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1.  Architecture of the Mediator Head module 
Nature  2011;475(7355):240-243.
Mediator is a key regulator of eukaryotic transcription1, connecting activators and repressors bound to regulatory DNA elements with RNA polymerase II (Pol II) 1-4. In the yeast S. cerevisiae, Mediator comprises 25 subunits with a total mass over 1 MDa 5,6, and is organized into three modules, termed Head, Middle/Arm and Tail 7-9. Our understanding of Mediator assembly and its role in regulating transcription has been impeded to date by limited structural information. Here, we report the crystal structure of the essential Mediator Head module (seven subunits, 223 kDa) at 4.3 Å resolution. Our structure reveals three distinct domains with the integrity of the complex centered on a bundle of ten helices from five different Head subunits. An intricate pattern of interactions within this helical bundle ensures stable assembly of the Head subunits, and provides the binding sites for general transcription factors (GTFs) and Pol II. Our structural and functional data suggest the Head module to juxtapose TFIIH and the carboxyl-terminal domain (CTD) of the largest subunit of Pol II, thereby facilitating CTD phosphorylation. Our results reveal architectural principles underlying the role of Mediator in the regulation of gene expression.
doi:10.1038/nature10162
PMCID: PMC4109712  PMID: 21725323
2.  Climate Change and the Integrity of Science 
Gleick, PH | Adams, RM | Amasino, RM | Anders, E | Anderson, DJ | Anderson, WW | Anselin, LE | Arroyo, MK | Asfaw, B | Ayala, FJ | Bax, A | Bebbington, AJ | Bell, G | Bennett, MVL | Bennetzen, JL | Berenbaum, MR | Berlin, OB | Bjorkman, PJ | Blackburn, E | Blamont, JE | Botchan, MR | Boyer, JS | Boyle, EA | Branton, D | Briggs, SP | Briggs, WR | Brill, WJ | Britten, RJ | Broecker, WS | Brown, JH | Brown, PO | Brunger, AT | Cairns, J | Canfield, DE | Carpenter, SR | Carrington, JC | Cashmore, AR | Castilla, JC | Cazenave, A | Chapin, FS | Ciechanover, AJ | Clapham, DE | Clark, WC | Clayton, RN | Coe, MD | Conwell, EM | Cowling, EB | Cowling, RM | Cox, CS | Croteau, RB | Crothers, DM | Crutzen, PJ | Daily, GC | Dalrymple, GB | Dangl, JL | Darst, SA | Davies, DR | Davis, MB | De Camilli, PV | Dean, C | Defries, RS | Deisenhofer, J | Delmer, DP | Delong, EF | Derosier, DJ | Diener, TO | Dirzo, R | Dixon, JE | Donoghue, MJ | Doolittle, RF | Dunne, T | Ehrlich, PR | Eisenstadt, SN | Eisner, T | Emanuel, KA | Englander, SW | Ernst, WG | Falkowski, PG | Feher, G | Ferejohn, JA | Fersht, A | Fischer, EH | Fischer, R | Flannery, KV | Frank, J | Frey, PA | Fridovich, I | Frieden, C | Futuyma, DJ | Gardner, WR | Garrett, CJR | Gilbert, W | Goldberg, RB | Goodenough, WH | Goodman, CS | Goodman, M | Greengard, P | Hake, S | Hammel, G | Hanson, S | Harrison, SC | Hart, SR | Hartl, DL | Haselkorn, R | Hawkes, K | Hayes, JM | Hille, B | Hökfelt, T | House, JS | Hout, M | Hunten, DM | Izquierdo, IA | Jagendorf, AT | Janzen, DH | Jeanloz, R | Jencks, CS | Jury, WA | Kaback, HR | Kailath, T | Kay, P | Kay, SA | Kennedy, D | Kerr, A | Kessler, RC | Khush, GS | Kieffer, SW | Kirch, PV | Kirk, K | Kivelson, MG | Klinman, JP | Klug, A | Knopoff, L | Kornberg, H | Kutzbach, JE | Lagarias, JC | Lambeck, K | Landy, A | Langmuir, CH | Larkins, BA | Le Pichon, XT | Lenski, RE | Leopold, EB | Levin, SA | Levitt, M | Likens, GE | Lippincott-Schwartz, J | Lorand, L | Lovejoy, CO | Lynch, M | Mabogunje, AL | Malone, TF | Manabe, S | Marcus, J | Massey, DS | McWilliams, JC | Medina, E | Melosh, HJ | Meltzer, DJ | Michener, CD | Miles, EL | Mooney, HA | Moore, PB | Morel, FMM | Mosley-Thompson, ES | Moss, B | Munk, WH | Myers, N | Nair, GB | Nathans, J | Nester, EW | Nicoll, RA | Novick, RP | O’Connell, JF | Olsen, PE | Opdyke, ND | Oster, GF | Ostrom, E | Pace, NR | Paine, RT | Palmiter, RD | Pedlosky, J | Petsko, GA | Pettengill, GH | Philander, SG | Piperno, DR | Pollard, TD | Price, PB | Reichard, PA | Reskin, BF | Ricklefs, RE | Rivest, RL | Roberts, JD | Romney, AK | Rossmann, MG | Russell, DW | Rutter, WJ | Sabloff, JA | Sagdeev, RZ | Sahlins, MD | Salmond, A | Sanes, JR | Schekman, R | Schellnhuber, J | Schindler, DW | Schmitt, J | Schneider, SH | Schramm, VL | Sederoff, RR | Shatz, CJ | Sherman, F | Sidman, RL | Sieh, K | Simons, EL | Singer, BH | Singer, MF | Skyrms, B | Sleep, NH | Smith, BD | Snyder, SH | Sokal, RR | Spencer, CS | Steitz, TA | Strier, KB | Südhof, TC | Taylor, SS | Terborgh, J | Thomas, DH | Thompson, LG | Jian, RTT | Turner, MG | Uyeda, S | Valentine, JW | Valentine, JS | Van Etten, JL | Van Holde, KE | Vaughan, M | Verba, S | Von Hippel, PH | Wake, DB | Walker, A | Walker, JE | Watson, EB | Watson, PJ | Weigel, D | Wessler, SR | West-Eberhard, MJ | White, TD | Wilson, WJ | Wolfenden, RV | Wood, JA | Woodwell, GM | Wright, HE | Wu, C | Wunsch, C | Zoback, ML
Science (New York, N.Y.)  2010;328(5979):689-690.
