Flexibility of centromere and kinetochore structures

Centromeres, and the kinetochores that assemble on them, are essential for accurate chromosome segregation. Diverse centromere organization patterns and kinetochore structures have evolved in eukaryotes ranging from yeast to humans. In addition, centromere DNA and kinetochore position can vary even within individual cells. This flexibility is manifested in several ways: centromere DNA sequences evolve rapidly, kinetochore positions shift in response to altered chromosome structure, and kinetochore complex numbers change in response to fluctuations in kinetochore protein levels. Despite their differences, all of these diverse structures promote efficient chromosome segregation. This robustness is inherent to chromosome segregation mechanisms and balances genome stability with adaptability. In this review, we explore the mechanisms and consequences of centromere and kinetochore flexibility as well as the benefits and limitations of different experimental model systems for their study.     

Prediction of midbody, centrosome and kinetochore proteins based on gene ontology information

In the process of cell division, a great deal of proteins is assembled into three distinct organelles, namely midbody, centrosome and kinetochore. Knowing the localization of microkit (midbody, centrosome and kinetochore) proteins will facilitate drug target discovery and provide novel insights into understanding their functions. In this study, a support vector machine (SVM) model, MicekiPred, was presented to predict the localization of microkit proteins based on gene ontology (GO) information. A total accuracy of 77.51% was achieved using the jackknife cross-validation. This result shows that the model will be an effective complementary tool for future experimental study. The prediction model and dataset used in this article can be freely downloaded from http://cobi.uestc.edu.cn/people/hlin/tools/MicekiPred/.     

Phylogenetic and structural analysis of centromeric DNA and kinetochore proteins

Background  

Kinetochores are large multi-protein structures that assemble on centromeric DNA (CEN DNA) and mediate the binding of chromosomes to microtubules. Comprising 125 base-pairs of CEN DNA and 70 or more protein components, Saccharomyces cerevisiae kinetochores are among the best understood. In contrast, most fungal, plant and animal cells assemble kinetochores on CENs that are longer and more complex, raising the question of whether kinetochore architecture has been conserved through evolution, despite considerable divergence in CEN sequence.     

Elastomeric proteins: biological roles, structures and mechanisms

Elastomeric proteins are able to withstand significant deformations without rupture before returning to their original state when the stress is removed. Although elastomeric proteins differ considerably in their amino acid sequence, they all have a complex domain structure and share two common properties. Namely, they contain elastomeric domains, comprised of repeated sequences, and additional domains that form intermolecular crosslinks. Furthermore, several protein contain beta-turns as a structural motif within the elastomeric domains.     

Columba: an integrated database of proteins,structures, and annotations

Background  

Structural and functional research often requires the computation of sets of protein structures based on certain properties of the proteins, such as sequence features, fold classification, or functional annotation. Compiling such sets using current web resources is tedious because the necessary data are spread over many different databases. To facilitate this task, we have created COLUMBA, an integrated database of annotations of protein structures.     

Insulin-like growth factor binding proteins: new proteins, new functions.

The insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), and IGFBP proteases regulate somatic growth and cellular proliferation both in vivo and in vitro. IGFs are potent mitogens whose actions are determined by the availability of free IGFs to interact with IGF receptors. IGFBPs comprise a family of six proteins that bind IGFs with high affinity and specificity and thereby regulate IGF-dependent actions. IGFBPs have recently emerged as IGF-independent regulators of cell growth. Cleavage of IGFBPs by specific proteases modulate levels of free IGFs and IGFBPs and thereby their actions. IGFBP-related proteins (IGFBP-rPs) bind IGFs with low affinity and also play important roles in cell growth and differentiation. The GH-IGF-IGFBP axis is complex and powerful. Future research on its physiology promises exciting insights into cell biology as well as therapies for diseases such as cancer and diabetes mellitus.     

Identification of microtubule-associated proteins in the centrosome, spindle, and kinetochore of the early Drosophila embryo

We have developed affinity chromatography methods for the isolation of microtubule-associated proteins (MAPs) from soluble cytoplasmic extracts and have used them to analyze the cytoskeleton of the early Drosophila embryo. More than 50 Drosophila embryo proteins bind to microtubule affinity columns. To begin to characterize these proteins, we have generated individual mouse polyclonal antibodies that specifically recognize 24 of them. As judged by immunofluorescence, some of the antigens localize to the mitotic spindle in the early Drosophila embryo, while others are present in centrosomes, kinetochores, subsets of microtubules, or a combination of these structures. Since 20 of the 24 antibodies stain microtubule structures, it is likely that most of the proteins that bind to our columns are associated with microtubules in vivo. Very few MAPS seem to be identically localized in the cell, indicating that the microtubule cytoskeleton is remarkably complex.     

Small-molecule natural products: new structures, new activities

A wide variety of novel small-molecule natural products has recently been reported. These compounds were isolated from marine and terrestrial sources, and from a variety of animals, plants and microorganisms. With the breadth of diversity represented in these bioactive small molecules, the future of natural product drug discovery looks bright.     

