Monday, June 1, 2009

Hot off the presses! Jun 01 Nat Cell Biol

The Jun 01 issue of the Nat Cell Biol is now up on Pubget (About Nat Cell Biol): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

  • How robust is your data?
    - Nat Cell Biol 11(6):667 (2009)
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  • New fashion models
    - Nat Cell Biol 11(6):668 (2009)
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  • STIMulating store-operated Ca2+ entry
    - Nat Cell Biol 11(6):669-677 (2009)
    Calcium influx through plasma membrane store-operated Ca2+ (SOC) channels is triggered when the endoplasmic reticulum (ER) Ca2+ store is depleted — a homeostatic Ca2+ signalling mechanism that remained enigmatic for more than two decades. RNA-interference (RNAi) screening and molecular and cellular physiological analysis recently identified STIM1 as the mechanistic 'missing link' between the ER and the plasma membrane. STIM proteins sense the depletion of Ca2+ from the ER, oligomerize, translocate to junctions adjacent to the plasma membrane, organize Orai or TRPC (transient receptor potential cation) channels into clusters and open these channels to bring about SOC entry.
  • Wicked views on stem cell news
    - Nat Cell Biol 11(6):678-679 (2009)
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  • Inverted rod nuclei see the light
    - Nat Cell Biol 11(6):680-681 (2009)
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  • MicroRNA-mediated regulation of synaptic palmitoylation: shrinking fat spines
    - Nat Cell Biol 11(6):681-682 (2009)
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  • Research highlights
    - Nat Cell Biol 11(6):684 (2009)
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  • Live-imaging of single stem cells within their niche reveals that a U3snoRNP component segregates asymmetrically and is required for self-renewal in Drosophila
    - Nat Cell Biol 11(6):685-693 (2009)
    Stem cells generate self-renewing and differentiating progeny over many rounds of asymmetric divisions. How stem cell growth rate and size are maintained over time remains unknown. We isolated mutations in a Drosophila melanogaster gene, wicked (wcd), which induce premature differentiation of germline stem cells (GSCs). Wcd is a member of the U3 snoRNP complex required for pre-ribosomal RNA maturation. This general function of Wcd contrasts with its specific requirement for GSC self-renewal. However, live imaging of GSCs within their niche revealed a pool of Wcd-forming particles that segregate asymmetrically into the GSCs on mitosis, independently of the Dpp signal sent by the niche. A fraction of Wcd also segregated asymmetrically in dividing larval neural stem cells (NSCs). In the absence of Wcd, NSCs became smaller and produced fewer neurons. Our results show that regulation of ribosome synthesis is a crucial parameter for stem cell maintenance and function.
  • p53 controls cancer cell invasion by inducing the MDM2-mediated degradation of Slug
    - Nat Cell Biol 11(6):694-704 (2009)
    The tumour suppressor p53 is known to prevent cancer progression by inhibiting proliferation and inducing apoptosis of tumour cells. Slug, an invasion promoter, exerts its effects by repressing E-cadherin transcription. Here we show that wild-type p53 (wtp53) suppresses cancer invasion by inducing Slug degradation, whereas mutant p53 may stabilize Slug protein. In non-small-cell lung cancer (NSCLC), mutation of p53 correlates with low MDM2, high Slug and low E-cadherin expression. This expression profile is associated with poor overall survival and short metastasis-free survival in patients with NSCLC. wtp53 upregulates MDM2 and forms a wtp53–MDM2–Slug complex that facilitates MDM2-mediated Slug degradation. Downregulation of Slug by wtp53 or MDM2 enhances E-cadherin expression and represses cancer cell invasiveness. In contrast, mutant p53 inactivates Slug degradation and leads to Slug accumulation and increased cancer cell invasiveness. Our findings indicate that! wtp53 and p53 mutants may differentially control cancer invasion and metastasis through the p53–MDM2–Slug pathway.
