Hot off the presses! Oct 01 Nat Cell Biol

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Latest Articles Include:

• Milestones in light microscopy
  - Nat Cell Biol 11(10):1165 (2009)
  
• Receptor-like kinases shape the plant
  - Nat Cell Biol 11(10):1166-1173 (2009)
  To generate the various tissues and organs that build up the adult body, plants and animals require organized formative cell divisions and correct cell specification. In plants, these processes are controlled mainly by phytohormones and transcriptional networks. Recently, ligand–receptor-like kinase signalling pathways have been revealed as additional potentially crucial regulators of cell specification in plants. We review here the importance of such signalling cascades for plant growth and development, and we discuss, where possible, similarities to well-investigated cascades in animals.
• A bacterial virulence factor that dissipates tension
  - Nat Cell Biol 11(10):1174-1175 (2009)
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• NURD keeps chromatin young
  - Nat Cell Biol 11(10):1176-1177 (2009)
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• A motor driving PTEN
  - Nat Cell Biol 11(10):1177-1179 (2009)
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• Research highlights
  - Nat Cell Biol 11(10):1180 (2009)
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• C/EBP and couple interfollicular keratinocyte proliferation arrest to commitment and terminal differentiation
  - Nat Cell Biol 11(10):1181-1190 (2009)
  The transcriptional regulators that couple interfollicular basal keratinocyte proliferation arrest to commitment and differentiation are yet to be identified. Here we report that the basic region leucine zipper transcription factors C/EBP and C/EBP are co-expressed in basal keratinocytes, and are coordinately upregulated as keratinocytes exit the basal layer and undergo terminal differentiation. Mice lacking both C/EBP and in the epidermis showed increased proliferation of basal keratinocytes and impaired commitment to differentiation. This led to ectopic expression of keratin 14 (K14) and Np63 in suprabasal cells, decreased expression of spinous and granular layer proteins, parakeratosis and defective epidermal water barrier function. Knock-in mutagenesis revealed that C/EBP-E2F interaction was required for control of interfollicular epidermis (IFE) keratinocyte proliferation, but not for induction of spinous and granular layer markers, whereas C/EBP DNA binding was ! required for Np63 downregulation and K1/K10 induction. Finally, loss of C/EBP/ induced stem cell gene expression signatures in the epidermis. C/EBPs, therefore, couple basal keratinocyte cell cycle exit to commitment to differentiation through E2F repression and DNA binding, respectively, and may act to restrict the epidermal stem cell compartment.
• MyosinV controls PTEN function and neuronal cell size
  - Nat Cell Biol 11(10):1191-1196 (2009)
  The tumour suppressor PTEN can inhibit cell proliferation and migration as well as control cell growth, in different cell types1. PTEN functions predominately as a lipid phosphatase, converting PtdIns(3,4,5)P3 to PtdIns(4,5)P2, thereby antagonizing PI(3)K (phosphoinositide 3-kinase) and its established downstream effector pathways2. However, much is unclear concerning the mechanisms that regulate PTEN movement to the cell membrane, which is necessary for its activity towards PtdIns(3,4,5)P3 (Refs 3, 4, 5). Here we show a requirement for functional motor proteins in the control of PI3K signalling, involving a previously unknown association between PTEN and myosinV. FRET (Förster resonance energy transfer) measurements revealed that PTEN interacts directly with myosinV, which is dependent on PTEN phosphorylation mediated by CK2 and/or GSK3. Inactivation of myosinV-transport function in neurons increased cell size, which, in line with known attributes of PTEN-loss6, 7, r! equired PI(3)K and mTor. Our data demonstrate a myosin-based transport mechanism that regulates PTEN function, providing new insights into the signalling networks regulating cell growth.