Tubulin interaction with kinetochore proteins: analysis by in vitro assembly and chemical cross-linking

The sera from patients with the CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) variation of the autoimmune disease scleroderma contain autoantibodies that specifically recognize the kinetochore by immunofluorescence. Two major antigens of molecular masses 18 and 80 kD are consistently identified by Western blotting of proteins of isolated chromosomes using CREST sera. In this paper, the possible roles that these two proteins play in the interaction of metaphase chromosomes with tubulin and microtubules are examined using two different procedures. In one set of experiments. Chinese hamster ovary (CHO) chromosomes were extracted with 1-2 M NaCl before incubating with phosphocellulose-purified tubulin under in vitro microtubule assembly conditions. After this treatment, the kinetochores of the residual chromosome scaffolds can still initiate the in vitro assembly of microtubules. Immunoblots of the chromosome scaffold proteins demonstrate that the 18-kD protein has been solubilized by the 1-2 M NaCl extraction, suggesting that this protein is not essential for microtubule assembly at the kinetochore. In a second approach, tubulin was covalently cross-linked to kinetochores of CHO chromosomes using the reversible cross-linking reagent dithiobis (succinimidyl propionate). After DNase I digestion, the chromosomes were solubilized and subjected to anti-tubulin affinity chromatography. Tubulin-kinetochore protein complexes were specifically eluted and analyzed by PAGE and immunoblotting with scleroderma CREST serum. Only a small number of proteins were eluted from the antitubulin affinity column as shown by Coomassie Blue-stained gels. In addition to tubulin, an 80-kD polypeptide, bands at 110 and 24 kD, as well as a faint band at 54 kD, can be resolved. Several minor bands can also be seen in silver-stained gels. The 80-kD protein band from whole metaphase chromosomes reacted with scleroderma CREST serum by immunoblotting and therefore probably represents the major centromere antigen CENP-B. This report provides evidence for a specific protein complex on metaphase chromosomes that is contiguous with kinetochore-bound tubulin and may be involved in microtubule-kinetochore interactions during mitosis.     

Iron-sulfur proteins: ancient structures, still full of surprises

This article is a survey of the properties and functions of Fe-S proteins under the following headings: sulfur and iron; iron-sulfur clusters; evolution of cofactor use; early observations; complex and extended clusters; sulfur exchange and core interconversions; synthesis and biosynthesis of Fe-S clusters; functions of Fe-S clusters: electron transfer, electron delocalization, spin states and magnetism, covalency of sulfur bonds; non-electron transfer functions of Fe-S clusters: substrate binding and catalysis, regulatory and sensing functions.     

Sos7, an essential component of the conserved Schizosaccharomyces pombe Ndc80-MIND-Spc7 complex, identifies a new family of fungal kinetochore proteins

Chromosome segregation is powered by the kinetochore, a large macromolecular structure assembled on centromeric chromatin. Attachment of sister chromatids to microtubules is mediated by the highly conserved tripartite KMN (acronym for KNL-1-Mis12-Ndc80) kinetochore network. In the fission yeast Schizosaccharomyces pombe, the equivalent complex is called NMS (Ndc80-MIND-Spc7). Here, we show that not all components of the NMS complex had been identified previously. A 10th NMS component exists, the essential Sos7 protein, which is a genetic and physical interaction partner of Spc7. The analysis of sos7 kinetochore-null mutant yeast strains demonstrated that Sos7 is central to NMS function. In particular, Sos7 is required for kinetochore targeting of Spc7 as well as components of the MIND complex. sos7 mutant strains show severe chromosome missegregation phenotypes and have compromised microtubule-kinetochore interactions. Sos7 is the founding member of a functionally conserved fungal kinetochore family not present in the point centromere carrying Saccharomycotina clusters, suggesting that the new Sos7 family might be a signature motif of fungi with regional centromeres.     

Dynein at the kinetochore: Timing,Interactions and Functions

Kinetochores have been proposed to play multiple roles in mitotic chromosome alignment, including initial microtubule (MT) capture, monitoring MT attachments, prometaphase and anaphase chromosome movement and tension generation at metaphase. In addition, kinetochores are essential components of the spindle assembly checkpoint (SAC), and couple chromosome alignment with SAC silencing at metaphase. Although the molecular details of these activities remain under investigation, cytoplasmic dynein has been implicated in several aspects of MT and SAC regulation. Recent work clarifies the contribution of dynein to MT interactions and to events that drive anaphase onset. This review summarizes these studies and provides new models for dynein function.     