  • A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis
    - Nat Cell Biol 11(6):705-716 (2009)
    The microRNA pathway has been implicated in the regulation of synaptic protein synthesis and ultimately in dendritic spine morphogenesis, a phenomenon associated with long-lasting forms of memory. However, the particular microRNAs (miRNAs) involved are largely unknown. Here we identify specific miRNAs that function at synapses to control dendritic spine structure by performing a functional screen. One of the identified miRNAs, miR-138, is highly enriched in the brain, localized within dendrites and negatively regulates the size of dendritic spines in rat hippocampal neurons. miR-138 controls the expression of acyl protein thioesterase 1 (APT1), an enzyme regulating the palmitoylation status of proteins that are known to function at the synapse, including the alpha13 subunits of G proteins (Galpha13). RNA-interference-mediated knockdown of APT1 and the expression of membrane-localized Galpha13 both suppress spine enlargement caused by inhibition of miR-138, suggesting t! hat APT1-regulated depalmitoylation of Galpha13 might be an important downstream event of miR-138 function. Our results uncover a previously unknown miRNA-dependent mechanism in neurons and demonstrate a previously unrecognized complexity of miRNA-dependent control of dendritic spine morphogenesis.
  • The mitotic kinesin-14 Ncd drives directional microtubule–microtubule sliding
    - Nat Cell Biol 11(6):717-723 (2009)
    During mitosis and meiosis, the bipolar spindle facilitates chromosome segregation through microtubule sliding as well as microtubule growth and shrinkage1. Kinesin-14, one of the motors involved, causes spindle collapse in the absence of kinesin-5 (Refs 2, 3), participates in spindle assembly4 and modulates spindle length5. However, the molecular mechanisms underlying these activities are not known. Here, we report that Drosophila melanogaster kinesin-14 (Ncd) alone causes sliding of anti-parallel microtubules but locks together (that is, statically crosslinks) those that are parallel. Using single molecule imaging we show that Ncd diffuses along microtubules in a tail-dependent manner and switches its orientation between sliding microtubules. Our results show that kinesin-14 causes sliding and expansion of an anti-parallel microtubule array by dynamic interactions through the motor domain on the one side and the tail domain on the other. This mechanism accounts for t! he roles of kinesin-14 in spindle organization.
  • The kinesin-14 Klp2 organizes microtubules into parallel bundles by an ATP-dependent sorting mechanism
    - Nat Cell Biol 11(6):724-730 (2009)
    The dynamic organization of microtubules into parallel arrays allows interphase cells to set up multi-lane highways for intracellular transport and M-phase cells to build the mitotic and meiotic spindles. Here we show that a minimally reconstituted system composed of Klp2, a kinesin-14 from the fission yeast Schizosaccharomyces pombe, together with microtubules assembled from purified S. pombe tubulin, autonomously assembles bundles of parallel microtubules. Bundles form by an ATP-dependent sorting mechanism that requires the full-length Klp2 motor. By this mechanism, antiparallel-overlapped microtubules slide over one another until they dissociate from the bundles, whereas parallel-overlapped microtubules are selectively trapped by an energy-dissipating force-balance mechanism. Klp2-driven microtubule sorting provides a robust pathway for the organization of microtubules into parallel arrays. In vivo evidence indicates that Klp2 is required for the proper organization! of S. pombe interphase microtubules into bipolar arrays of parallel-overlapped microtubules1, 2, 3, 4, suggesting that kinesin-14-dependent microtubule sorting may have wide biological importance.
  • Local auxin biosynthesis modulates gradient-directed planar polarity in Arabidopsis
    - Nat Cell Biol 11(6):731-738 (2009)
    The coordination of cell polarity within the plane of a single tissue layer (planar polarity) is a crucial task during development of multicellular organisms. Mechanisms underlying establishment of planar polarity, however, differ substantially between plants and animals1, 2, 3. In Arabidopsis thaliana, planar polarity of root-hair positioning along epidermal cells is coordinated towards maximum concentration of an auxin gradient in the root tip3, 4, 5. This gradient has been hypothesized to be sink-driven6 and computational modelling suggests that auxin efflux carrier activity may be sufficient to generate the gradient in the absence of auxin biosynthesis in the root7. Here, we demonstrate that the Raf-like kinase CONSTITUTIVE TRIPLE RESPONSE1 (CTR1; Refs 8, 9) acts as a concentration-dependent repressor of a biosynthesis-dependent auxin gradient that modulates planar polarity in the root tip. We analysed auxin biosynthesis and concentration gradients in a variety of ! root-hair-position mutants affected in CTR1 activity, auxin biosynthesis and transport. Our results reveal that planar polarity relies on influx- and efflux-carrier-mediated auxin redistribution from a local biosynthesis maximum. Thus, a local source of auxin biosynthesis contributes to gradient homeostasis during long-range coordination of cellular morphogenesis.
