Changes in the Nuclear Envelope Environment Affect Spindle Pole Body Duplication in Saccharomyces cerevisiae

The Saccharomyces cerevisiae nuclear membrane is part of a complex nuclear envelope environment also containing chromatin, integral and peripheral membrane proteins, and large structures such as nuclear pore complexes (NPCs) and the spindle pole body. To study how properties of the nuclear membrane affect nuclear envelope processes, we altered the nuclear membrane by deleting the SPO7 gene. We found that spo7Δ cells were sickened by the mutation of genes coding for spindle pole body components and that spo7Δ was synthetically lethal with mutations in the SUN domain gene MPS3. Mps3p is required for spindle pole body duplication and for a variety of other nuclear envelope processes. In spo7Δ cells, the spindle pole body defect of mps3 mutants was exacerbated, suggesting that nuclear membrane composition affects spindle pole body function. The synthetic lethality between spo7Δ and mps3 mutants was suppressed by deletion of specific nucleoporin genes. In fact, these gene deletions bypassed the requirement for Mps3p entirely, suggesting that under certain conditions spindle pole body duplication can occur via an Mps3p-independent pathway. These data point to an antagonistic relationship between nuclear pore complexes and the spindle pole body. We propose a model whereby nuclear pore complexes either compete with the spindle pole body for insertion into the nuclear membrane or affect spindle pole body duplication by altering the nuclear envelope environment.THE nuclear envelope is composed of distinct outer and inner nuclear membranes. The outer nuclear membrane is continuous with the endoplasmic reticulum. The inner nuclear membrane is associated with a unique set of proteins, some of which mediate interactions between the nuclear envelope and chromatin (reviewed in Zhao et al. 2009). Nuclear pore complexes traverse both membranes and allow transport of proteins and solutes between the cytoplasm and the nucleus. The inner and outer nuclear membranes fuse in the region surrounding each nuclear pore complex.In animal cells, the nuclear envelope disassembles as cells enter mitosis and reassembles upon mitotic exit. Nuclear envelope breakdown allows the association of chromosomes with spindle microtubules, which are nucleated from centrosomes that reside in the cytoplasm. In contrast, certain types of fungi, such as the budding yeast Saccharomyces cerevisiae, undergo closed mitosis, where the nuclear envelope remains intact throughout the entire cell cycle. Closed mitosis is possible because the yeast centrosome-equivalent, the spindle pole body (SPB), is embedded in the nuclear envelope, allowing the SPB to nucleate both cytoplasmic and nuclear microtubules.SPB duplication requires a mechanism for inserting the new SPB into the nuclear envelope (reviewed in Jaspersen and Winey 2004). The new SPB begins to form in late G₁/early S phase as satellite material deposited on the cytoplasmic face of an electron-dense region of the nuclear envelope, called the half-bridge. The satellite material matures into a duplication plaque, which is then inserted into the nuclear membrane and becomes the daughter SPB. Many genes are known to be required for SPB duplication, and this process has been carefully examined cytologically (Rose and Fink 1987; Winey et al. 1991, 1993; Spang et al. 1995; Bullitt et al. 1997; Adams and Kilmartin 1999; Elliott et al. 1999; Schramm et al. 2000; Jaspersen et al. 2002; Nishikawa et al. 2003; Araki et al. 2006). However, the exact mechanisms by which SPB duplication and insertion occur remain a mystery.Equally unclear is how nuclear pore complexes are inserted into an intact nuclear envelope (reviewed in Hetzer and Wente 2009). For both the SPB and nuclear pore complexes, the inner and outer nuclear membranes must fuse to allow insertion into the nuclear envelope. Yeast and vertebrate nuclear pore complexes each have four pore membrane (POM) nucleoporins containing transmembrane domains. Other nucleoporins have motifs with potential for bending membranes or sensing membrane curvature. Thus, certain nuclear pore complex components may have the ability to alter the nuclear membrane or stabilize