Human Shugoshin Mediates Kinetochore-Driven Formation of Kinetochore Microtubules

The conserved protein Shugoshin (Sgo) plays a role in the maintenance of centromeric cohesion in mitosis and meiosis. Human Shugoshin (hSgo) was first identified as an overexpressed protein in breast cancers. Here we demonstrate that hSgo mediates kinetochore-driven formation of kinetochore microtubules (MTs) during bipolar spindle assembly. The regulated overexpression of full-length hSgo, or of truncated proteins containing both the conserved N-terminal coiled-coil domain and C-terminal basic domain, resulted in hSgo localization at centromere at early mitosis and was associated with aberrant nucleation and formation of bundles of kinetochore MTs. The mid-portion of hSgo, between the N- and C-terminal domains, includes both a functional domain for centromeric cohesion and a regulatory domain for spindle assembly. The cells overexpressing natural alternative splicing isoforms, which are almost completely defective for the mid-portion of the hSgo protein, showed premature centromere separation (PCS) and aberrant MT connections. These isoforms are mildly overexpressed in HEK293 cells. On the other hand, cells expressing a truncated protein, defective in the lysine-rich region of the mid-portion, arrested at mitosis due to persistent abnormal MT connections and not because of PCS. Aberrant MT connections were predominantly observed in spindle regions where chromosomes were clustered. Interestingly, we also found that hSgo is rapidly exchanged at kinetochores at early mitosis. Based on these results, we conclude that hSgo may be diffusible and have a role in proper kinetochores-MTs attachment.     

Ndc80复合体在外层动粒中的作用

动粒是参与有丝分裂过程中染色体分离的蛋白的附着支架。结构保守的Ndc80复合体位于动粒的外层,连接动粒和微管,与动粒-微管连接的稳定性有关。Aurora B/Ipl1激酶参与纠正动粒-微管的错误连接。Ndc80复合体对纺锤体组装检查点的功能非常重要。本文主要介绍了Ndc80复合体的研究进展。     

Molecular Dynamics Simulations of DNA-Polycation Complex Formation

Complexes formed from DNA and polycations are of interest because of their potential use in gene therapy; however, there remains a lack of understanding of the structure and formation of DNA-polycation complexes at atomic scale. In this work, molecular dynamics simulations of the DNA duplex d(CGCGAATTCGCG) in the presence of polycation chains are carried out to shed light on the specific atomic interaction that result in complex formation. The structures of complexes formed from DNA with polyethylenimine, which is considered one of the most promising DNA vector candidates, and a second polycation, poly-L-lysine, are compared. After an initial separation of ∼50 Å, the DNA and polycation come together and form a stable complex within 10 ns. The DNA does not undergo any major structural changes on complexation and remains in the B-form. In the formed complex, the charged amine groups of the polycation mainly interact with DNA phosphate groups, with polycation intrusion into the major and minor grooves dependent on the identity and charge state of the polycation. The ability of the polycation to effectively neutralize the charge of the DNA phosphate groups and the resulting influence on the DNA helix interaction are discussed.     

Kinetochore Biorientation in Saccharomyces cerevisiae Requires a Tightly Folded Conformation of the Ndc80 Complex

Accurate transmission of genetic material relies on the coupling of chromosomes to spindle microtubules by kinetochores. These linkages are regulated by the conserved Aurora B/Ipl1 kinase to ensure that sister chromatids are properly attached to spindle microtubules. Kinetochore–microtubule attachments require the essential Ndc80 complex, which contains two globular ends linked by large coiled-coil domains. In this study, we isolated a novel ndc80 mutant in Saccharomyces cerevisiae that contains mutations in the coiled-coil domain. This ndc80 mutant accumulates erroneous kinetochore–microtubule attachments, resulting in misalignment of kinetochores on the mitotic spindle. Genetic analyses with suppressors of the ndc80 mutant and in vitro cross-linking experiments suggest that the kinetochore misalignment in vivo stems from a defect in the ability of the Ndc80 complex to stably fold at a hinge in the coiled coil. Previous studies proposed that the Ndc80 complex can exist in multiple conformations: elongated during metaphase and bent during anaphase. However, the distinct functions of individual conformations in vivo are unknown. Here, our analysis revealed a tightly folded conformation of the Ndc80 complex that is likely required early in mitosis. This conformation is mediated by a direct, intracomplex interaction and involves a greater degree of folding than the bent form of the complex at anaphase. Furthermore, our results suggest that this conformation is functionally important in vivo for efficient error correction by Aurora B/Ipl1 and, consequently, to ensure proper kinetochore alignment early in mitosis.     