  • The Patched dependence receptor triggers apoptosis through a DRAL–caspase-9 complex
    - Nat Cell Biol 11(6):739-746 (2009)
    Sonic hedgehog (Shh) and its main receptor, Patched (Ptc), are implicated in both neural development and tumorigenesis1, 2. Besides its classic morphogenic activity, Shh is also a survival factor3, 4. Along this line, Ptc has been shown to function as a dependence receptor; it induces apoptosis in the absence of Shh, whereas its pro-apoptotic activity is blocked in the presence of Shh5. Here we show that, in the absence of its ligand, Ptc interacts with the adaptor protein DRAL (downregulated in rhabdomyosarcoma LIM-domain protein; also known as FHL2). DRAL is required for the pro-apoptotic activity of Ptc both in immortalized cells and during neural tube development in chick embryos. We demonstrate that, in the absence of Shh, Ptc recruits a protein complex that includes DRAL, one of the caspase recruitment (CARD)-domain containing proteins TUCAN (family member, 8) or NALP1 (NLR family, pyrin domain containing 1) and apical caspase-9. Ptc triggers caspase-9 activation! and enhances cell death through a caspase-9-dependent mechanism. Thus, we propose that in the absence of its ligand Shh the dependence receptor Ptc serves as the anchor for a caspase-activating complex that includes DRAL, and caspase-9.
  • The bioenergetic and antioxidant status of neurons is controlled by continuous degradation of a key glycolytic enzyme by APC/C–Cdh1
    - Nat Cell Biol 11(6):747-752 (2009)
    Neurons are known to have a lower glycolytic rate than astrocytes and when stressed they are unable to upregulate glycolysis1 because of low Pfkfb3 (6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase-3) activity2. This enzyme generates fructose-2,6-bisphosphate (F2,6P2)3, the most potent activator of 6-phosphofructo-1-kinase (Pfk1; ref. 4), a master regulator of glycolysis5. Here, we show that Pfkfb3 is absent from neurons in the brain cortex and that Pfkfb3 in neurons is constantly subject to proteasomal degradation by the action of the E3 ubiquitin ligase6, anaphase-promoting complex/cyclosome (APC/C)–Cdh1. By contrast, astrocytes have low APC/C–Cdh1 activity and therefore Pfkfb3 is present in these cells. Upregulation of Pfkfb3 by either inhibition of Cdh1 or overexpression of Pfkfb3 in neurons resulted in the activation of glycolysis. This, however, was accompanied by a marked decrease in the oxidation of glucose through the pentose phosphate pathway (a meta! bolic route involved in the regeneration of reduced glutathione7) resulting in oxidative stress and apoptotic death. Thus, by actively downregulating glycolysis by APC/C–Cdh1, neurons use glucose to maintain their antioxidant status at the expense of its utilization for bioenergetic purposes.
  • Replication stress induces sister-chromatid bridging at fragile site loci in mitosis
    - Nat Cell Biol 11(6):753-760 (2009)
    Several inherited syndromes in humans are associated with cancer predisposition. The gene products defective in two of these disorders, BLM (a helicase defective in Bloom's syndrome)1 and FANC A–N (defective in Fanconi anaemia)2, associate in a multienzyme complex called BRAFT3. How these proteins suppress tumorigenesis remains unclear, although both conditions are associated with chromosome instability. Here we show that the Fanconi anaemia proteins FANCD2 and FANCI specifically associate with common fragile site loci irrespective of whether the chromosome is broken. Unexpectedly, these loci are frequently interlinked through BLM-associated ultra-fine DNA bridges4 (UFBs) even as cells traverse mitosis. Similarly to fragile site expression5, fragile site bridging is induced after partial inhibition of DNA replication. We propose that, after replication stress, sister chromatids are interlinked by replication intermediates primarily at genetic loci with intrinsic repl! ication difficulties, such as fragile sites. In Bloom's syndrome cells, inefficient resolution of DNA linkages at fragile sites gives rise to increased numbers of anaphase UFBs and micronuclei containing fragile site DNA. Our data have general implications concerning the contribution of fragile site loci to chromosomal instability and tumorigenesis.