particular membrane conformations (Devos et al. 2004, 2006; Alber et al. 2007; Drin et al. 2007). It is interesting to note that, in S. cerevisiae, nuclear pore complexes are enriched in the vicinity of the SPB (Heath et al. 1995; Winey et al. 1997; Adams and Kilmartin 1999), but the significance of this phenomenon is not known. The SPB and nuclear pore complexes share at least two common components, the integral membrane protein Ndc1p and the small calcium-binding protein Cdc31p (Chial et al. 1998; Fischer et al. 2004). Ndc1p is thought to play a role in insertion of both SPBs and nuclear pore complexes into the nuclear membrane.SUN domain proteins are a conserved family of inner nuclear membrane proteins that interact with specific outer nuclear membrane proteins to form a physical bridge across the nuclear envelope (reviewed in Hiraoka and Dernburg 2009; Razafsky and Hodzic 2009). One of the components of the S. cerevisiae SPB is the SUN domain protein Mps3p. The N terminus of Mps3p is in the nucleoplasm, while the C terminus, containing the SUN domain, is found in the space between the inner and outer nuclear membranes. In addition to the SPB, Mps3p localizes to multiple foci at the nuclear periphery, and these two pools of Mps3p have distinct functions (Jaspersen et al. 2002, 2006; Nishikawa et al. 2003). At the SPB, Mps3p is required for half-bridge formation and early steps of SPB duplication, and cells compromised for Mps3p function accumulate in mitosis with a single SPB and a monopolar spindle (Jaspersen et al. 2002; Nishikawa et al. 2003). At the nuclear periphery, Mps3p is involved in tethering telomeres to the nuclear envelope in mitosis and meiosis, sequestering DNA double-strand breaks away from recombination factors, and associating with soluble chromatin proteins (Antoniacci et al. 2004, 2007; Bupp et al. 2007; Conrad et al. 2007, 2008; Oza et al. 2009; Schober et al. 2009).While many structural features of the yeast nucleus have been identified, little is known about how the physical properties of the nuclear membrane contribute to processes that occur at the nuclear envelope. As noted above, resident proteins of the nuclear envelope may affect nuclear membrane properties. In addition, the nuclear membrane is affected by altering lipid biosynthesis, for example, by inactivating the phosphatidic acid (PA) phosphohydrolase Pah1p or by inactivating the phosphates complex, made of Spo7p and Nem1p, which activates Pah1p. In the absence of Spo7p, Nem1p, or Pah1p, cells exhibit nuclear envelope extensions and extensive ER membrane sheets, and they also have altered membrane lipid composition, including a decrease in phosphatidylcholine and an increase in PA, phosphatidylethanolamine, and phosphatidylinositol (Siniossoglou et al. 1998; Santos-Rosa et al. 2005; Campbell et al. 2006; Han et al. 2006). These three proteins are unique among phospholipid biosynthesis proteins in their ability to affect nuclear morphology upon gene disruption (Han et al. 2008). A similar phenotype was seen upon overexpression of DGK1, which counteracts the activity of Pah1p by converting diacylglycerol to PA, leading to an increase in PA levels at the nuclear envelope (Han et al. 2008). Consistent with a conserved role for Pah1p in regulating nuclear envelope processes, deletion of either NEM1 or SPO7 is synthetically lethal with deletions of certain nucleoporin genes (Siniossoglou et al. 1998), and inactivation of the PAH1 homolog in Caenorhabditis elegans, LPIN-1, results in defects in nuclear envelope disassembly and reassembly (Golden et al. 2009; Gorjanacz and Mattaj 2009).To identify processes that are affected by altered nuclear membrane properties, we screened for pathways that are compromised in spo7Δ cells. We found that SPO7 inactivation strongly influences the SPB. By screening for proteins that could alleviate spo7Δ-induced SPB defects, we uncovered an unexpected inhibitory role for nucleoporins in SPB function, revealing that nuclear pore complexes, or components thereof, act antagonistically to the SPB in the nuclear envelope. Taken together, our findings indicate that the nuclear envelope environment is important for the function of protein complexes and biological processes occurring at the nuclear periphery.     

