Distinct chromosome segregation roles for spindle checkpoint proteins

The spindle checkpoint plays a central role in the fidelity of chromosome transmission by ensuring that anaphase is initiated only after kinetochore-microtubule associations of all sister chromatid pairs are complete. In this study, we find that known spindle checkpoint proteins do not contribute equally to chromosome segregation fidelity in Saccharomyces cerevisiae. Loss of Bub1 or Bub3 protein elicits the largest effect. Analysis of Bub1p reveals the presence of two molecular functions. An N-terminal 608-amino acid (nonkinase) portion of the protein supports robust checkpoint activity, and, as expected, contributes to chromosome segregation. A C-terminal kinase-encoding segment independently contributes to chromosome segregation through an unknown mechanism. Both molecular functions depend on association with Bub3p. A 156-amino acid fragment of Bub1p functions in Bub3p binding and in kinetochore localization by one-hybrid assay. An adjacent segment is required for Mad1p binding, detected by deletion analysis and coimmunoprecipitation. Finally, overexpression of wild-type BUB1 or MAD3 genes leads to chromosome instability. Analysis of this activity indicates that the Bub3p-binding domain of Bub1p contributes to this phenotype through disruption of checkpoint activity as well as through introduction of kinetochore or spindle damage.     

Structural and regulatory roles of muscle ankyrin repeat protein family in skeletal muscle

The biological response of muscle to eccentric contractions (ECs) results in strengthening and protection from further injury. However, the cellular basis for this response remains unclear. Previous studies identified the muscle ankyrin repeat protein (MARP) family, consisting of cardiac ankyrin repeat protein (CARP), ankyrin repeat domain 2/ankyrin repeat protein with PEST and proline-rich region (Ankrd2/Arpp), and diabetes-associated ankyrin repeat protein (DARP), as rapidly and specifically upregulated in mice after a single bout of EC. To determine the role of these genes in skeletal muscle, a survey of skeletal muscle structural and functional characteristics was performed on mice lacking all three MARP family members (MKO). There was a slight trend toward MKO muscles having a slower fiber type distribution but no differences in muscle fiber size. Single MKO fibers were less stiff, tended to have longer resting sarcomere lengths, and expressed a longer isoform of titin than their wild-type counterparts, indicating that these proteins may play a role in the passive mechanical behavior of muscle. Finally, MKO mice showed a greater degree of torque loss after a bout of ECs compared with wild-type mice, although they recovered from the injury with the same or even improved time course. This recovery was associated with enhanced expression of the muscle regulatory genes MyoD and muscle LIM protein (MLP), suggesting that the MARP family may play both important structural and gene regulatory roles in skeletal muscle. CARP; Ankrd2; Arpp; DARP; eccentric contractions; titin     

Structural and functional analyses of mutant Fur proteins with impaired regulatory function.

Vibrio anguillarum Fur mutants, 775met9 and 775met11, were characterized. V. anguillarum 775met9 had a change of D to G at position 104 located in the carboxy terminus resulting in impaired Fur activity.Computer analysis predicts perturbation of an alpha-helix in the carboxy terminus which may interfere with Fur protein conformation. Strain 775met11 had a change in the start codon resulting in no protein synthesis. The mutants are unstable, and reversion to the wild type occurs frequently.     

Acentrosomal spindle assembly and maintenance in Caenorhabditis elegans oocytes requires a kinesin-12 nonmotor microtubule interaction domain

During the meiotic divisions in oocytes, microtubules are sorted and organized by motor proteins to generate a bipolar spindle in the absence of centrosomes. In most organisms, kinesin-5 family members crosslink and slide microtubules to generate outward force that promotes acentrosomal spindle bipolarity. However, the mechanistic basis for how other kinesin families act on acentrosomal spindles has not been explored. We investigated this question in Caenorhabditis elegans oocytes, where kinesin-5 is not required to generate outward force and the kinesin-12 family motor KLP-18 instead performs this function. Here we use a combination of in vitro biochemical assays and in vivo mutant analysis to provide insight into the mechanism by which KLP-18 promotes acentrosomal spindle assembly. We identify a microtubule binding site on the C-terminal stalk of KLP-18 and demonstrate that a direct interaction between the KLP-18 stalk and its adaptor protein MESP-1 activates nonmotor microtubule binding. We also provide evidence that this C-terminal domain is required for KLP-18 activity during spindle assembly and show that KLP-18 is continuously required to maintain spindle bipolarity. This study thus provides new insight into the construction and maintenance of the oocyte acentrosomal spindle as well as into kinesin-12 mechanism and regulation.     