doi:10.1126/science.328.5979.689
PMCID: PMC5125622  PMID: 20448167
3.  Stable chromosome condensation revealed by chromosome conformation capture 
Cell  2015;163(4):934-946.
SUMMARY
Chemical cross-linking and DNA sequencing have revealed regions of intra-chromosomal interaction, referred to as topologically associating domains (TADs), interspersed with regions of little or no interaction, in interphase nuclei. We find that TADs and the regions between them correspond with the bands and interbands of polytene chromosomes of Drosophila. We further establish the conservation of TADs between polytene and diploid cells of Drosophila. From direct measurements on light micrographs of polytene chromosomes, we then deduce the states of chromatin folding in the diploid cell nucleus. Two states of folding, fully extended fibers containing regulatory regions and promoters, and fibers condensed up to ten-fold containing coding regions of active genes, constitute the euchromatin of the nuclear interior. Chromatin fibers condensed up to 30-fold, containing coding regions of inactive genes, represent the heterochromatin of the nuclear periphery. A convergence of molecular analysis with direct observation thus reveals the architecture of interphase chromosomes.
doi:10.1016/j.cell.2015.10.026
PMCID: PMC4639323  PMID: 26544940
4.  A severe neurological complication of influenza in a previously well child 
BMJ Case Reports  2014;2014:bcr2014206930.
We describe a case of a 3-year-old girl who was admitted with encephalopathy and a right-sided hemiparesis secondary to acute influenza A. She was up-to-date with the Australian National Immunisation Program (which does not routinely include the seasonal influenza vaccine). After initial treatment with intravenous antimicrobials and acyclovir, a brain and spinal cord MRI demonstrated extensive focal necrotic and haemorrhagic changes in keeping with acute necrotising encephalopathy (ANE). She was started on a course of oseltamivir and intravenous pulse methylprednisolone, followed by an oral weaning regimen of prednisolone. After an intense period of rehabilitation, she has made a remarkable recovery. Genetic testing has since confirmed that this girl has the RANBP2 gene mutation, which leads to increased susceptibility of developing ANE. This case report highlights ANE as a rare but severe complication of influenza, the unfortunate complication of having the RANBP2 mutation and the importance of paediatric influenza vaccination.
doi:10.1136/bcr-2014-206930
PMCID: PMC4208126  PMID: 25342191
5.  A mixed methods study of age at diagnosis and diagnostic odyssey for Duchenne muscular dystrophy 
European Journal of Human Genetics  2015;23(10):1294-1300.
The delayed diagnosis of Duchenne muscular dystrophy (DMD) may be an ongoing problem internationally. We aimed to ascertain age at diagnosis and explore parents' experiences of the diagnosis of DMD in Australia. Using mixed methods, data were collected from laboratory and clinical record audits of testing for DMD in Victoria and Tasmania, interviews and a national survey of parents regarding their experiences from first noticing symptoms to receiving a diagnosis. The audits revealed that the median age at diagnosis for DMD was 5 years (n=49 during 2005–2010); this age had not changed substantially over this period. Fourteen parents interviewed reported age at diagnosis ranging from 2 to 8 years with a 6 month to 4 year delay between initial concerns about their child's development and receiving the DMD diagnosis. Sixty-two survey respondents reported the median age at diagnosis was 3 years and 9 months, while the median age when symptoms were noticed was 2 years and 9 months. Parents experienced many emotions in their search for a diagnosis and consulted with a wide range of health professionals. Half the survey respondents felt that their child could have been diagnosed earlier. Despite advances in testing technologies and increasing awareness of DMD, the age at diagnosis has remained constant in Australia. This mixed methods study shows that this diagnostic delay continues to have a negative impact on parents' experiences, places families at risk of having a second affected child and may have a deleterious effect on affected children's treatment.
doi:10.1038/ejhg.2014.301
PMCID: PMC4592094  PMID: 25626706
6.  Cells must express components of the planar cell polarity system and extracellular matrix to support cytonemes 
eLife  null;5:e18979.