• Regulation of endoplasmic reticulum stress response by a BBF2H7-mediated Sec23a pathway is essential for chondrogenesis
  - Nat Cell Biol 11(10):1197-1204 (2009)
  Many tissues have a specific signal transduction system for endoplasmic reticulum (ER) dysfunction; however, the mechanisms underlying the ER stress response in cartilage remain unclear. BBF2H7 (BBF2 human homologue on chromosome 7), an ER-resident basic leucine zipper transcription factor, is activated in response to ER stress1 and is highly expressed in chondrocytes. In this study, we generated Bbf2h7-/- mice to assess the in vivo function of BBF2H7. The mice showed severe chondrodysplasia and died by suffocation shortly after birth because of an immature chest cavity. The cartilage showed a lack of typical columnar structure in the proliferating zone and a decrease in the size of the hypertrophic zone, resulting in a significant reduction of extracellular matrix proteins. Interestingly, proliferating chondrocytes showed abnormally expanded ER, containing aggregated type II collagen (Col2) and cartilage oligomeric matrix protein (COMP). We identified Sec23a, which en! codes a coat protein complex II component responsible for protein transport from the ER to the Golgi2, 3, as a target of BBF2H7, which directly bound to a CRE-like sequence in the promoter region of Sec23a to activate its transcription. When Sec23a was introduced to Bbf2h7-/- chondrocytes, the impaired transport and secretion of cartilage matrix proteins was totally restored, indicating that by activating protein secretion the BBF2H7–Sec23a pathway has a crucial role in chondrogenesis. Our findings provide a new link by which ER stress is converted to signalling for the activation of ER-to-Golgi trafficking.
• Signalling mediated by the endoplasmic reticulum stress transducer OASIS is involved in bone formation
  - Nat Cell Biol 11(10):1205-1211 (2009)
  Eukaryotic cells have signalling pathways from the endoplasmic reticulum (ER) to cytosol and nuclei, to avoid excess accumulation of unfolded proteins in the ER. We previously identified a new type of ER stress transducer, OASIS, a bZIP (basic leucine zipper) transcription factor, which is a member of the CREB/ATF family and has a transmembrane domain1, 2, 3, 4, 5, 6. OASIS is processed by regulated intramembrane proteolysis (RIP) in response to ER stress, and is highly expressed in osteoblasts. OASIS-/- mice exhibited severe osteopenia, involving a decrease in type I collagen in the bone matrix and a decline in the activity of osteoblasts, which showed abnormally expanded rough ER, containing of a large amount of bone matrix proteins. Here we identify the gene for type 1 collagen, Col1a1, as a target of OASIS, and demonstrate that OASIS activates the transcription of Col1a1 through an unfolded protein response element (UPRE)-like sequence in the osteoblast-specific Co! l1a1 promoter region. Moreover, expression of OASIS in osteoblasts is induced by BMP2 (bone morphogenetic protein 2), the signalling of which is required for bone formation. Additionally, RIP of OASIS is accelerated by BMP2 signalling, which causes mild ER stress. Our studies show that OASIS is critical for bone formation through the transcription of Col1a1 and the secretion of bone matrix proteins, and they reveal a new mechanism by which ER stress-induced signalling mediates bone formation.
• The bacterial virulence factor InlC perturbs apical cell junctions and promotes cell-to-cell spread of Listeria
  - Nat Cell Biol 11(10):1212-1218 (2009)
  Several pathogenic bacteria, including Listeria monocytogenes, use an F-actin motility process to spread between mammalian cells1. Actin 'comet tails' propel Listeria through the cytoplasm, resulting in bacteria-containing membrane protrusions that are internalized by neighbouring cells. The mechanism by which Listeria overcomes cortical tension to generate protrusions is unknown. Here, we identify bacterial and host proteins that directly regulate protrusions. We show that efficient spreading between polarized epithelial cells requires the secreted Listeria virulence protein InlC (internalin C). We next identify the mammalian adaptor protein Tuba as a ligand of InlC. InlC binds to a carboxy-terminal SH3 domain in Tuba, which normally engages the human actin regulatory protein N-WASP2. InlC promotes protrusion formation by inhibiting Tuba and N-WASP activity, probably by impairing binding of N-WASP to the Tuba SH3 domain. Tuba and N-WASP are known to control the struct! ure of apical junctions in epithelial cells3. We demonstrate that, by inhibiting Tuba and N-WASP, InlC makes taut apical junctions become slack. Experiments with myosin II inhibitors indicate that InlC-mediated perturbation of apical junctions accounts for the role of this bacterial protein in protrusion formation. Collectively, our results suggest that InlC promotes bacterial dissemination by relieving cortical tension, thereby enhancing the ability of motile bacteria to deform the plasma membrane into protrusions.