Dynamical roles of biological regulatory circuits

Regulatory circuits are found at the basis of all non-trivial dynamical properties of biological networks. More specifically, positive circuits are involved in the generation of multiple differentiated states, whereas negative circuits can generate cyclic or homeostatic behaviours. These notions are briefly reviewed, from initial biological formulations to mathematical formalisations, further encompassing their application to the design of synthetic regulatory systems. Finally, current challenges for the analysis of increasingly complex regulatory networks are indicated, as well as prospects for our understanding of development and evolution.     

G-quadruplexes and their regulatory roles in biology

‘If G-quadruplexes form so readily in vitro, Nature will have found a way of using them in vivo’ (Statement by Aaron Klug over 30 years ago).During the last decade, four-stranded helical structures called G-quadruplex (or G4) have emerged from being a structural curiosity observed in vitro, to being recognized as a possible nucleic acid based mechanism for regulating multiple biological processes in vivo. The sequencing of many genomes has revealed that they are rich in sequence motifs that have the potential to form G-quadruplexes and that their location is non-random, correlating with functionally important genomic regions. In this short review, we summarize recent evidence for the in vivo presence and function of DNA and RNA G-quadruplexes in various cellular pathways including DNA replication, gene expression and telomere maintenance. We also highlight remaining open questions that will have to be addressed in the future.     

The roles of sterol regulatory element-binding proteins in the transactivation of the rat ATP citrate-lyase promoter

ATP citrate-lyase (ACL) is a key enzyme supplying acetyl-CoA for fatty acid and cholesterol synthesis. Its expression is drastically up-regulated when an animal is fed a low fat, high carbohydrate diet after prolonged fasting. In this report, we describe the role of sterol regulatory element-binding proteins (SREBPs) in the transactivation of the rat ACL promoter. ACL promoter activity was markedly stimulated by the overexpression of SREBP-1a and, to a lesser extent, by SREBP-2 in Alexander human hepatoma cells. The promoter elements responsive to SREBPs were located within the 55-base pair sequences from -114 to -60. The gel mobility shift assay revealed four SREBP-1a binding sites in this region. Of these four elements, the -102/-94 region, immediately upstream of the inverted Y-box, and the -70/-61 region, just adjacent to Sp1 binding site, played critical roles in SREBPs-mediated stimulation. The mutation in the inverted Y-box and the coexpression of dominant negative nuclear factor-Y (NF-Y) significantly attenuated the transactivation by SREBP-1a, suggesting that NF-Y binding is a prerequisite for SREBPs to activate the ACL promoter. However, the multiple Sp1 binding sites did not affect the transactivation of the ACL promoter by SREBPs. The binding affinity of SREBP-1a to SREs of the ACL promoter also was much higher than that of SREBP-2. The transactivation potencies of the chimeric SREBPs, of which the activation domains (70 amino acids of the amino terminus) were derived from the different species of their carboxyl-terminal region, were similar to those of SREBPs corresponding to their carboxyl termini. Therefore, it is suggested that the carboxyl-terminal portions of SREBPs containing DNA binding domains are important in determining their transactivation potencies to a certain promoter.     

Global versus local regulatory roles for Lrp-related proteins: Haemophilus influenzae as a case study

Lrp (leucine-responsive regulatory protein) plays a global regulatory role in Escherichia coli, affecting expression of dozens of operons. Numerous lrp-related genes have been identified in different bacteria and archaea, including asnC, an E. coli gene that was the first reported member of this family. Pairwise comparisons of amino acid sequences of the corresponding proteins shows an average sequence identity of only 29% for the vast majority of comparisons. By contrast, Lrp-related proteins from enteric bacteria show more than 97% amino acid identity. Is the global regulatory role associated with E. coli Lrp limited to enteric bacteria? To probe this question we investigated LrfB, an Lrp-related protein from Haemophilus influenzae that shares 75% sequence identity with E. coli Lrp (highest sequence identity among 42 sequences compared). A strain of H. influenzae having an lrfB null allele grew at the wild-type growth rate but with a filamentous morphology. A comparison of two-dimensional (2D) electrophoretic patterns of proteins from parent and mutant strains showed only two differences (comparable studies with lrp(+) and lrp E. coli strains by others showed 20 differences). The abundance of LrfB in H. influenzae, estimated by Western blotting experiments, was about 130 dimers per cell (compared to 3,000 dimers per E. coli cell). LrfB expressed in E. coli replaced Lrp as a repressor of the lrp gene but acted only to a limited extent as an activator of the ilvIH operon. Thus, although LrfB resembles Lrp sufficiently to perform some of its functions, its low abundance is consonant with a more local role in regulating but a few genes, a view consistent with the results of the 2D electrophoretic analysis. We speculate that an Lrp having a global regulatory role evolved to help enteric bacteria adapt to their ecological niches and that it is unlikely that Lrp-related proteins in other organisms have a broad regulatory function.     