Dynamics of centromere and kinetochore proteins; implications for checkpoint signaling and silencing

BACKGROUND: The mitotic checkpoint prevents the onset of anaphase before all chromosomes are attached to spindle microtubules. The checkpoint is thought to act by the catalytic generation at unattached kinetochores of a diffusible "wait signal" that prevents anaphase. Mad2 and Cdc20, two candidate proteins for components of a diffusible wait signal, have previously been shown to be recruited to and rapidly released from unattached kinetochores. RESULTS: Fluorescence recovery after photobleaching demonstrated that Mad1, Bub1, and a portion of Mad2, all essential mitotic-checkpoint components, are stably bound elements of unattached kinetochores (as are structural centromere components such as Centromere protein C [CENP-C]). After microtubule attachment, Mad1 and Mad2 are released from kinetochores and relocalize to spindle poles, whereas Bub1 remains at kinetochores. CONCLUSIONS: A long residence time at kinetochores identifies Bub1, Mad1, and a portion of Mad2 as part of a catalytic platform that recruits, activates, and releases a diffusible wait signal that is partly composed of the rapidly exchanging portion of Mad2. The release of Mad1 and Mad2, but not Bub1, from kinetochores upon attachment separates the elements of this "catalytic platform" and thereby silences generation of the anaphase inhibitor despite continued rapid cycling of Mad2 at spindle poles.     

Merotelic kinetochore orientation, aneuploidy, and cancer

Accurate chromosome segregation in mitosis is crucial to maintain a diploid chromosome number. A majority of cancer cells are aneuploid and chromosomally unstable, i.e. they tend to gain and lose chromosomes at each mitotic division. Chromosome mis-segregation can arise when cells progress through mitosis with mis-attached kinetochores. Merotelic kinetochore orientation, a type of mis-attachment in which a single kinetochore binds microtubules from two spindle poles rather than just one, can represent a particular threat for dividing cells, as: (i) it occurs frequently in early mitosis; (ii) it is not detected by the spindle assembly checkpoint (unlike other types of mis-attachments); (iii) it can lead to chromosome mis-segregation, and, hence, aneuploidy. A number of studies have recently started to unveil the cellular and molecular mechanisms involved in merotelic kinetochore formation and correction. Here, I review these studies and discuss the relevance of merotelic kinetochore orientation in cancer cell biology.     

Three-dimensional structures of fibrillar Sm proteins: Hfq and other Sm-like proteins

SET domain lysine methyltransferases are known to catalyze site and state-specific methylation of lysine residues in histones that is fundamental in epigenetic regulation of gene activation and silencing in eukaryotic organisms. Here we report the three-dimensional solution structure of the SET domain histone lysine methyltransferase (vSET) from Paramecium bursaria chlorella virus 1 bound to cofactor S-adenosyl-L-homocysteine and a histone H3 peptide containing mono-methylated lysine 27. The dimeric structure, mimicking an enzyme/cofactor/substrate complex, yields the structural basis of the substrate specificity and methylation multiplicity of the enzyme. Our results from mutagenesis and enzyme kinetics analyses argue that a general base mechanism is less likely for lysine methylation by SET domains; and that the only invariant active site residue tyrosine 105 in vSET facilitates methyl transfer from cofactor to the substrate lysine by aligning intermolecular interactions in the lysine access channel of the enzyme.     

Acceptor-photobleaching FRET analysis of core kinetochore and NAC proteins in living human cells

Faithful chromatin segregation is mediated and controlled by the kinetochore protein network which assembles at centromeres. In this study, the neighbourhood relations of inner kinetochore and nucleosome-associated complex (NAC) proteins were analysed in living human interphase cells by acceptor photobleaching FRET. The data indicate that CENP-U is in close vicinity to CENP-I as well as to CENP-B and that CENP-M is close to CENP-T. This article has been submitted as a contribution to the Festschrift entitled “Uncovering cellular sub-structures by light microscopy” in honour of Professor Cremer’s 65th birthday.     

Spindle microtubules generate tension-dependent changes in the distribution of inner kinetochore proteins

The kinetochore forms a dynamic interface with microtubules from the mitotic spindle. Live-cell light microscopy-based observations on the dynamic structural changes within the kinetochore suggest that molecular rearrangements within the kinetochore occur upon microtubule interaction. However, the source of these rearrangements is still unclear. In this paper, we analyze vertebrate kinetochore ultrastructure by immunoelectron microscopy (EM) in the presence or absence of tension from spindle microtubules. We found that the inner kinetochore region defined by CENP-A, CENP-C, CENP-R, and the C-terminal domain of CENP-T is deformed in the presence of tension, whereas the outer kinetochore region defined by Ndc80, Mis12, and CENP-E is not stretched even under tension. Importantly, based on EM, fluorescence microscopy, and in vitro analyses, we demonstrated that the N and C termini of CENP-T undergo a tension-dependent separation, suggesting that CENP-T elongation is at least partly responsible for changes in the shape of the inner kinetochore.     

Flexible structures of SIBLING proteins, bone sialoprotein, and osteopontin

Bone sialoprotein (BSP) and osteopontin (OPN) are two members of the SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family of genetically related proteins that are clustered on human chromosome 4. We present evidence that this entire family is the result of duplication and subsequent divergent evolution of a single ancient gene. The solution structures of these two post-translationally modified recombinant proteins were solved by one dimensional proton NMR and transverse relaxation times. The polypeptide backbones of both free BSP and OPN rapidly sample an ensemble of conformations consistent with them both being completely unstructured in solution. This flexibility appears to enable these relatively small glycoproteins to rapidly associate with a number of different binding partners including other proteins as well as the mineral phase of bones and teeth. These proteins often function by bridging two proteins of fixed structures into a biologically active complex.