Drosophila dorsal air sac development depends on Decapentaplegic (Dpp) and Fibroblast growth factor (FGF) proteins produced by the wing imaginal disc and transported by cytonemes to the air sac primordium (ASP). Dpp and FGF signaling in the ASP was dependent on components of the planar cell polarity (PCP) system in the disc, and neither Dpp- nor FGF-receiving cytonemes extended over mutant disc cells that lacked them. ASP cytonemes normally navigate through extracellular matrix (ECM) composed of collagen, laminin, Dally and Dally-like (Dlp) proteins that are stratified in layers over the disc cells. However, ECM over PCP mutant cells had reduced levels of laminin, Dally and Dlp, and whereas Dpp-receiving ASP cytonemes navigated in the Dally layer and required Dally (but not Dlp), FGF-receiving ASP cytonemes navigated in the Dlp layer, requiring Dlp (but not Dally). These findings suggest that cytonemes interact directly and specifically with proteins in the stratified ECM.
DOI: http://dx.doi.org/10.7554/eLife.18979.001
eLife digest
The embryos of animals develop in a controlled manner that ensures that their tissues and organs form properly and at the right time. These processes depend on molecules called morphogens that are distributed throughout the embryo in specific ways and that are dispersed via extensions that protrude from the surfaces of cells. These extensions, called cytonemes, transport the morphogens across the distances that separate cells and transfer these molecules to target cells via direct contact. However, it was not known how cytonemes navigate to their targets.
The fruit fly Drosophila is commonly used to investigate how animals develop organs and tissues. Previous studies have shown that the development of one of the fly’s organs – the air sac primordium –relies on morphogens transported by cytonemes.Now, Huang and Kornberg reveal that these cytonemes navigate to their targets by using the composition of the mesh-like framework – referred to as the extracellular matrix – that surrounds animal tissues as a guide. Further experiments showed that the extracellular matrix between the cells that produce the morphogens and the cells of the air sac primordium is roughly arranged into layers. These layers contain different molecules and the cytonemes navigate within specific layers.
These findings reinforce the idea that the extracellular space is organized and regulated, and show that the extracellular matrix is essential for developmental signaling. Future challenges include understanding how the layers of the extracellular matrix form and how information is encoded in these layers for the cytonemes to decipher as they navigate to their targets.
DOI: http://dx.doi.org/10.7554/eLife.18979.002
doi:10.7554/eLife.18979
PMCID: PMC5030081  PMID: 27591355
cytoneme; Heparan sulfate proteoglycan; planar cell polarity; extracellular matrix; D. melanogaster
7.  Deconvolution method for specific and nonspecific binding of ligand to multi-protein complex by native mass spectrometry 
Analytical chemistry  2015;87(16):8541-8546.
In native mass spectrometry, it has been difficult to discriminate between specific binding of a ligand to a multi-protein complex from the nonspecific interactions. Here, we present a deconvolution model that consists of two levels of data reduction. At the first level, the apparent association binding constants are extracted from the measured intensities of the target/ligand complexes by varying ligand concentration. At the second level, two functional forms representing the specific- and non-specific binding events are fit to the binding constants obtained from the first level of modeling. Using this approach, we found that an inverse power distribution described nonspecific binding of α-amanitin to yeast RNA polymerase II. Moreover, treating the concentration of the multi-protein complex as a fitting parameter reduced the impact of inaccuracies in this experimental measurement on the apparent association constants. This model provides an improved way of separating specific and non-specific binding to large, multi-protein complexes in native mass spectrometry.
doi:10.1021/acs.analchem.5b02258
PMCID: PMC4714924  PMID: 26189511
8.  Uncoupling promoter opening from start site scanning 
Molecular cell  2015;59(1):133-138.
SUMMARY
Whereas RNA polymerase II (pol II) transcription start sites (TSSs) occur about 30–35 bp downstream of the TATA box in metazoans, TSSs are located 40–120 bp downstream in S. cerevisiae. Promoter melting begins about 12 bp downstream in all eukaryotes, so pol II is presumed to “scan” further downstream before starting transcription in yeast. Here we report that removal of the kinase complex TFIIK from TFIIH shifts the TSS in a yeast system upstream to the location observed in metazoans. Conversely, moving the normal TSS to an upstream location enables a high level of TFIIK-independent transcription in the yeast system. We distinguish two stages of the transcription initiation process: bubble formation by TFIIH, which fills the pol II active center with single stranded DNA; and subsequent scanning downstream, also driven by TFIIH, which requires displacement of the initial bubble. Omission of TFIIK uncouples the two stages of the process.