• Intracellular fluid flow in rapidly moving cells
  - Nat Cell Biol 11(10):1219-1224 (2009)
  Cytosolic fluid dynamics have been implicated in cell motility1, 2, 3, 4, 5 because of the hydrodynamic forces they induce and because of their influence on transport of components of the actin machinery to the leading edge. To investigate the existence and the direction of fluid flow in rapidly moving cells, we introduced inert quantum dots into the lamellipodia of fish epithelial keratocytes and analysed their distribution and motion. Our results indicate that fluid flow is directed from the cell body towards the leading edge in the cell frame of reference, at about 40% of cell speed. We propose that this forward-directed flow is driven by increased hydrostatic pressure generated at the rear of the cell by myosin contraction, and show that inhibition of myosin II activity by blebbistatin reverses the direction of fluid flow and leads to a decrease in keratocyte speed. We present a physical model for fluid pressure and flow in moving cells that quantitatively accounts! for our experimental data.
• The planar cell polarity effector Fuz is essential for targeted membrane trafficking, ciliogenesis and mouse embryonic development
  - Nat Cell Biol 11(10):1225-1232 (2009)
  The planar cell polarity (PCP) signalling pathway is essential for embryonic development because it governs diverse cellular behaviours, and 'core PCP' proteins, such as Dishevelled and Frizzled, have been extensively characterized1, 2, 3, 4. By contrast, the 'PCP effector' proteins, such as Intu and Fuz, remain largely unstudied5, 6. These proteins are essential for PCP signalling, but they have never been investigated in mammals and their cell biological activities remain entirely unknown. We report here that Fuz mutant mice show neural tube defects, skeletal dysmorphologies and Hedgehog signalling defects stemming from disrupted ciliogenesis. Using bioinformatics and imaging of an in vivo mucociliary epithelium, we established a central role for Fuz in membrane trafficking, showing that Fuz is essential for trafficking of cargo to basal bodies and to the apical tips of cilia. Fuz is also essential for exocytosis in secretory cells. Finally, we identified a Rab-relat! ed small GTPase as a Fuz interaction partner that is also essential for ciliogenesis and secretion. These results are significant because they provide new insights into the mechanisms by which developmental regulatory systems such as PCP signalling interface with fundamental cellular systems such as the vesicle trafficking machinery.
• Listeria monocytogenes ActA-mediated escape from autophagic recognition
  - Nat Cell Biol 11(10):1233-1240 (2009)
  Autophagy degrades unnecessary organelles and misfolded protein aggregates1, as well as cytoplasm-invading bacteria2. Nevertheless, the bacteria Listeria monocytogenes efficiently escapes autophagy3, 4. We show here that recruitment of the Arp2/3 complex and Ena/VASP, via the bacterial ActA protein, to the bacterial surface disguises the bacteria from autophagic recognition, an activity that is independent of the ability to mediate bacterial motility. L. monocytogenes expressing ActA mutants that lack the ability to recruit the host proteins initially underwent ubiquitylation, followed by recruitment of p62 (also known as SQSTM1) and LC3, before finally undergoing autophagy. The ability of ActA to mediate protection from ubiquitylation was further demonstrated by generating aggregate-prone GFP–ActA–Q79C and GFP–ActA–170* chimaeras, consisting of GFP (green fluorescent protein), the ActA protein and segments of polyQ5 or Golgi membrane protein GCP170 (ref. 6). G! FP–ActA–Q79C and GFP–ActA–170* formed aggregates in the host cell cytoplasm, however, these ActA-containing aggregates were not targeted for association with ubiquitin and p62. Our findings indicate that ActA-mediated host protein recruitment is a unique bacterial disguise tactic to escape from autophagy.