Peptide Derived from HIV-1 TAT Protein Destabilizes a Monolayer of Endothelial Cells in an in Vitro Model of the Blood-Brain Barrier and Allows Permeation of High Molecular Weight Proteins

Most chemotherapeutic agents are blood-brain barrier (BBB) impermeants. HIV-1-derived TAT protein variants contain a transmembrane domain, which may enable them to cross the BBB and reach the brain. Here we synthesized CAYGRKKRRQRRR, a peptide containing a cysteine moiety attached to the N terminus of the transmembrane domain (C-TAT peptide), and studied its effects in an in vitro BBB model, which we found to reflect penetration by a receptor-independent pathway. Incubation of the brain capillary endothelial cell monolayer with 0.3–0.6 μmol/ml of this C-TAT peptide, for a period of 1–2 h, destabilizes brain capillary endothelial cell monolayer and introduces the ability of impermeant therapeutic agents including high molecular weight proteins to penetrate it substantially. The cysteinyl moiety at position 1 of the C-TAT peptide contributes largely to the destabilizing potency and the penetration efficacy of impermeant substances. The destabilizing effect was reversed using heparin. In summary, experimental conditions allowing a significant increase in entry of impermeant low and high molecular weight substances from the luminal (blood) to the abluminal side (brain) were found in an in vitro BBB model reflecting in vivo protein penetrability by a receptor-independent pathway.     

Genomic variation in the vomeronasal receptor gene repertoires of inbred mice

ABSTRACT: BACKGROUND: Vomeronasal receptors (VRs), expressed in sensory neurons of the vomeronasal organ, are thought to bind pheromones and mediate innate behaviours. The mouse reference genome has over 360 functional VRs arranged in highly homologous clusters, but the vast majority are of unknown function. Differences in these receptors within and between closely related species of mice are likely to underpin a range of behavioural responses. To investigate these differences, we interrogated the VR gene repertoire from 17 inbred strains of mice using massively parallel sequencing. RESULTS: Approximately half of the 6222 VR genes that we investigated could be successfully resolved, and those that were unambiguously mapped resulted in an extremely accurate dataset. Collectively VRs have over twice the coding sequence variation of the genome average; but we identify striking non-random distribution of these variants within and between genes, clusters, clades and functional classes of VRs. We show that functional VR gene repertoires differ considerably between different Mus subspecies and species, suggesting these receptors may play a role in mediating behavioural adaptations. Finally, we provide evidence that widely-used, highly inbred laboratory-derived strains have a greatly reduced, but not entirely redundant capacity for differential pheromone-mediated behaviours. CONCLUSIONS: Together our results suggest that the unusually variable VR repertoires of mice have a significant role in encoding differences in olfactory-mediated responses and behaviours. Our dataset has expanded over nine fold the known number of mouse VR alleles, and will enable mechanistic analyses into the genetics of innate behavioural differences in mice.     

A p38 MAPK-CREB pathway functions to pattern mesoderm in Xenopus

Dorsal-ventral patterning is specified by signaling centers secreting antagonizing morphogens that form a signaling gradient. Yet, how morphogen gradient is translated intracellularly into fate decisions remains largely unknown. Here, we report that p38 MAPK and CREB function along the dorsal-ventral axis in mesoderm patterning. We find that the phosphorylated form of CREB (S133) is distributed in a gradient along the dorsal-ventral mesoderm axis and that the p38 MAPK pathway mediates the phosphorylation of CREB. Knockdown of CREB prevents chordin expression and mesoderm dorsalization by the Spemann organizer, whereas ectopic expression of activated CREB-VP16 chimera induces chordin expression and dorsalizes mesoderm. Expression of high levels of p38 activator, MKK6E or CREB-VP16 in embryos converts ventral mesoderm into a dorsal organizing center. p38 MAPK and CREB function downstream of maternal Wnt/β-catenin and the organizer-specific genes siamois and goosecoid. At low expression levels, MKK6E induces expression of lateral genes without inducing the expression of dorsal genes. Loss of CREB or p38 MAPK activity enables the expansion of the ventral homeobox gene vent1 into the dorsal marginal region, preventing the lateral expression of Xmyf5. Overall, these data indicate that dorsal-ventral mesoderm patterning is regulated by differential p38/CREB activities along the axis.     