Graphical abstract
doi:10.1016/j.molcel.2015.05.021
PMCID: PMC4490988  PMID: 26073544
9.  ALG1-CDG: Clinical and molecular characterization of 39 unreported patients 
Human mutation  2016;37(7):653-660.
Congenital disorders of glycosylation (CDG) arise from pathogenic mutations in over one hundred genes leading to impaired protein or lipid glycosylation. ALG1 encodes a β1,4 mannosyltransferase that catalyzes the addition of the first of nine mannose moieties to form a dolichol-lipid linked oligosaccharide intermediate (DLO) required for proper N-linked glycosylation. ALG1 mutations cause a rare autosomal recessive disorder termed ALG1-CDG. To date thirteen mutations in eighteen patients from fourteen families have been described with varying degrees of clinical severity. We identified and characterized thirty-nine previously unreported cases of ALG1-CDG from thirty-two families and add twenty-six new mutations. Pathogenicity of each mutation was confirmed based on its inability to rescue impaired growth or hypoglycosylation of a standard biomarker in an alg1-deficient yeast strain. Using this approach we could not establish a rank order comparison of biomarker glycosylation and patient phenotype, but we identified mutations with a lethal outcome in the first two years of life. The recently identified protein-linked xeno-tetrasaccharide biomarker, NeuAc-Gal-GlcNAc2, was seen in all twenty-seven patients tested. Our study triples the number of known patients and expands the molecular and clinical correlates of this disorder.
doi:10.1002/humu.22983
PMCID: PMC4907823  PMID: 26931382
CDG; Asparagine-linked glycosylation protein 1; Carbohydrate-deficient transferrin; Xeno-tetrasaccharide
10.  Corneal Nerve Structure and Function After Long-Term Wear of Fluid-Filled Scleral Lens 
Cornea  2015;34(4):427-432.
Purpose
To determine if long-term wear of a fluid-filled scleral lens alters basal tear production, corneal sensation, corneal nerve density and corneal nerve morphology in two disease categories.
Methods
Patients recruited from the Prosthetic Replacement of the Ocular Surface Ecosystem (PROSE) treatment program at Weill Cornell Medical College were categorized into two groups: distorted corneas (DC) or ocular surface disease (OSD). We measured tear production, central corneal sensation, sub-basal nerve density and tortuosity, and stromal nerve thickness before and after long-term wear of the prosthetic device used in PROSE treatment, defined as at least 60 days of wear for a minimum of eight hours a day.
Results
Twenty patients were included in the study. After long-term wear of the prosthetic device, tear production decreased in patients with DC (21.2±8.5 mm to 10.4±4.6 mm; P < 0.0001), but did not change in patients with OSD (7.5±5.2 mm to 8.7±7.2 mm; P = 0.71). Corneal sensation increased in the DC group (45.6±9.2 mm to 55.0±5.6 mm; P < 0.05). There was no significant change in sensation in patients with OSD (45.0±8.7 mm to 49.1±14.8 mm; P = 0.37). Sub-basal nerve density, sub-basal nerve tortuosity, and stromal nerve thickness remained unchanged in both DC and OSD groups after long-term wear (P > 0.05)
Conclusions
Patients with DC had significantly reduced basal tear production and increased corneal sensation after long-term wear of the scleral lens, but patients with OSD did not show any changes in tear production or corneal sensation.
doi:10.1097/ICO.0000000000000381
PMCID: PMC4383242  PMID: 25710510
scleral lens; PROSE; corneal nerves; lacrimal functional unit; ocular surface disease; corneal ectasia; tear production; corneal sensation; confocal
11.  Validation of Endothelin B Receptor Antibodies Reveals Two Distinct Receptor-related Bands on Western Blot 
Analytical biochemistry  2014;468:28-33.
Antibodies are important tools for the study of protein expression, but are often used without full validation. In this study, we use Western blots to characterize antibodies targeted to the N- (NT) or C-termini (CT) and the second (IL2) or third intracellular (IL3) loops of the endothelin B receptor (ETB). The IL2-targeted antibody accurately detected endogenous ETB expression in rat brain and cultured rat astrocytes by labeling a 50kD band, the expected weight of full-length ETB. However, this antibody failed to detect transfected ETB in HEK293 cultures. In contrast, the NT-targeted antibody accurately detected endogenous ETB in rat astrocyte cultures and transfected ETB in HEK293 cultures by labeling a 37 kD band, but failed to detect endogenous ETB in rat brain. Bands detected by the CT-targeted or IL3-targeted antibodies were found to be unrelated to ETB. Our findings show that functional ETB receptors can be detected at 50 kD or 37 kD on Western blot, with drastic differences in antibody affinity for these bands. The 37 kD band likely reflects ETB receptor processing, which appears to be dependent on cell type and/or culture condition.
doi:10.1016/j.ab.2014.09.004
PMCID: PMC4362934  PMID: 25232999
Endothelin B receptor; antibody; Western; immunoblot
12.  Real-Time Observation of the Initiation of RNA Polymerase II Transcription 
Nature  2015;525(7568):274-277.
