Cryo-electron tomography: The challenge of doing structural biology in situ

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.     

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.     

Cohesin, condensin, and the intramolecular centromere loop together generate the mitotic chromatin spring

Sister chromatid cohesion provides the mechanistic basis, together with spindle microtubules, for generating tension between bioriented chromosomes in metaphase. Pericentric chromatin forms an intramolecular loop that protrudes bidirectionally from the sister chromatid axis. The centromere lies on the surface of the chromosome at the apex of each loop. The cohesin and condensin structural maintenance of chromosomes (SMC) protein complexes are concentrated within the pericentric chromatin, but whether they contribute to tension-generating mechanisms is not known. To understand how pericentric chromatin is packaged and resists tension, we map the position of cohesin (SMC3), condensin (SMC4), and pericentric LacO arrays within the spindle. Condensin lies proximal to the spindle axis and is responsible for axial compaction of pericentric chromatin. Cohesin is radially displaced from the spindle axis and confines pericentric chromatin. Pericentric cohesin and condensin contribute to spindle length regulation and dynamics in metaphase. Together with the intramolecular centromere loop, these SMC complexes constitute a molecular spring that balances spindle microtubule force in metaphase.     

Kinetochores and chromatin diminution in early embryos of Parascaris univalens

In Parascaris the mitotic chromosomes of gonial germline cells are holocentric and possess a continuous kinetochore along their entire length. By contrast, in meiotic cells, the centromeric activity is restricted to the heterochromatic tips where direct insertion of spindle microtubules into chromatin without any kinetochore plate is seen. In the presomatic cells of early embryos, which undergo heterochromatin elimination, only euchromatin shows kinetic activity. After developing a technique to separate the very resistant egg shell from the embryos, we studied the cell divisions during early embryogenesis by immunochemical and EM approaches. The results reported here show that in presomatic cells microtubules bind only the euchromatin where a continuous kinetochore plate is present. We also report observations suggesting that the binding of the long kinetochores to the mitotic spindle initiates to a limited number of sites and extends along the entire length, during chromosome condensation. The existence of different centromere stages in different cell types, rends Parascaris chromosomes a very good model to study centromere organization.     

Centromeres and kinetochores: from epigenetics to mitotic checkpoint signaling

The centromere is a chromosomal locus that ensures delivery of one copy of each chromosome to each daughter at cell division. Efforts to understand the nature and specification of the centromere have demonstrated that this central element for ensuring inheritance is itself epigenetically determined. The kinetochore, the protein complex assembled at each centromere, serves as the attachment site for spindle microtubules and the site at which motors generate forces to power chromosome movement. Unattached kinetochores are also the signal generators for the mitotic checkpoint, which arrests mitosis until all kinetochores have correctly attached to spindle microtubules, thereby representing the major cell cycle control mechanism protecting against loss of a chromosome (aneuploidy).     

Epigenetics regulate centromere formation and kinetochore function

The eukaryote centromere was initially defined cytologically as the primary constriction on vertebrate chromosomes and functionally as a chromosomal feature with a relatively low recombination frequency. Structurally, the centromere is the foundation for sister chromatid cohesion and kinetochore formation. Together these provide the basis for interaction between chromosomes and the mitotic spindle, allowing the efficient segregation of sister chromatids during cell division. Although centromeric (CEN) DNA is highly variable between species, in all cases the functional centromere forms in a chromatin domain defined by the substitution of histone H3 with the centromere specific H3 variant centromere protein A (CENP-A), also known as CENH3. Kinetochore formation and function are dependent on a variety of regional epigenetic modifications that appear to result in a loop chromatin conformation providing exterior CENH3 domains for kinetochore construction, and interior heterochromatin domains essential for sister chromatid cohesion. In addition pericentric heterochromatin provides a structural element required for spindle assembly checkpoint function. Advances in our understanding of CENH3 biology have resulted in a model where kinetochore location is specified by the epigenetic mark left after dilution of CENH3 to daughter DNA strands during S phase. This results in a self-renewing and self-reinforcing epigenetic state favorable to reliably mark centromere location, as well as to provide the optimal chromatin configuration for kinetochore formation and function.     

Pericentric chromatin is an elastic component of the mitotic spindle

BACKGROUND: Prior to chromosome segregation, the mitotic spindle bi-orients and aligns sister chromatids along the metaphase plate. During metaphase, spindle length remains constant, which suggests that spindle forces (inward and outward) are balanced. The contribution of microtubule motors, regulators of microtubule dynamics, and cohesin to spindle stability has been previously studied. In this study, we examine the contribution of chromatin structure on kinetochore positioning and spindle-length control. After nucleosome depletion, by either histone H3 or H4 repression, spindle organization was examined by live-cell fluorescence microscopy. RESULTS: Histone repression led to a 2-fold increase in sister-centromere separation and an equal increase in metaphase spindle length. Histone H3 repression does not impair kinetochores, whereas H4 repression disrupts proper kinetochore function. Deletion of outward force generators, kinesins Cin8p and Kip1p, shortens the long spindles observed in histone-repressed cells. Oscillatory movements of individual sister chromatid pairs are not altered after histone repression. CONCLUSIONS: The increase in spindle length upon histone repression and restoration of wild-type spindle length by the loss of plus-end-directed motors suggests that during metaphase, centromere separation and spindle length are governed in part by the stretching of pericentric chromatin. Chromatin is an elastic molecule that is stretched in direct opposition to the outward force generators Cin8p and Kip1p. Thus, we assign a new role to chromatin packaging as an integral biophysical component of the mitotic apparatus.     

