Analysis of a spindle pole body mutant reveals a defect in biorientation and illuminates spindle forces

The spindle pole body (SPB) is the microtubule organizing center in Saccharomyces cerevisiae. An essential task of the SPB is to ensure assembly of the bipolar spindle, which requires a proper balancing of forces on the microtubules and chromosomes. The SPB component Spc110p connects the ends of the spindle microtubules to the core of the SPB. We previously reported the isolation of a mutant allele spc110-226 that causes broken spindles and SPB disintegration 30 min after spindle formation. By live cell imaging of mutant cells with green fluorescent protein (GFP)-Tub1p or Spc97p-GFP, we show that spc110-226 mutant cells have early defects in spindle assembly. Short spindles form but do not advance to the 1.5-microm stage and frequently collapse. Kinetochores are not arranged properly in the mutant cells. In 70% of the cells, no stable biorientation occurs and all kinetochores are associated with only one SPB. Examination of the SPB remnants by electron microscopy tomography and fluorescence microscopy revealed that the Spc110-226p/calmodulin complex is stripped off of the central plaque of the SPB and coalesces to from a nucleating structure in the nucleoplasm. The central plaque components Spc42p and Spc29p remain behind in the nuclear envelope. The delamination is likely due to a perturbed interaction between Spc42p and Spc110-226p as detected by fluorescence resonance energy transfer analysis. We suggest that the force exerted on the SPB by biorientation of the chromosomes pulls the Spc110-226p out of the SPB; removal of force exerted by coherence of the sister chromatids reduced fragmentation fourfold. Removal of the forces exerted by the cytoplasmic microtubules had no effect on fragmentation. Our results provide insights into the relative contributions of the kinetochore and cytoplasmic microtubules to the forces involved in formation of a bipolar spindle.     

The role of mitochondrial DNA in the development of type 2 diabetes caused by fetal malnutrition

Epidemiological studies have revealed strong and reproducible links between indices of poor fetal growth and susceptibility to the development of glucose intolerance and insulin resistance syndrome in adult life. To explain these associations, the thrifty phenotype hypothesis has been proposed. Mitochondrial DNA abnormalities have been known to cause insulin deficiency, insulin resistance and diabetes mellitus. In this review, we propose that mitochondrial dysfunction is a link between malnutrition during early life and disease in adult life. The potential mechanism for mitochondrial dysfunction will be focused on availability of the taurine and nucleotides, and imprinting on the genes.     

Caveolin-2 regulation of the cell cycle in response to insulin in Hirc-B fibroblast cells

The regulatory function of caveolin-2 in cell cycle regulation by insulin was investigated in human insulin receptor-overexpressed rat 1 fibroblast (Hirc-B) cells. Insulin increased induction of the caveolin-2 gene in a time-dependent manner. Direct interaction between ERK and caveolin-2 was confirmed by immunoprecipitation and phosphorylated ERK increased the specific interaction in response to insulin. That insulin induced their nuclear co-localization over time was demonstrated by immunofluorescence microscopy. Insulin increased the S phase in the cell cycle by 6-fold. When recombinant caveolin-1 was transiently expressed, a decrease in the S phase was detected by flow-cytometry. The results indicate that the up-regulation of caveolin-2 in response to insulin activates the downstream signal cascades in the cell cycle, chiefly the increased phosphorylation of ERK, the nuclear translocation of phosphorylated ERK, and the subsequent activation of G0/G1 to S phase transition of the cell cycle. The results also suggest that DNA synthesis and the activation of the cell cycle by insulin are achieved concomitantly with an increase in the interaction between caveolin-2 and phosphorylated ERK, and the nuclear translocation of that complex. Taken together, we conclude that caveolin-2 positively regulates the insulin-induced cell cycle through activation of and direct interaction with ERK in Hirc-B cells.     

Effects of insulin and transgenic overexpression of UDP-glucose pyrophosphorylase on UDP-glucose and glycogen accumulation in skeletal muscle fibers

UDP-glucose (UDP-Glc) and glycogen levels in skeletal muscle fibers of defined fiber type were measured using microanalytical methods. Infusing rats with insulin increased glycogen in both Type I and Type II fibers. Insulin was without effect on UDP-Glc in Type I fibers but decreased UDP-Glc by 35-40% in Type IIA/D and Type IIB fibers. The reduction in UDP-Glc suggested that UDP-Glc pyrophosphorylase (PPL) activity might limit glycogen synthesis in response to insulin. To explore this possibility, we generated mice overexpressing a UDP-Glc PPL transgene in skeletal muscle. The transgene increased both UDP-Glc PPL activity and levels of UDP-Glc in skeletal muscles by approximately 3-fold. However, overexpression of UDP-Glc PPL was without effect on either the levels of skeletal muscle glycogen or glucose tolerance in vivo. The transgene was also without effect on either control or insulin-stimulated rates of (14)C-glucose incorporation into glycogen in muscles incubated in vitro. The results indicate that UDP-Glc PPL activity is not limiting for glycogen synthesis.     