Toward a PKB inhibitor: modification of a selective PKA inhibitor by rational design

Protein kinase B/Akt (PKB) is an anti-apoptotic protein kinase that has strongly elevated activity in human malignancies. We therefore initiated a program to develop PKB inhibitors, "Aktstatins". We screened about 500 compounds for PKB inhibitors, using a radioactive assay and an ELISA assay that we established for this purpose. These compounds were produced as combinatorial libraries, designed using the structure of the selective PKA inhibitor H-89 as a starting point. We have identified a successful lead compound, which inhibits PKB activity in vitro and in cells overexpressing active PKB. The new compound shows reversed selectivity to H-89: In contrast to H-89, which inhibits PKA 70 times better than PKB, the new compound, NL-71-101, inhibits PKB 2.4-fold better than PKA. The new compound, but not H-89, induces apoptosis in tumor cells in which PKB is amplified. We have identified structural features in NL-71-101 that are significant for the specificity and that can be used for future development and optimization of PKB inhibitors.     

Dab2 inhibits the cholesterol-dependent activation of JNK by TGF-β

Transforming growth factor-β (TGF-β) ligands activate Smad-mediated and noncanonical signaling pathways in a cell context–dependent manner. Localization of signaling receptors to distinct membrane domains is a potential source of signaling output diversity. The tumor suppressor/endocytic adaptor protein disabled-2 (Dab2) was proposed as a modulator of TGF-β signaling. However, the molecular mechanism(s) involved in the regulation of TGF-β signaling by Dab2 were not known. Here we investigate these issues by combining biophysical studies of the lateral mobility and endocytosis of the type I TGF-β receptor (TβRI) with TGF-β phosphoprotein signaling assays. Our findings demonstrate that Dab2 interacts with TβRI to restrict its lateral diffusion at the plasma membrane and enhance its clathrin-mediated endocytosis. Small interfering RNA–mediated knockdown of Dab2 or Dab2 overexpression shows that Dab2 negatively regulates TGF-β–induced c-Jun N-terminal kinase (JNK) activation, whereas activation of the Smad pathway is unaffected. Moreover, activation of JNK by TGF-β in the absence of Dab2 is disrupted by cholesterol depletion. These data support a model in which Dab2 regulates the domain localization of TβRI in the membrane, balancing TGF-β signaling via the Smad and JNK pathways.     

Similar cation channels mediate protection from cerebellar exitotoxicity by exercise and inheritance

Exercise and inherited factors both affect recovery from stroke and head injury, but the underlying mechanisms and interconnections between them are yet unknown. Here, we report that similar cation channels mediate the protective effect of exercise and specific genetic background in a kainate injection model of cerebellar stroke. Microinjection to the cerebellum of the glutamatergic agonist, kainate, creates glutamatergic excito\xE2\x80\x90toxicity characteristic of focal stroke, head injury or alcoholism. Inherited protection and prior exercise were both accompanied by higher cerebellar expression levels of the Kir6.1 ATP-dependent potassium channel in adjacent Bergmann glia, and voltage-gated KVbeta2 and cyclic nucleotide-gated cation HCN1 channels in basket cells. Sedentary FVB/N and exercised C57BL/6 mice both expressed higher levels of these cation channels compared to sedentary C57BL/6 mice, and were both found to be less sensitive to glutamate toxicity. Moreover, blocking ATP-dependent potassium channels with Glibenclamide enhanced kainate-induced cell death in cerebellar slices from the resilient sedentary FVB/N mice. Furthermore, exercise increased the number of acetylcholinesterase-positive fibres in the molecular layer, reduced cerebellar cytokine levels and suppressed serum acetylcholinesterase activity, suggesting anti-inflammatory protection by enhanced cholinergic signalling. Our findings demonstrate for the first time that routine exercise and specific genetic backgrounds confer protection from cerebellar glutamatergic damages by similar molecular mechanisms, including elevated expression of cation channels. In addition, our findings highlight the involvement of the cholinergic anti-inflammatory pathway in insult-inducible cerebellar processes. These mechanisms are likely to play similar roles in other brain regions and injuries as well, opening new venues for targeted research efforts.