Where is the right path heading from the centromere to spindle microtubules?

The kinetochore is a large protein complex that ensures accurate chromosome segregation during mitosis by connecting the centromere and spindle microtubules. One of the kinetochore sub-complexes, the constitutive centromere-associated network (CCAN), associates with the centromere and recruits another sub-complex, the KMN (KNL1, Mis12, and Ndc80 complexes) network (KMN), which binds to spindle microtubules. The CCAN-KMN interaction is mediated by two parallel pathways (CENP-C- and CENP-T-pathways) in the kinetochore, which bridge the centromere and microtubules. Here, we discuss dynamic protein-interaction changes in the two pathways that couple the centromere with spindle microtubules during mitotic progression.     

Incorporation of Drosophila CID/CENP-A and CENP-C into centromeres during early embryonic anaphase

The centromere/kinetochore complex is indispensable for accurate segregation of chromosomes during cell divisions when it serves as the attachment site for spindle microtubules. Centromere identity in metazoans is believed to be governed by epigenetic mechanisms, because the highly repetitive centromeric DNA is neither sufficient nor required for specifying the assembly site of the kinetochore. A candidate for an epigenetic mark is the centromere-specific histone H3 variant CENP-A that replaces H3 in alternating blocks of chromatin exclusively in active centromeres. CENP-A acts as an initiator of kinetochore assembly, but the detailed dynamics of the deposition of metazoan CENP-A and of other constitutive kinetochore components are largely unknown. Here we show by quantitative fluorescence measurements in living early embryos that functional fluorescent fusion proteins of the Drosophila CENP-A and CENP-C homologs are rapidly incorporated into centromeres during anaphase. This incorporation is independent of ongoing DNA synthesis and pulling forces generated by the mitotic spindle, but strictly coupled to mitotic progression. Thus, our findings uncover a strikingly dynamic behavior of centromere components in anaphase.     

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

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.     

Structure of centromere chromatin: from nucleosome to chromosomal architecture

The centromere is essential for the segregation of chromosomes, as it serves as attachment site for microtubules to mediate chromosome segregation during mitosis and meiosis. In most organisms, the centromere is restricted to one chromosomal region that appears as primary constriction on the condensed chromosome and is partitioned into two chromatin domains: The centromere core is characterized by the centromere-specific histone H3 variant CENP-A (also called cenH3) and is required for specifying the centromere and for building the kinetochore complex during mitosis. This core region is generally flanked by pericentric heterochromatin, characterized by nucleosomes containing H3 methylated on lysine 9 (H3K9me) that are bound by heterochromatin proteins. During mitosis, these two domains together form a three-dimensional structure that exposes CENP-A-containing chromatin to the surface for interaction with the kinetochore and microtubules. At the same time, this structure supports the tension generated during the segregation of sister chromatids to opposite poles. In this review, we discuss recent insight into the characteristics of the centromere, from the specialized chromatin structures at the centromere core and the pericentromere to the three-dimensional organization of these regions that make up the functional centromere.     

着丝粒核小体结构研究进展

着丝粒是构成真核生物染色体的必需元件。在细胞有丝分裂或减数分裂时,微管通过动粒与染色体着丝粒连接,参与细胞分裂的染色体分离与分配过程,使染色体平均分配到子细胞中。构成着丝粒的基本单位是着丝粒特异的核小体,与常规核小体不同的是着丝粒核小体中的组蛋白H3被其变种——着丝粒组蛋白H3所替换。最近几年,着丝粒核小体的结构成为细胞生物学研究的热点之一。该文综述了最近在多种真核生物研究中,通过体外和体内实验,提出的着丝粒核小体结构的八聚体、六聚体、同型四聚体以及半八聚体模型,并对着丝粒核小体结构的动态模型与功能的关系进行了探讨。     

Focus on the centre: the role of chromatin on the regulation of centromere identity and function

The centromere is a specialised chromosomal structure that regulates faithful chromosome segregation during cell division, as it dictates the site of assembly of the kinetochore, a critical structure that mediates binding of chromosomes to the spindle, monitors bipolar attachment and pulls chromosomes to the poles during anaphase. Identified more than a century ago as the primary constriction of condensed metaphase chromosomes, the centromere remained elusive to molecular characterisation for many years owed to its unusual enrichment in highly repetitive satellite DNA sequences, except in budding yeast. In the last decade, our understanding of centromere structure, organisation and function has increased tremendously. Nowadays, we know that centromere identity is determined epigenetically by the formation of a unique type of chromatin, which is characterised by the presence of the centromere‐specific histone H3 variant CenH3, originally called CENP‐A, which replaces canonical histone H3 at centromeres. CenH3‐chromatin constitutes the physical and functional foundation for kinetochore assembly. This review explores recent studies addressing the structural and functional characterisation of CenH3‐chromatin, its assembly and propagation during mitosis, and its contribution to kinetochore assembly.     