不良孕产夫妇着丝粒-动粒复合体的细胞遗传学研究

为研究不良孕产夫妇染色体着丝粒-动粒复合体(centromerekinetochorecomplex,CKC)变异与不良孕产的相关性,探索不良孕产中非整倍体形成的细胞遗传学基础,应用改良的着丝粒点-核仁组织区(Cd-NOR)同步银染技术,分别对53对不明原因的不良孕产夫妇和57对已生育正常儿的正常夫妇外周血淋巴细胞染色体CKC变异类型及频率进行研究和分析.结果发现,不良孕产夫妇其小Cd、Cd消失、Cd迟滞和Cd-NOR融合频率均较正常对照组明显增高,两者相比有显著性差异(P＜0.05).CKC变异频率增高可能是导致不良孕产非整倍体形成的主要原因之一。 Abstract:To search the cytogenctic mechanism of adverse pregnancy,a study was carried out on 110 couples,57 of them with unexplained adverse pregnancy and 57 served as a control.A technique for the simultaneous staining of both nucleolar organizer regions and kinetochores of human chromosomes with silver was used.The results showed that the variations of chromosomal centromere kinetochore complex (CKC) in couples with adverse pregnancy were significantly high than of the control.The variations of CKC may be the main reason for the chromosomal nondisjunction during meiosis that is attributed to the adverse pregnancy.     

不良孕产夫妇着丝粒—动粒复合体的细胞遗传学研究

为研究不良孕产夫妇着丝粒－动粒复合体（centromere kinetochore complex,CKC)变异与不良孕产的相关性,探索不良孕产中非整倍体表成的细胞遗传学基础,应用改良的着丝粒点－核仁组织区（Cd-NOR)同步银染技术,分别对53对不明原因的不良孕产夫妇和57对已生育正常儿的正常夫妇外周血淋巴细胞染色体CKC变异类型及频率进行研究和分析。结果发现,不良孕产夫妇其小Cd,Cd消失、Cd迟滞和Cd-NOR融合频率均较正常对照组明显增高,两相比有显性差异（P＜0．05）。CKC变异频率增高可能是导致不良孕产非整倍体形成的主要原因之一。     

Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice

The DELLA protein SLENDER RICE1 (SLR1) is a repressor of gibberellin (GA) signaling in rice (Oryza sativa), and most of the GA-associated responses are induced upon SLR1 degradation. It is assumed that interaction between GIBBERELLIN INSENSITIVE DWARF1 (GID1) and the N-terminal DELLA/TVHYNP motif of SLR1 triggers F-box protein GID2-mediated SLR1 degradation. We identified a semidominant dwarf mutant, Slr1-d4, which contains a mutation in the region encoding the C-terminal GRAS domain of SLR1 (SLR1^G576V). The GA-dependent degradation of SLR1^G576V was reduced in Slr1-d4, and compared with SLR1, SLR1^G576V showed reduced interaction with GID1 and almost none with GID2 when tested in yeast cells. Surface plasmon resonance of GID1-SLR1 and GID1-SLR1^G576V interactions revealed that the GRAS domain of SLR1 functions to stabilize the GID1-SLR1 interaction by reducing its dissociation rate and that the G576V substitution in SLR1 diminishes this stability. These results suggest that the stable interaction of GID1-SLR1 through the GRAS domain is essential for the recognition of SLR1 by GID2. We propose that when the DELLA/TVHYNP motif of SLR1 binds with GID1, it enables the GRAS domain of SLR1 to interact with GID1 and that the stable GID1-SLR1 complex is efficiently recognized by GID2.     

Mathematical Model of the Formation of Morphogen Gradients Through Membrane-Associated Non-receptors

The importance of morphogens is a central concept in developmental biology. Multiple-fate patterning and the robustness of the morphogen gradient are essential for embryo development. The ways by which morphogens diffuse from a local source to form long distance gradients can differ from one morphogen to the other, and for the same morphogen in different organs. This paper will study the mechanism by which morphogens diffuse through the aid of membrane-associated non-receptors and will investigate how the membrane-associated non-receptors help the morphogen to form long distance gradients and to achieve good robustness. Such a mechanism has been reported for some morphogens that are rapidly turned over. We will establish a set of reaction-diffusion equations to model the dynamical process of morphogen gradient formation. Under the assumption of rapid morphogen degradation, we discuss the existence, uniqueness, local stability, approximation solution, and the robustness of the steady-state gradient. The results in this paper show that when the morphogen is rapidly turned over, diffusion of the morphogen through membrane-associated non-receptors is a possible strategy to form a long distance multiple-fate gradient that is locally stable and is robust against the changes in morphogen synthesis rate.     

Probing a Structural Model of the Nuclear Pore Complex Channel through Molecular Dynamics

The central pore of a nuclear pore complex (NPC) is filled with unstructured proteins that contain many FG-repeats separated by hydrophilic regions. An example of such protein is nsp1. By simulating an array of nsp1 segments, we identified, in an earlier study, a spontaneously formed brushlike structure that promises to explain selective transport in the NPC channel. Here we report four (350,000 atom, 200 ns) simulations probing this structure via its interaction with transport receptor NTF2 as well as with an inert protein. NTF2 dimers are observed to gradually enter the brush, but the inert protein is not. Both NTF2 and the inert protein are found to bind to FG-repeats, but binding periods lasted more briefly for the inert protein. A simulation also investigated the behavior of a brush made of mutant nsp1 that is known to be less effective in NPC-selective transport, finding that this brush does not attract NTF2.     