Biochemical and structural studies have shown that the initiation of RNA polymerase II (pol II) transcription proceeds in the following stages: assembly of pol II with general transcription factors (GTFs) and promoter DNA in a “closed” preinitiation complex (PIC)1,2; unwinding about 15 bp of the promoter DNA to form an “open” complex3,4; scanning downstream to a transcription start site; synthesis of a short transcript, believed to be about 10 nucleotides; and promoter escape. We have assembled a 32-protein, 1.5 megadalton PIC5 derived from Saccharomyces cerevisiae and observed subsequent initiation processes in real time with optical tweezers6. Contrary to expectation, scanning driven by transcription factor IIH (TFIIH)7-12 entailed the rapid opening of an extended bubble, averaging 85 bp, accompanied by the synthesis of a transcript up to the entire length of the extended bubble, followed by promoter escape. PICs that failed to achieve promoter escape nevertheless formed open complexes and extended bubbles, which collapsed back to closed or open complexes, resulting in repeated futile scanning.
doi:10.1038/nature14882
PMCID: PMC4624315  PMID: 26331540
13.  Bicoid gradient formation and function in the Drosophila pre-syncytial blastoderm 
eLife  null;5:e13222.
Bicoid (Bcd) protein distributes in a concentration gradient that organizes the anterior/posterior axis of the Drosophila embryo. It has been understood that bcd RNA is sequestered at the anterior pole during oogenesis, is not translated until fertilization, and produces a protein gradient that functions in the syncytial blastoderm after 9–10 nuclear divisions. However, technical issues limited the sensitivity of analysis of pre-syncytial blastoderm embryos and precluded studies of oocytes after stage 13. We developed methods to analyze stage 14 oocytes and pre-syncytial blastoderm embryos, and found that stage 14 oocytes make Bcd protein, that bcd RNA and Bcd protein distribute in matching concentration gradients in the interior of nuclear cycle 2–6 embryos, and that Bcd regulation of target gene expression is apparent at nuclear cycle 7, two cycles prior to syncytial blastoderm. We discuss the implications for the generation and function of the Bcd gradient.
DOI: http://dx.doi.org/10.7554/eLife.13222.001
eLife digest
As an embryo develops, a single cell transforms into a collection of different types of cells. One protein that is crucial for this process in fruit fly embryos is Bicoid. Thirty years ago, scientists discovered that Bicoid protein is concentrated at the head end of the embryo and gradually decreases in amount towards the rear end. This concentration gradient of Bicoid protein organizes the embryo body and regulates the expression of many genes, thus directing the cells to develop different identities.
Several assumptions had been made about how this gradient is established. It was thought that in the unfertilized egg, the mRNA molecules that will be translated to produce Bicoid proteins are stored in an inactive state in the region of the egg that later develops into the embryo’s head. In the embryo, the mRNA molecules were believed to remain in the head region while being translated, with the newly formed proteins then gradually spreading from this site to create the Bicoid gradient. It was also thought that no Bicoid proteins are stored in the unfertilized egg. However, no known methods were sensitive enough to investigate these assumptions.
Now, using newer and more sensitive methods, Ali-Murthy and Kornberg show that Bicoid protein is present in the unfertilized fruit fly egg in the same region as the mRNA molecules that make Bicoid. Furthermore, the Bicoid gradient forms when the embryo has fewer than 32 nuclei, much earlier in development than previously thought. The Bicoid protein also does not appear to spread passively towards the rear of the embryo, but is transported in a more orchestrated manner.
Overall, Ali-Murthy and Kornberg’s results suggest that the early fruit fly embryo is more organized and actively regulated than had been previously understood. This paves the way for further studies that use sensitive techniques to investigate this early stage of development.
DOI: http://dx.doi.org/10.7554/eLife.13222.002
doi:10.7554/eLife.13222
PMCID: PMC4786422  PMID: 26883601
drosophila; embryo; oocyte; bicoid; krüppel; D. melanogaster
14.  A naturally-monomeric infrared fluorescent protein for protein labeling in vivo 
Nature methods  2015;12(8):763-765.
Infrared fluorescent proteins (IFPs) provide an additional color to GFP and its red homologs in protein labeling. Based on structural analysis of the dimer interface, a monomeric bateriophytochrome is identified from a sequence database, and is engineered into a naturally-monomeric IFP (mIFP). We demonstrate that mIFP correctly labels proteins in live Drosophila and zebrafish requiring no exogenous cofactor, and will thus be useful in molecular, cell and developmental biology.
doi:10.1038/nmeth.3447
PMCID: PMC4521985  PMID: 26098020
15.  Paracrine signaling mediated at cell-cell contacts 
Recent findings in several organ systems show that cytoneme-mediated signaling transports signaling proteins along cellular extensions and targets cell-to-cell exchanges to synaptic contacts. This mechanism of paracrine signaling may be a general one that is used by many (or all) cell types in many (or all) organs. We briefly review these findings in this perspective. We also describe the properties of several signaling systems that have previously been interpreted to support a passive diffusion mechanism of signaling protein dispersion, but can now be understood in the context of the cytoneme mechanism.