Dynein-dependent transport of spindle assembly checkpoint proteins off kinetochores toward spindle poles

A predominant mechanism of spindle assembly checkpoint (SAC) silencing is dynein-mediated transport of certain kinetochore proteins along microtubules. There are still conflicting data as to which SAC proteins are dynein cargoes. Using two ATP reduction assays, we found that the core SAC proteins Mad1, Mad2, Bub1, BubR1, and Bub3 redistributed from attached kinetochores to spindle poles, in a dynein-dependent manner. This redistribution still occurred in metaphase-arrested cells, at a time when the SAC should be satisfied and silenced. Unexpectedly, we found that a pool of Hec1 and Mis12 also relocalizes to spindle poles, suggesting KMN components as additional dynein cargoes. The potential significance of these results for SAC silencing is discussed.     

A regulatory model for spindle function during mitosis

Structural information on the mitotic spindle of Saccharomyces cerevisiae obtained from isolated whole mount preparations has shown that the spindle undergoes a two-fold increase in length whilst comprising only a single microtubule continuous between the two spindle pole bodies. Further data from immunofluorescence microscopy on the timing of anaphase B has suggested that microtubules do not directly produce the required force, but instead have a more passive role. Here a regulatory function for spindle microtubules during mitosis is explored and the existence of a non-microtubule force-generating system is postulated. Thus it is suggested that the continuous microtubules govern the velocity of anaphase B by providing a resistive force that is itself regulated by the number of microtubules and their rate of polymerization. On this basis a model for the forces acting on a spindle pole body during anaphase is proposed.     

Structural and regulatory functions of keratins

The diversity of epithelial functions is reflected by the expression of distinct keratin pairs that are responsible to protect epithelial cells against mechanical stress and to act as signaling platforms. The keratin cytoskeleton integrates these functions by forming a supracellular scaffold that connects at desmosomal cell-cell adhesions. Multiple human diseases and murine knockouts in which the integrity of this system is destroyed testify to its importance as a mechanical stabilizer in certain epithelia. Yet, surprisingly little is known about the precise mechanisms responsible for assembly and disease pathology. In addition to these structural aspects of keratin function, experimental evidence accumulating in recent years has led to a much more complex view of the keratin cytoskeleton. Distinct keratins emerge as highly dynamic scaffolds in different settings and contribute to cell size determination, translation control, proliferation, cell type-specific organelle transport, malignant transformation and various stress responses. All of these properties are controlled by highly complex patterns of phosphorylation and molecular associations.     

Differential roles of p39Mos-Xp42Mpk1 cascade proteins on Raf1 phosphorylation and spindle morphogenesis in Xenopus oocytes

Fully-grown G2-arrested Xenopus oocytes resume meiosis upon hormonal stimulation. Resumption of meiosis is characterized by germinal vesicle breakdown, chromosome condensation, and organization of a bipolar spindle. These cytological events are accompanied by activation of MPF and the p39(Mos)-MEK1-Xp42(Mpk1)-p90(Rsk) pathways. The latter cascade is activated upon p39(Mos) accumulation. Using U0126, a MEK1 inhibitor, and p39(Mos) antisense morpholino and phosphorothioate oligonucleotides, we have investigated the role of the members of the p39(Mos)-MEK1-Xp42(Mpk1)-p90(Rsk) in spindle morphogenesis. First, we have observed at a molecular level that prevention of p39(Mos) accumulation always led to MEK1 phosphorylation defects, even when meiosis was stimulated through the insulin Ras-dependent pathway. Moreover, we have observed that Raf1 phosphorylation that occurs during meiosis resumption was dependent upon the activity of MEK1 or Xp42(Mpk1) but not p90(Rsk). Second, inhibition of either p39(Mos) accumulation or MEK1 inhibition led to the formation of a cytoplasmic aster-like structure that was associated with condensed chromosomes. Spindle morphogenesis rescue experiments using constitutively active Rsk and purified murine Mos protein suggested that p39(Mos) or p90(Rsk) alone failed to promote meiotic spindle organization. Our results indicate that activation of the p39(Mos)-MEK1-Xp42(Mpk1)-p90(Rsk) pathway is required for bipolar organization of the meiotic spindle at the cortex.     