Structure of Soybean Serine Acetyltransferase and Formation of the Cysteine Regulatory Complex as a Molecular Chaperone

Serine acetyltransferase (SAT) catalyzes the limiting reaction in plant and microbial biosynthesis of cysteine. In addition to its enzymatic function, SAT forms a macromolecular complex with O-acetylserine sulfhydrylase. Formation of the cysteine regulatory complex (CRC) is a critical biochemical control feature in plant sulfur metabolism. Here we present the 1.75–3.0 Å resolution x-ray crystal structures of soybean (Glycine max) SAT (GmSAT) in apoenzyme, serine-bound, and CoA-bound forms. The GmSAT-serine and GmSAT-CoA structures provide new details on substrate interactions in the active site. The crystal structures and analysis of site-directed mutants suggest that His¹⁶⁹ and Asp¹⁵⁴ form a catalytic dyad for general base catalysis and that His¹⁸⁹ may stabilize the oxyanion reaction intermediate. Glu¹⁷⁷ helps to position Arg²⁰³ and His²⁰⁴ and the β1c-β2c loop for serine binding. A similar role for ionic interactions formed by Lys²³⁰ is required for CoA binding. The GmSAT structures also identify Arg²⁵³ as important for the enhanced catalytic efficiency of SAT in the CRC and suggest that movement of the residue may stabilize CoA binding in the macromolecular complex. Differences in the effect of cold on GmSAT activity in the isolated enzyme versus the enzyme in the CRC were also observed. A role for CRC formation as a molecular chaperone to maintain SAT activity in response to an environmental stress is proposed for this multienzyme complex in plants.     

Differential Complex Formation via Paralogs in the Human Sin3 Protein Interaction Network

1. Download : Download high-res image (68KB)
2. Download : Download full-size image

Highlights

• •Sin3 paralog identity influences Sin3 complex composition.
• •Chemical cross-linking mass spectrometry identifies domains in SIN3A and SIN3B that mediate complex formation.
• •Complex subunit homology to yeast Sin3 complex components may assist in defining distinct forms of the Sin3 complex in humans.
• •A nuclear import signal within SIN3B is identified via chemical cross-linking mass spectrometry.

     

Microtubule Attachment and Centromeric Tension Shape the Protein Architecture of the Human Kinetochore

1. Download : Download high-res image (260KB)
2. Download : Download full-size image

     

Production and Initial Characterization of Dad1p,a Component of the Dam1-DASH Kinetochore Complex

In all dividing eukaryotic cells, the mitotic spindle (composed primarily of microtubules) must interact with chromosomes through a complex protein assembly called the kinetochore. In Saccharomyces cerevisiae, the Dam1-DASH complex plays an important role in promoting attachment between the kinetochore and the mitotic spindle. It also actively participates in the physical separation of sister chromatids in anaphase. Understanding the biochemical mechanisms used by Dam1-DASH has been facilitated by bacterial co-expression of the ten Dam1-DASH genes, which results in the production of a heterodecameric protein complex that can be studied in vitro. However, individual protein subunits are not soluble when expressed in E. coli, thus precluding analysis of the nature of the interaction between subunits and an examination of the assembly of the functional complex. In this paper, we describe the expression, solubilization, purification and refolding of Dad1p, one of the Dam1-DASH complex subunits. In addition, we show that Dad1p, when isolated in this manner forms dimers and/or tetramers, dependent upon protein concentration. This work provides an important tool for studying the Dam1-DASH complex that was previously unavailable, and provides an avenue of investigation for understanding how the individual heterodecamers associate with each other to facilitate chromosome segregation.     

Modular Assembly of RWD Domains on the Mis12 Complex Underlies Outer Kinetochore Organization

1. Download : Download high-res image (154KB)
2. Download : Download full-size image

     

Molecular Model for the C-type Lectin Domain of Human Thrombomodulin

Thrombomodulin (TM) is a multi-modular membrane receptor (557 residues) present on the surface of endothelial cells. TM binds thrombin (T) and this complex promotes downregulation of the coagulation cascade via activation of protein C and delay fibrinolysis through activation of the thrombin-activatable fibrinolysis inhibitor (TAFI). The N-term region (155 residues) of TM possesses the signature of the C-type lectin domain. This module seems required for constitutive internalization of the T-TM complex, plays a role in the modulation of cell growth and may direct soluble forms of TM (or T-TM) to specific regions of the vasculature during inflammation and in a variety of vascular disorders. The understanding of this domain is however limited and structural information would contribute to the design of new experiments aiming at characterizing its functions. We have developed a 3D model for the lectin domain of TM using prediction-based threading and comparative model building. The X-ray structures of lithostathine (LIT), mannose-binding protein (MBP) and E-selectin (ESL) were used as initial templates. Despite a sequence identity of about 28 % between TM and LIT (best score) it is possible to build an accurate 3D model for TM. The TM lectin domain contains two α-helices, two β-sheets and a compact hydrophobic/aromatic core. The disulfide bridging pattern of TM has not been reported experimentally but the model proposes the formation of four disulfide bonds between C12-C17, C34-C149, C78-C115 and C119-C140. Based on the model, potential binding sites are proposed.