Application of logistic regression to case-control association studies involving two causative loci

Models in which two susceptibility loci jointly influence the risk of developing disease can be explored using logistic regression analysis. Comparison of likelihoods of models incorporating different sets of disease model parameters allows inferences to be drawn regarding the nature of the joint effect of the loci. We have simulated case-control samples generated assuming different two-locus models and then analysed them using logistic regression. We show that this method is practicable and that, for the models we have used, it can be expected to allow useful inferences to be drawn from sample sizes consisting of hundreds of subjects. Interactions between loci can be explored, but interactive effects do not exactly correspond with classical definitions of epistasis. We have particularly examined the issue of the extent to which it is helpful to utilise information from a previously identified locus when investigating a second, unknown locus. We show that for some models conditional analysis can have substantially greater power while for others unconditional analysis can be more powerful. Hence we conclude that in general both conditional and unconditional analyses should be performed when searching for additional loci.     

Mono-, bi-, or tridentate ligands? The labeling of peptides with 99mTc-carbonyls

The labeling of targeting peptides with (99m)Tc is a useful concept for the diagnosis of various diseases such as cancer. Although in research for at least one decade, only a very few radiopharmaceuticals based on peptides are in clinical use. The difficulty of labeling, and the resulting authenticity of the new vector, is largely responsible for this observation. In this overview, we present an alternate strategy based on the organometallic fac-[(99m)Tc(CO)(3)](+) core for introducing (99m)Tc in biomolecules in general and in peptides in particular. The three coordination sites available in [(99m)Tc(OH(2))(3)(CO)(3)](+) can be occupied with many different ligand types, pendant to a biomolecule and serving as the anchor group for labeling. This makes the appropriate choice difficult. We intend to present some useful concepts for the practice. Monodentate chelators are robust but bear the risk of multiple binding of biomolecules. Coordinating a bidentate ligand of choice prior to labeling bypasses this problem and enables a systematic drug discovery by variation of the bidentate ligand. Bidentate ligands attached to the biomolecule are stronger but occasionally require protection of the remaining site by a monodentate ligand. Both approaches refer to a mixed-ligand [2+1] approach. Tridentate chelators are the most efficient but need some protecting group chemistry in order to achieve selectivity for the coupling process. Examples with cysteine and histidine are presented. This article aims to provide versatile and reproducible approaches for the labeling of biomolecules while not focusing on particular systems. It should be left to the readers to derive a strategy for their own peptide.     

The future of association studies: gene-based analysis and replication

Historically, association tests were limited to single variants, so that the allele was considered the basic unit for association testing. As marker density increases and indirect approaches are used to assess association through linkage disequilibrium, association is now frequently considered at the haplotypic level. We suggest that there are difficulties in replicating association findings at the single-nucleotide-polymorphism (SNP) or the haplotype level, and we propose a shift toward a gene-based approach in which all common variation within a candidate gene is considered jointly. Inconsistencies arising from population differences are more readily resolved by use of a gene-based approach rather than either a SNP-based or a haplotype-based approach. A gene-based approach captures all of the potential risk-conferring variations; thus, negative findings are subject only to the issue of power. In addition, chance findings due to multiple testing can be readily accounted for by use of a genewide-significance level. Meta-analysis procedures can be formalized for gene-based methods through the combination of P values. It is only a matter of time before all variation within genes is mapped, at which point the gene-based approach will become the natural end point for association analysis and will inform our search for functional variants relevant to disease etiology.     