doi:10.1002/bies.201400122
PMCID: PMC4407995  PMID: 25476066
cytonemes; filopodia; morphogen; paracrine signaling; synapse; TGF-β
16.  Cytonemes and the dispersion of morphogens 
Filopodia are cellular protrusions that have been implicated in many types of mechanosensory activities. Morphogens are signaling proteins that regulate the patterned development of embryos and tissues. Both have long histories that date to the beginnings of cell and developmental biology in the early 20th century, but recent findings tie specialized filopodia called cytonemes to morphogen movement and morphogen signaling. This review explores the conceptual and experimental background for a model of paracrine signaling in which the exchange of morphogens between cells is directed to sites where cytonemes directly link cells that produce morphogens to cells that receive and respond to them.
doi:10.1002/wdev.151
PMCID: PMC4199865  PMID: 25186102
morphogen; cytoneme; developmental organizer; signaling center
17.  Developmental compartments in the larval trachea of Drosophila 
eLife  null;4:e08666.
The Drosophila tracheal system is a branched tubular network that forms in the embryo by a post-mitotic program of morphogenesis. In third instar larvae (L3), cells constituting the second tracheal metamere (Tr2) reenter the cell cycle. Clonal analysis of L3 Tr2 revealed that dividing cells in the dorsal trunk, dorsal branch and transverse connective branches respect lineage restriction boundaries near branch junctions. These boundaries corresponded to domains of gene expression, for example where cells expressing Spalt, Delta and Serrate in the dorsal trunk meet vein–expressing cells in the dorsal branch or transverse connective. Notch signaling was activated to one side of these borders and was required for the identity, specializations and segregation of border cells. These findings suggest that Tr2 is comprised of developmental compartments and that developmental compartments are an organizational feature relevant to branched tubular networks.
DOI: http://dx.doi.org/10.7554/eLife.08666.001
eLife digest
As a fruit fly develops, its cells may sort themselves into groups according to the type of cell that they will eventually become. Some groups form ‘developmental compartments’ that are separated by boundaries that cells cannot move across. All the descendants of a cell in a compartment will activate the same specific gene (called a ‘selector’ gene) that determines their identity and fate. Similar compartments also form in the developing hindbrains of mammals, but it is not clear how general this mechanism of tissue patterning is.
Fruit fly larvae undergo a physical transformation called metamorphosis to become adult fruit flies. Here, Rao et al. discover that the cells in the developing airways (or trachea) of the larvae at the start of metamorphosis are organised into compartments. At this stage the cells in the trachea start to divide and grow to make the adult tracheal system. The experiments show that these cells do not spread from one main branch of the tracheal system into another. Instead, the cells cluster in locations where the different branches meet on either side of a straight boundary.
The cells on each side of these boundaries activate different genes that regulate their identity and development. For example, cells in one branch of the system switch on a selector gene that makes a protein called Spalt. A pathway known as Notch signaling is activated by cells on the other side of a nearby boundary in a different branch of the tracheal system. This separation of Spalt production and Notch activation establishes a cell communication system that keeps the cells of the different compartments apart.
Rao et al.’s findings reveal a role for the Notch protein in regulating the organization of cells into compartments to form branches in fruit fly airways. A future challenge is to find out if Notch plays a similar role in other branched tissues, such as blood vessels.
DOI: http://dx.doi.org/10.7554/eLife.08666.002
doi:10.7554/eLife.08666
PMCID: PMC4718809  PMID: 26491942
developmental compartments; trachea; notch; D. melanogaster
18.  The contrasting roles of primary cilia and cytonemes in Hh signaling 
Developmental biology  2014;394(1):1-5.
Hedgehog (Hh) is a paracrine signaling protein with major roles in development and disease. In vertebrates and invertebrates, Hh signal transduction is carried out almost entirely by evolutionarily conserved components, and in both, intercellular movement of Hh is mediated by cytonemes – specialized filopodia that serve as bridges that bring distant cells into contact. A significant difference is the role of primary cilia, a slender, tubulin-based protuberance of many vertebrate cells. Although the primary cilium is essential for Hh signaling in cells that have one, most Drosophila cells lack a primary cilium. This perspective addresses the roles of primary cilia and cytonemes, and proposes that for Hh signaling, the role of primary cilia is to provide a specialized hydrophobic environment that hosts lipid-modified Hh and other components of Hh signal transduction after Hh has traveled from elsewhere in the cell. Implicit in this model is the idea that initial binding and uptake of Hh is independent of and segregated from the processes of signal transduction and activation.
doi:10.1016/j.ydbio.2014.07.015
PMCID: PMC4163518  PMID: 25072627
Hedgehog; primary cilium; cytoneme; lipid raft
19.  Molecular architecture of the yeast Mediator complex 
eLife  null;4:e08719.