• Oxidant-induced apoptosis is mediated by oxidation of the actin-regulatory protein cofilin
  - Nat Cell Biol 11(10):1241-1246 (2009)
  Physiological oxidants that are generated by activated phagocytes comprise the main source of oxidative stress during inflammation1, 2. Oxidants such as taurine chloramine (TnCl) and hydrogen peroxide (H2O2) can damage proteins and induce apoptosis, but the role of specific protein oxidation in this process has not been defined. We found that the actin-binding protein cofilin is a key target of oxidation. When oxidation of this single regulatory protein is prevented, oxidant-induced apoptosis is inhibited. Oxidation of cofilin causes it to lose its affinity for actin and to translocate to the mitochondria, where it induces swelling and cytochrome c release by mediating opening of the permeability transition pore (PTP). This occurs independently of Bax activation and requires both oxidation of cofilin Cys residues and dephosphorylation at Ser 3. Knockdown of endogenous cofilin using targeted siRNA inhibits oxidant-induced apoptosis, which is restored by re-expression of! wild-type cofilin but not by cofilin containing Cys to Ala mutations. Exposure of cofilin to TnCl results in intramolecular disulphide bonding and oxidation of Met residues to Met sulphoxide, but only Cys oxidation causes cofilin to induce mitochondrial damage.
• NEK11 regulates CDC25A degradation and the IR-induced G2/M checkpoint
  - Nat Cell Biol 11(10):1247-1253 (2009)
  DNA damage-induced cell-cycle checkpoints have a critical role in maintaining genomic stability1, 2. A key target of the checkpoints is the CDC25A (cell division cycle 25 homologue A) phosphatase, which is essential for the activation of cyclin-dependent kinases and cell-cycle progression3, 4, 5. To identify new genes involved in the G2/M checkpoint we performed a large-scale short hairpin RNA (shRNA) library screen. We show that NIMA (never in mitosis gene A)-related kinase 11 (NEK11) is required for DNA damage-induced G2/M arrest. Depletion of NEK11 prevents proteasome-dependent degradation of CDC25A, both in unperturbed and DNA-damaged cells. We show that NEK11 directly phosphorylates CDC25A on residues whose phosphorylation is required for -TrCP (-transducin repeat-containing protein)-mediated polyubiquitylation and degradation of CDC25A. Furthermore, we demonstrate that CHK1 (checkpoint kinase 1) directly activates NEK11 by phosphorylating it on Ser 273, indicatin! g that CHK1 and NEK11 operate in a single pathway that controls proteolysis of CDC25A. Taken together, these results demonstrate that NEK11 is an important component of the pathway enforcing the G2/M checkpoint, suggesting that genetic mutations in NEK11 may contribute to the development of human cancer.