GADD34 protein levels increase after transient ischemia in the cortex but not in the CA1 subfield: implications for post-ischemic recovery of protein synthesis in ischemia-resistant cells

Transient cerebral ischemia is a pathological process whereby an irreversible suppression of protein synthesis is believed to contribute to the extent of cell death in vulnerable neurons. Endoplasmic reticulum (ER) dysfunction has been identified as being responsible for ischemia-induced shut-down of translation. Recovery from ER dysfunction is facilitated by GADD34, a protein that dephosphorylates eukaryotic initiation factor (eIF)2alpha-P and thus reactivates protein synthesis. We investigated ischemia-induced changes in GADD34 levels in wild-type and Cu2+/Zn2+ SOD (SOD1) over-expressing rats. Transient global cerebral ischemia was induced by common carotid artery occlusion. Tissue samples were taken from the vulnerable hippocampal CA1 subfield and the resistant cerebral cortex of the right and left hemispheres for evaluation of changes in gadd34 mRNA and GADD34 protein levels. In wild-type animals, we found significantly lower GADD34 levels than in SOD1 transgenes but no differences in gadd34 mRNA levels, implying that superoxides regulate gadd34 translation. After ischemia, GADD34 protein levels were significantly increased in the cortex but not in the CA1 subfield, and these changes occurred earlier in SOD1 transgenic than in wild-type animals. The rise in gadd34 mRNA levels did not differ in the cortex and CA1 subfield, implying that gadd34 expression is regulated at the translational level.     

The Rapidly Evolving Centromere-Specific Histone Has Stringent Functional Requirements in Arabidopsis thaliana