The 21-subunit Mediator complex transduces regulatory information from enhancers to promoters, and performs an essential role in the initiation of transcription in all eukaryotes. Structural information on two-thirds of the complex has been limited to coarse subunit mapping onto 2-D images from electron micrographs. We have performed chemical cross-linking and mass spectrometry, and combined the results with information from X-ray crystallography, homology modeling, and cryo-electron microscopy by an integrative modeling approach to determine a 3-D model of the entire Mediator complex. The approach is validated by the use of X-ray crystal structures as internal controls and by consistency with previous results from electron microscopy and yeast two-hybrid screens. The model shows the locations and orientations of all Mediator subunits, as well as subunit interfaces and some secondary structural elements. Segments of 20–40 amino acid residues are placed with an average precision of 20 Å. The model reveals roles of individual subunits in the organization of the complex.
DOI: http://dx.doi.org/10.7554/eLife.08719.001
eLife digest
Inside a cell, proteins are made from instructions encoded by DNA. To produce a particular protein, a section of DNA within a gene is copied into a molecule of messenger ribonucleic acid (or mRNA). This process is called transcription and is carried out by an enzyme known as RNA polymerase.
Transcription begins in a region of DNA called a promoter, which is found at the start of the gene. RNA polymerase is brought to the DNA by many proteins, including the so-called Mediator complex. Mediator receives signals from within the cell and from the environment, processes the information, and instructs RNA polymerase whether to transcribe the gene or not. Mediator performs this important role in all organisms from yeast to humans, but it is not clear how it works. A crucial step towards the solution of this problem is to understand the three-dimensional structure of the complex.
Previous research using a technique called ‘electron microscopy’ showed that Mediator is composed of three modules, referred to as Head, Middle and Tail. The images from electron microscopy were not sufficiently detailed to reveal the organization of the proteins within these modules. An open-source Integrative Modeling Platform (IMP for short) was recently developed to arrive at structural models of large protein complexes from a combination of experimental data and computer models. Now, Robinson, Trnka, Pellarin et al. have used this platform to study the Mediator complex.
First, Robinson, Trnka, Pellarin et al. collected experimental data on the structure of the Mediator complex using two approaches called ‘chemical cross-linking’ and ‘mass spectrometry’. This data was combined with biochemical and structural information from previous studies to generate a three-dimensional model of the structure of the entire Mediator using IMP. The model is detailed enough to show the location and orientation of all the proteins in the complex. For example, a protein called Med17 connects the Head and Middle modules, while another subunit—known as Med14—spans the entire complex and makes extensive contacts with other proteins in all three modules.
DOI: http://dx.doi.org/10.7554/eLife.08719.002
doi:10.7554/eLife.08719
PMCID: PMC4631838  PMID: 26402457
RNA polymerase II; transcriptional regulation; cross-linking; mass spectrometry; modeling; S. cerevisiae
20.  The Environment of “Mycobacterium avium subsp. hominissuis” Microaggregates Induces Synthesis of Small Proteins Associated with Efficient Infection of Respiratory Epithelial Cells 
Infection and Immunity  2014;83(2):625-636.
“Mycobacterium avium subsp. hominissuis” is an opportunistic environmental pathogen that causes respiratory illness in immunocompromised patients, such as those with cystic fibrosis as well as other chronic respiratory diseases. Currently, there is no efficient approach to prevent or treat M. avium subsp. hominissuis infection in the lungs. During initial colonization of the airways, M. avium subsp. hominissuis forms microaggregates composed of 3 to 20 bacteria on human respiratory epithelial cells, which provides an environment for phenotypic changes leading to efficient mucosal invasion in vitro and in vivo. DNA microarray analysis was employed to identify genes associated with the microaggregate phenotype. The gene encoding microaggregate-binding protein 1 (MBP-1) (MAV_3013) is highly expressed during microaggregate formation. When expressed in noninvasive Mycobacterium smegmatis, MBP-1 increased the ability of the bacteria to bind to HEp-2 epithelial cells. Using anti-MBP-1 immune serum, microaggregate binding to HEp-2 cells was significantly reduced. By far-Western blotting, and verified by coimmunoprecipitation, we observed that MBP-1 interacts with the host cytoskeletal protein vimentin. As visualized by confocal microscopy, microaggregates, as well as MBP-1, induced vimentin polymerization at the site of bacterium-host cell contact. Binding of microaggregates to HEp-2 cells was inhibited by treatment with an antivimentin antibody, suggesting that MBP-1 expression is important for M. avium subsp. hominissuis adherence to the host cell. MBP-1 immune serum significantly inhibited M. avium subsp. hominissuis infection throughout the respiratory tracts of mice. This study characterizes a pathogenic mechanism utilized by M. avium subsp. hominissuis to bind and invade the host respiratory epithelium, suggesting new potential targets for the development of antivirulence therapy.
doi:10.1128/IAI.02699-14
PMCID: PMC4294266  PMID: 25422262
21.  Epigenomics: A Roadmap, But to Where? 
Science (New York, N.Y.)  2008;322(5898):43-44.
doi:10.1126/science.322.5898.43b
PMCID: PMC4479142  PMID: 18832628
22.  Intravenous immunoglobulins in liver transplant patients: Perspectives of clinical immune modulation 
World Journal of Hepatology  2015;7(11):1494-1508.