Australin: a chromosomal passenger protein required specifically for Drosophila melanogaster male meiosis

The chromosomal passenger complex (CPC), which is composed of conserved proteins aurora B, inner centromere protein (INCENP), survivin, and Borealin/DASRA, localizes to chromatin, kinetochores, microtubules, and the cell cortex in a cell cycle-dependent manner. The CPC is required for multiple aspects of cell division. Here we find that Drosophila melanogaster encodes two Borealin paralogues, Borealin-related (Borr) and Australin (Aust). Although Borr is a passenger in all mitotic tissues studied, it is specifically replaced by Aust for the two male meiotic divisions. We analyzed aust mutant spermatocytes to assess the effects of fully inactivating the Aust-dependent functions of the CPC. Our results indicate that Aust is required for sister chromatid cohesion, recruitment of the CPC to kinetochores, and chromosome alignment and segregation but not for meiotic histone phosphorylation or spindle formation. Furthermore, we show that the CPC is required earlier in cytokinesis than previously thought; cells lacking Aust do not initiate central spindle formation, accumulate anillin or actin at the cell equator, or undergo equatorial constriction.     

Heterochromatin tells CENP-A where to go

The centromere is the region of the chromosome where the kinetochore forms. Kinetochores are the attachment sites for spindle microtubules that separate duplicated chromosomes in mitosis and meiosis. Kinetochore formation depends on a special chromatin structure containing the histone H3 variant CENP-A. The epigenetic mechanisms that maintain CENP-A chromatin throughout the cell cycle have been studied extensively but little is known about the mechanism that targets CENP-A to naked centromeric DNA templates. In a recent report published in Science, such de novo centromere assembly of CENP-A is shown to be dependent on heterochromatin and the RNA interference pathway.     

Long-range communication between chromatin and microtubules in Xenopus egg extracts

The mitotic spindle of animal cells is a bipolar array of microtubules that guides chromosome segregation during cell division. It has been proposed that during spindle assembly chromatin can positively influence microtubule stability at a distance from its surface throughout its neighboring cytoplasm. However, such an "à distance" effect has never been visualized directly. Here, we have used centrosomal microtubules and chromatin beads to probe the regulation of microtubule behavior around chromatin in Xenopus egg extracts. We show that, in this system, chromatin does affect microtubule formation at a distance, inducing preferential orientation of centrosomal microtubules in its direction. Moreover, this asymmetric distribution of microtubules is translated into a directional migration of centrosomal asters toward chromatin and their steady-state repositioning within 10 microm of chromatin. To our knowledge, this is the first direct evidence of a long-range guidance effect at the sub-cellular level.     

At the right place at the right time: novel CENP-A binding proteins shed light on centromere assembly

Centromeres, the chromosomal loci that form the sites of attachment for spindle microtubules during mitosis, are identified by a unique chromatin structure generated by nucleosomes containing the histone H3 variant CENP-A. The apparent epigenetic mode of centromere inheritance across mitotic and meiotic divisions has generated much interest in how CENP-A assembly occurs and how structurally divergent centromeric nucleosomes can specify the centromere complex. Although a substantial number of proteins have been implicated in centromere assembly, factors that can bind CENP-A specifically and deliver nascent protein to the centromere were, thus far, lacking. Several recent reports on experiments in fission yeast and human cells have now shown significant progress on this problem. Here, we discuss these new developments and their implications for epigenetic centromere inheritance.     

Formation of a centromere-specific chromatin structure

The kinetochore is formed on centromeric DNA as a key interface with microtubules from the mitotic spindle to achieve accurate chromosome segregation during mitosis. However, in contrast to other regions of the chromosome, the position of the kinetochore is specified by sequence-independent epigenetic mechanisms. Most recent work on kinetochore specification has focused on the centromere-specific histone H3-variant CENP-A. Whereas CENP-A is an important epigenetic marker for the kinetochore specification, it is unclear how centromeric chromatin structure is organized. To understand centromeric chromatin structure, we focused on additional centromere proteins that have an intrinsic DNA binding activity and identified the DNA binding CENP-T-W-S-X complex. Tetramer formation of CENP-T-W-S-X is essential for functional kinetochore assembly in vertebrate cells. Our structural and biochemical analysis reveals that the CENP-T-W-S-X complex is composed of four histone-fold domains with structural similarity to nucleosomes and displays DNA supercoiling activity. These results suggest that the CENP-T-W-S-X complex forms a unique nucleosome-like structure at centromeric chromatin. In addition, CENP-S and CENP-X function at non-centromeric sites. The intriguing histone-like properties of these proteins suggest that they may form nucleosome-like structures at various genome loci, extending the chromatin code beyond classical histone variants.