Centromeres control chromosome inheritance in eukaryotes, yet their DNA structure and primary sequence are hypervariable. Most animals and plants have megabases of tandem repeats at their centromeres, unlike yeast with unique centromere sequences. Centromere function requires the centromere-specific histone CENH3 (CENP-A in human), which replaces histone H3 in centromeric nucleosomes. CENH3 evolves rapidly, particularly in its N-terminal tail domain. A portion of the CENH3 histone-fold domain, the CENP-A targeting domain (CATD), has been previously shown to confer kinetochore localization and centromere function when swapped into human H3. Furthermore, CENP-A in human cells can be functionally replaced by CENH3 from distantly related organisms including Saccharomyces cerevisiae. We have used cenh3-1 (a null mutant in Arabidopsis thaliana) to replace endogenous CENH3 with GFP-tagged variants. A H3.3 tail domain–CENH3 histone-fold domain chimera rescued viability of cenh3-1, but CENH3''s lacking a tail domain were nonfunctional. In contrast to human results, H3 containing the A. thaliana CATD cannot complement cenh3-1. GFP–CENH3 from the sister species A. arenosa functionally replaces A. thaliana CENH3. GFP–CENH3 from the close relative Brassica rapa was targeted to centromeres, but did not complement cenh3-1, indicating that kinetochore localization and centromere function can be uncoupled. We conclude that CENH3 function in A. thaliana, an organism with large tandem repeat centromeres, has stringent requirements for functional complementation in mitosis.CENTROMERES are essential for chromosome inheritance, because they nucleate kinetochores, the protein complexes on eukaryotic chromosomes that attach to spindle microtubules. Despite the essential requirement for centromeres in chromosome segregation, their DNA sequences and the sequences of kinetochore proteins are highly variable. Kinetochores in Saccharomyces cerevisiae and related budding yeasts assemble on small, unique centromere DNAs (125 bp in S. cerevisiae) (Meraldi et al. 2006). Centromere DNAs in the fission yeast Schizosaccharomyces pombe are larger, consisting of a central core sequence of 4–5 kb, which binds kinetochore proteins, flanked by large inverted repeats whose heterochromatic nature is important for centromere function (the total size of the S. pombe centromere DNA is 35–110 kb). At the other extreme from small yeast centromeres are holocentric organisms, such as Caenorhabditis elegans, in which kinetochore proteins bind along the entire length of mitotic chromosomes (Dernburg 2001). Most plants and animals have extremely large centromere DNA tracts consisting of megabases of simple tandem repeats. The repeat sequence evolves extremely rapidly, and only a small fraction of the repeat array is likely to be bound by kinetochore proteins. Furthermore, kinetochores can be nucleated by noncentromeric DNA sequences in plant and animal cells (Amor and Choo 2002; Nagaki et al. 2004; Nasuda et al. 2005; Heun et al. 2006; Wade et al. 2009). Despite these findings, the maintenance of massive centromere repeat arrays in both animal and plant taxa suggests that repeats are a central feature of centromere biology in these organisms.Although centromere DNAs are extremely diverse, all eukaryote kinetochores contain the centromere-specific histone H3 variant CENH3 (originally described as CENP-A in human) (Henikoff and Dalal 2005; Black and Bassett 2008). CENH3 replaces conventional H3 specifically in a subset of centromere nucleosomes. It is essential for kinetochore function in all eukaryotes where this requirement has been tested. Conventional histones are among the most conserved proteins in eukaryote genomes. In contrast, CENH3 is rapidly evolving. The C-terminal histone-fold domain, which complexes with other histones to form the globular nucleosome core, can be aligned with conventional H3''s but evolves rapidly and shows signatures of adaptive evolution in some residues (Malik and Henikoff 2001; Talbert et al. 2002; Cooper and Henikoff 2004). The N-terminal tail domain of conventional histone H3 protrudes from the nucleosome core and is not resolved in the structure solved by X-ray crystallography (Luger et al. 1997). In CENH3, the tail domain evolves so rapidly that its sequence can barely be aligned between closely related species.Experiments in yeast and in animals have delineated functionally important regions within CENH3. S. cerevisiae kinetochores contain only a single CENH3/Cse4p nucleosome (Furuyama and Biggins 2007). In S. cerevisiae Cse4p, amino acid residues required for normal function are distributed throughout the histone-fold domain (Keith et al. 1999). The N-terminal tail of Cse4p contains an essential region termed the END domain, but overexpression of a Cse4p lacking the tail altogether can rescue a cse4 deletion mutant (Chen et al. 2000; Morey et al. 2004). In Drosophila melanogaster cells, CENH3/Cid from the distantly related D. bipectinata did not localize to kinetochores unless a specific region of the histone-fold domain, loop 1, was swapped with the corresponding region from D. melanogaster CENH3/Cid (Vermaak et al. 2002). In human, the histone-fold domain is important for centromere targeting (Sullivan et al. 1994). The functionally important region within the histone-fold domain was further defined by inserting loop 1 and the α-2 helix from CENH3/CENP-A (termed the CENP-A targeting domain, or CATD) into conventional H3 (Black et al. 2004). H3 containing the CATD acquires several functions of CENP-A when expressed in human cells. It localizes to kinetochores, binds the kinetochore protein CENP-N, has a rigid secondary structure when assembled into nucleosomes, and can restore normal chromosome segregation in cells depleted for CENP-A using RNA interference (RNAi) (Black et al. 2004, 2007a,b; Carroll et al. 2009).Despite these extensive studies, questions about structure–function relationships within CENH3 remain. CENH3 function may differ between small yeast centromeres and the large tandem repeat centromeres of animals and plants, particularly because larger centromere DNAs are likely to contain many more CENH3 nucleosomes and may require a higher level of organization. Experiments in D. melanogaster and in human cells have used RNAi to downregulate the endogenous protein, and a conditional knockout has been made in chicken DT-40 cells (Blower and Karpen 2001; Goshima et al. 2003; Regnier et al. 2005; Black et al. 2007b). These experiments are challenging because CENH3 is very stable. If preexisting CENH3 is partitioned equally between duplicated sister centromeres, its amount will be approximately halved at each cell division. Therefore the protein may persist for many cell divisions after induction of RNAi, as shown by Western blots indicating that ∼10% of endogenous CENH3 remains in human cells subjected to two rounds of RNAi (Black et al. 2007b).We have chosen to study CENH3 in the model plant A. thaliana, which combines facile genetics and transgenesis with centromere DNA structure that is similar to most plants and animals (megabases of tandem repeats with a repeating unit of 178 bp) (Murata et al. 1994; Copenhaver et al. 1999). Although Drosophila and mouse CENH3 knockout mutants have been characterized (Howman et al. 2000; Blower et al. 2006), a large-scale structure–function analysis of CENH3 has not been attempted in these organisms. A cenh3 null mutant in A. thaliana allows us to completely replace the endogenous protein with transgenic variants (Ravi and Chan 2010). Here we report four major conclusions regarding CENH3 function in A. thaliana: (1) CENH3 function requires an N-terminal histone tail domain, although either the CENH3 tail or the H3 tail can support mitotic chromosome segregation. (2) Inserting the CENP-A targeting domain of CENH3 into H3 does not confer CENH3 function. (3) Complementation of cenh3 by heterologous CENH3 requires that the species of origin be closely related to A. thaliana. (4) Localization of a heterologous CENH3 protein to kinetochores in the presence of native CENH3 does not necessarily indicate that it can complement a cenh3 mutant. Overall, our results indicate that requirements for CENH3 function in A. thaliana are more stringent that those obtained in human cells. They underscore the usefulness of comparative studies of centromere function using genetically tractable experimental organisms.