Novel roles for saccharomyces cerevisiae mitotic spindle motors.

The single cytoplasmic dynein and five of the six kinesin-related proteins encoded by Saccharomyces cerevisiae participate in mitotic spindle function. Some of the motors operate within the nucleus to assemble and elongate the bipolar spindle. Others operate on the cytoplasmic microtubules to effect spindle and nuclear positioning within the cell. This study reveals that kinesin-related Kar3p and Kip3p are unique in that they perform roles both inside and outside the nucleus. Kar3p, like Kip3p, was found to be required for spindle positioning in the absence of dynein. The spindle positioning role of Kar3p is performed in concert with the Cik1p accessory factor, but not the homologous Vik1p. Kar3p and Kip3p were also found to overlap for a function essential for the structural integrity of the bipolar spindle. The cytoplasmic and nuclear roles of both these motors could be partially substituted for by the microtubule-destabilizing agent benomyl, suggesting that these motors perform an essential microtubule-destabilizing function. In addition, we found that yeast cell viability could be supported by as few as two microtubule-based motors: the BimC-type kinesin Cin8p, required for spindle structure, paired with either Kar3p or Kip3p, required for both spindle structure and positioning.     

Structural and functional roles of ether lipids

Ether lipids, such as plasmalogens, are peroxisomederived glycerophospholipids in which the hydrocarbon chain at the sn-1 position of the glycerol backbone is attached by an ether bond, as opposed to an ester bond in the more common diacyl phospholipids. This seemingly simple biochemical change has profound structural and functional implications. Notably, the tendency of ether lipids to form non-lamellar inverted hexagonal structures in model membranes suggests that they have a role in facilitating membrane fusion processes. Ether lipids are also important for the organization and stability of lipid raft microdomains, cholesterol-rich membrane regions involved in cellular signaling. In addition to their structural roles, a subset of ether lipids are thought to function as endogenous antioxidants, and emerging studies suggest that they are involved in cell differentiation and signaling pathways. Here, we review the biology of ether lipids and their potential significance in human disorders, including neurological diseases, cancer, and metabolic disorders.     

巨核细胞生成调控相关细胞因子及其作用研究进展

造血干细胞分化生成巨核细胞是一个十分复杂的过程,包括造血干细胞动员及其向巨核系祖细胞分化,巨核系祖细胞增殖、分化生成未成熟巨核细胞,巨核细胞的成熟和血小板释放等过程。研究发现,造血干细胞动员及其向各系细胞分化的大部分过程都在一种称为"龛"的结构中进行,多种龛内信号分子参与了造血干细胞的动员和分化调控。该文对造血干细胞龛内参与造血干细胞动员和分化生成巨核细胞的几种重要细胞因子及其调控作用进行综述。     

Telomeres and centromeres have interchangeable roles in promoting meiotic spindle formation

Telomeres and centromeres have traditionally been considered to perform distinct roles. During meiotic prophase, in a conserved chromosomal configuration called the bouquet, telomeres gather to the nuclear membrane (NM), often near centrosomes. We found previously that upon disruption of the fission yeast bouquet, centrosomes failed to insert into the NM at meiosis I and nucleate bipolar spindles. Hence, the trans-NM association of telomeres with centrosomes during prophase is crucial for efficient spindle formation. Nonetheless, in approximately half of bouquet-deficient meiocytes, spindles form properly. Here, we show that bouquet-deficient cells can successfully undergo meiosis using centromere–centrosome contact instead of telomere–centrosome contact to generate spindle formation. Accordingly, forced association between centromeres and centrosomes fully rescued the spindle defects incurred by bouquet disruption. Telomeres and centromeres both stimulate focal accumulation of the SUN domain protein Sad1 beneath the centrosome, suggesting a molecular underpinning for their shared spindle-generating ability. Our observations demonstrate an unanticipated level of interchangeability between the two most prominent chromosomal landmarks.