Shortage of appropriate donor grafts is the foremost current problem in organ transplantation. As a logical consequence, waiting times have extended and pretransplant mortality rates were significantly increasing. The implementation of a priority-based liver allocation system using the model of end-stage liver disease (MELD) score helped to reduce waiting list mortality in liver transplantation (LT). However, due to an escalating organ scarcity, pre-LT MELD scores have significantly increased and liver recipients became more complex in recent years. This has finally led to posttransplant decreasing survival rates, attributed mainly to elevated rates of infectious and immunologic complications. To meet this challenging development, an increasing number of extended criteria donor grafts are currently accepted, which may, however, aggravate the patients’ infectious and immunologic risk profiles. The administration of intravenous immunoglobulins (IVIg) is an established treatment in patients with immune deficiencies and other antibody-mediated diseases. In addition, IVIg was shown to be useful in treatment of several disorders caused by deterioration of the cellular immune system. It proved to be effective in preventing hyperacute rejection in highly sensitized kidney and heart transplants. In the liver transplant setting, the administration of specific Ig against hepatitis B virus is current standard in post-LT antiviral prophylaxis. The mechanisms of action of IVIg are complex and not fully understood. However, there is increasing experimental and clinical evidence that IVIg has an immuno-balancing impact by a combination of immuno-supporting and immuno-suppressive properties. It may be suggested that, especially in the context of a worsening organ shortage with all resulting clinical implications, liver transplant patients should benefit from immuno-regulatory capabilities of IVIg. In this review, perspectives of immune modulation by IVIg and impact on outcome in liver transplant patients are described.
doi:10.4254/wjh.v7.i11.1494
PMCID: PMC4462688  PMID: 26085909
Intravenous immunoglobulins; Immune modulation; Hyperimmunoglobulin; Model of end-stage liver disease; Liver transplantation
23.  The dynamics of SecM-induced translational stalling 
Cell reports  2014;7(5):1521-1533.
Summary
SecM is an E. coli secretion monitor capable of stalling translation on the prokaryotic ribosome without co-factors. Biochemical and structural studies have demonstrated that the SecM nascent chain interacts with the 50S subunit exit tunnel to inhibit peptide bond formation. However, the timescales and pathways of stalling on a mRNA remain undefined. To provide a dynamic mechanism for stalling, we directly tracked the dynamics of elongation on ribosomes translating the SecM stall sequence (FSTPVWISQAQGIRAGP) using single-molecule fluorescence techniques. Within one minute, three peptide-ribosome interactions work cooperatively over the last 5 codons of the SecM sequence, leading to severely impaired elongation rates beginning from the terminal proline and lasting 4 codons. Our results suggest that stalling is tightly linked to the dynamics of elongation and underscore the roles that the exit tunnel and nascent chain play in controlling fundamental steps in translation.
doi:10.1016/j.celrep.2014.04.033
PMCID: PMC4059775  PMID: 24836001
24.  Sequence-dependent elongation dynamics on macrolide-bound ribosomes 
Cell reports  2014;7(5):1534-1546.
SUMMARY
The traditional view of macrolide antibiotics as plugs inside the ribosomal nascent peptide exit tunnel (NPET) has lately been challenged in favor of a more complex, heterogeneous mechanism, where drug-peptide interactions determine the fate of a translating ribosome. To investigate these highly dynamic processes, we applied single-molecule tracking of elongating ribosomes during inhibition of elongation by erythromycin of several nascent chains, including ErmCL and H-NS, which were shown to be respectively sensitive and resistant to erythromycin. Peptide sequence-specific changes were observed in translation elongation dynamics in the presence of a macrolide-obstructed NPET. Elongation rates were not severely inhibited in general by the presence of the drug; instead, stalls or pauses were observed as abrupt events. The dynamic pathways of nascent-chain dependent elongation pausing in the presence of macrolides determine the fate of the translating ribosome - stalling or read-through.
doi:10.1016/j.celrep.2014.04.034
PMCID: PMC4387896  PMID: 24836000
25.  Communicating by touch – neurons are not alone 
Trends in cell biology  2014;24(6):370-376.
Summary
Long distance cell-cell communication is essential for organ development and function. Whereas neurons communicate at long distances by transferring signals at sites of direct contact (i.e. at synapses), it has been presumed that the only way other cell types signal is by dispersing signals through extracellular fluid - indirectly. Recent evidence from Drosophila suggests that non-neuronal cells also exchange signaling proteins at sites of direct contact, even when long distances separate the cells. Here, we review contact-mediated signaling in neurons and discuss how this signaling mechanism is shared by other cell types.
doi:10.1016/j.tcb.2014.01.003
PMCID: PMC4037336  PMID: 24560610
cytoneme; synapse; morphogen; neuron

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