373–381) make an important advance by demonstrating that SAC release depends on molecular rearrangements within the kinetochore rather than tension-produced stretch between sister kinetochores.

Ultrastructure of Nocodazole-Treated PtK1 Kinetochore after High-Pressure Freezing and Freeze-Substitution

Accurate chromosome segregation is critical to the long-term survival of all organisms. In eukarotes, the process is dependent upon a dynamic interaction between microtubules and kinetochores [1, 2]. Recent work has demonstrated that kinetochores in S. cerevisiae are composed of over 60 molecular components arranged into at least 14 different complexes [3]. Currently there is only a rudimentary understanding of how these components are arranged at the ultrastructural level. Classical serial section studies of conventionally fixed specimens established the tri-laminar model for vertebrate kinetochores consisting of an inner plate, translucent zone, and an outer plate [4]. Prior to attaching microtubules the outer plate has a robust fibrous corona on its distal surface. Electron tomographic reconstructions indicated a complex pattern of fibrous arrangements in the outer plate that sometimes formed parallel rows and discrete unit substructures [5]. Studies using high-pressure freezing (HPF) and freezesubstitution (FS) revealed that outer plate is a delicate fibrous mat structure composed of a fiberlike element running parallel to the chromatin surface and fibers radiating from the heterochromatin [6]. The mat is wider than the plate observed in conventional preparations, and the translucent middle layer is largely absent. The corona appears as a ribosome-exclusion zone apparently occupied by very fine fibers. These data demonstrate kinetochore sensitivity to general collapse and condensation when using conventional fixation methods.