• Brassinosteroid signal transduction from cell-surface receptor kinases to nuclear transcription factors
  - Nat Cell Biol 11(10):1254-1260 (2009)
  Brassinosteroid (BR) regulates gene expression and plant development through a receptor kinase-mediated signal transduction pathway1. Despite the identification of many components of this pathway, it remains unclear how the BR signal is transduced from the cell surface to the nucleus2. Here we describe a complete BR signalling pathway by elucidating key missing steps. We show that phosphorylation of BSK1 (BR-signalling kinase 1) by the BR receptor kinase BRI1 (BR-insensitive 1) promotes BSK1 binding to the BSU1 (BRI1 suppressor 1) phosphatase, and BSU1 inactivates the GSK3-like kinase BIN2 (BR-insensitive 2) by dephosphorylating a conserved phospho-tyrosine residue (pTyr 200). Mutations that affect phosphorylation/dephosphorylation of BIN2 pTyr200 (bin2-1, bin2-Y200F and quadruple loss-of-function of BSU1-related phosphatases) support an essential role for BSU1-mediated BIN2 dephosphorylation in BR-dependent plant growth. These results demonstrate direct sequential BR ! activation of BRI1, BSK1 and BSU1, and inactivation of BIN2, leading to accumulation of unphosphorylated BZR (brassinazole resistant) transcription factors in the nucleus. This study establishes a fully connected BR signalling pathway and provides new insights into the mechanism of GSK3 regulation.
• Ageing-related chromatin defects through loss of the NURD complex
  - Nat Cell Biol 11(10):1261-1267 (2009)
  Physiological and premature ageing are characterized by multiple defects in chromatin structure and accumulation of persistent DNA damage. Here we identify the NURD chromatin remodelling complex as a key modulator of these ageing-associated chromatin defects. We demonstrate loss of several NURD components during premature and normal ageing and we find an ageing-associated reduction in HDAC1 activity. Silencing of individual NURD subunits recapitulated chromatin defects associated with ageing and we provide evidence that structural chromatin defects precede DNA damage accumulation. These results outline a molecular mechanism for chromatin defects during ageing.
• The non-coding RNA of the multidrug resistance-linked vault particle encodes multiple regulatory small RNAs
  - Nat Cell Biol 11(10):1268-1271 (2009)
  Vault particles are conserved organelles implicated in multidrug resistance and intracellular transport. They contain three different proteins and non-coding vault RNAs (vRNAs). Here we show that human vRNAs produce several small RNAs (svRNAs) by mechanisms different from those in the canonical microRNA (miRNA) pathway. At least one of these svRNAs, svRNAb, associates with Argonaute proteins to guide sequence-specific cleavage and regulate gene expression similarly to miRNAs. We demonstrate that svRNAb downregulates CYP3A4, a key enzyme in drug metabolism. Our findings expand the repertoire of small regulatory RNAs and assign, for the first time, a function to vRNAs that may help explain the association between vault particles and drug resistance.
• Persistent DNA damage signalling triggers senescence-associated inflammatory cytokine secretion
  - Nat Cell Biol 11(10):1272 (2009)
  Introduction ; published online 13 July 2009; corrected after print 24 August 2009 In the version of this article initially published online and in print, the labelling of the radiation doses in Fig. 1a was reversed. The correct version of this figure is shown below. This error has been corrected in both the HTML and PDF versions of the article.
• Multivesicular bodies associate with components of miRNA effector complexes and modulate miRNA activity
  - Nat Cell Biol 11(10):1272 (2009)
  Introduction ; published online 16 August 2009; corrected after print 8 September 2009 In the version of this article initially published online and in print, the images in the left column were not included in the merge column. The correct version of this figure is shown below. This error has been corrected in both the HTML and PDF versions of the article.
• Axin determines cell fate by controlling the p53 activation threshold after DNA damage
  - Nat Cell Biol 11(10):1272 (2009)
  Introduction ; published online 23 August 2009; corrected after print 28 August 2009 In the version of this article initially published online and in print, the word Tip60 was incorrectly used instead of Pirh2 in the legend for Fig. 2b. This error has been corrected in the HTML and PDF versions of the article.
• mRNA decay turns on apoptosis
  - Nat Cell Biol 11(10):1272 (2009)
  Introduction In the September Research highlight 'mRNA decay turns on apoptosis' a couple of sentences have been changed to the following: "This effect requires the activation of its endoribonuclease activity. Based on recent structural data, chronic ER stress could induce IRE1 higher order oligomers, which may display a higher promiscuity towards mRNA." This has been corrected in both the HTML and PDF versions of the article.