  • The FANC pathway and BLM collaborate during mitosis to prevent micro-nucleation and chromosome abnormalities
    - Nat Cell Biol 11(6):761-768 (2009)
    Loss-of-function of caretaker genes characterizes a group of cancer predisposition diseases that feature cellular hypersensitivity to DNA damage and chromosome fragility; this group includes Fanconi anaemia and Bloom syndrome1, 2, 3. The products of the 13 FANC genes4 (mutated in Fanconi anaemia), which constitute the 'FANC' pathway, and BLM (the RecQ helicase mutated in Bloom syndrome) are thought to collaborate during the S phase of the cell cycle, preventing chromosome instability. Recently, BLM has been implicated in the completion of sister chromatid separation during mitosis, a complex process in which precise regulation and execution is crucial to preserve genomic stability. Here we show for the first time a role for the FANC pathway in chromosome segregation during mitotic cell division. FANCD2, a key component of the pathway, localizes to discrete spots on mitotic chromosomes. FANCD2 chromosomal localization is responsive to replicative stress and specifically! targets aphidicolin (APH)-induced chromatid gaps and breaks. Our data indicate that the FANC pathway is involved in rescuing abnormal anaphase and telophase (ana-telophase) cells, limiting aneuploidy and reducing chromosome instability in daughter cells. We further address a cooperative role for the FANC pathway and BLM in preventing micronucleation, through FANC-dependent targeting of BLM to non-centromeric abnormal structures induced by replicative stress. We reveal new crosstalk between FANC and BLM proteins, extending their interaction beyond the S-phase rescue of damaged DNA5, 6 to the safeguarding of chromosome stability during mitosis.
  • Deficiency of MIP/MTMR14 phosphatase induces a muscle disorder by disrupting Ca2+ homeostasis
    - Nat Cell Biol 11(6):769-776 (2009)
    The intracellular Ca2+ concentration ([Ca2+]i) in skeletal muscles must be rapidly regulated during the excitation-contraction-relaxation process1. However, the signalling components involved in such rapid Ca2+ movement are not fully understood. Here we report that mice deficient in the newly identified PtdInsP (phosphatidylinositol phosphate) phosphatase MIP/MTMR14 (muscle-specific inositol phosphatase) show muscle weakness and fatigue. Muscles isolated from MIP/MTMR14-/- mice produced less contractile force, had markedly prolonged relaxation and showed exacerbated fatigue relative to normal muscles. Further analyses revealed that MIP/MTMR14 deficiency resulted in spontaneous Ca2+ leakage from the internal store — the sarcoplasmic reticulum. This was attributed to decreased metabolism (dephosphorylation) and the subsequent accumulation of MIP/MTMR14 substrates, especially PtdIns(3,5)P2 and PtdIns (3,4)P2. Furthermore, we found that PtdIns(3,5)P2 and PtdIns(3,4)P2 bo! und to, and directly activated, the Ca2+ release channel (ryanodine receptor 1, RyR1) of the sarcoplasmic reticulum. These studies provide the first evidence that finely controlled PtdInsP levels in muscle cells are essential for maintaining Ca2+ homeostasis and muscle performance.
  • TGF-beta signalling is regulated by Schnurri-2-dependent nuclear translocation of CLIC4 and consequent stabilization of phospho-Smad2 and 3
    - Nat Cell Biol 11(6):777-784 (2009)
    CLIC4 (chloride intracellular channel 4), a multifunctional protein that traffics between the cytoplasm and nucleus, interacts with Schnurri-2, a transcription factor in the bone morphogenetic protein (BMP) signalling pathway. Here we show that transforming growth factor beta (TGF-beta) promotes the expression of CLIC4 and Schnurri-2 as well as their association in the cytoplasm and their translocation to the nucleus. In the absence of CLIC4 or Schnurri-2, TGF-beta signalling is abrogated. Direct nuclear targeting of CLIC4 enhances TGF-beta signalling and removes the requirement for Schnurri-2. Nuclear CLIC4 associates with phospho (p)-Smad2 and p-Smad3, protecting them from dephosphorylation by nuclear phosphatases. An intact TGF-beta signalling pathway is essential for CLIC4-mediated growth-arrest. These results newly identify Schnurri-2 and CLIC4 as modifiers of TGF-beta signalling through their stabilization of p-Smad2 and 3 in the nucleus.

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