Publications

2013
Pan, G, Feng Y, Ambegaonkar AA, Sun G, Huff M, Rauskolb C, Irvine KD.  2013.  Signal transduction by the Fat cytoplasmic domain.. Development. AbstractWebsite
The large atypical cadherin Fat is a receptor for both Hippo and planar cell polarity (PCP) pathways. Here we investigate the molecular basis for signal transduction downstream of Fat by creating targeted alterations within a genomic construct that contains the entire fat locus, and by monitoring and manipulating the membrane localization of the Fat pathway component Dachs. We establish that the human Fat homolog FAT4 lacks the ability to transduce Hippo signaling in Drosophila, but can transduce Drosophila PCP signaling. Targeted deletion of conserved motifs identifies a four amino acid C-terminal motif that is essential for aspects of Fat-mediated PCP, and other internal motifs that contribute to Fat-Hippo signaling. Fat-Hippo signaling requires the Drosophila Casein kinase 1_ encoded by discs overgrown (Dco), and we characterize candidate Dco phosphorylation sites in the Fat intracellular domain (ICD), the mutation of which impairs Fat-Hippo signaling. Through characterization of Dachs localization and directed membrane targeting of Dachs, we show that localization of Dachs influences both the Hippo and PCP pathways. Our results identify a conservation of Fat-PCP signaling mechanisms, establish distinct functions for different regions of the Fat ICD, support the correlation of Fat ICD phosphorylation with Fat-Hippo signaling, and confirm the importance of Dachs membrane localization to downstream signaling pathways.
Reddy, BVVG, Irvine KD.  2013.  Regulation of Hippo Signaling by EGFR-MAPK Signaling through Ajuba Family Proteins.. Developmental Cell. 24:459-471. AbstractWebsite
EGFR and Hippo signaling pathways both control growth and, when dysregulated, contribute to tumorigenesis. We find that EGFR activates the Hippo pathway transcription factor Yorkie and demonstrate that Yorkie is required for the influence of EGFR on cell proliferation in Drosophila. EGFR regulates Yorkie through the influence of its Ras-MAPK branch on the Ajuba LIM protein Jub. Jub is epistatic to EGFR and Ras for Yorkie regulation, Jub is subject to MAPK-dependent phosphorylation, and EGFR-Ras-MAPK signaling enhances Jub binding to the Yorkie kinase Warts and the adaptor protein Salvador. An EGFR-Hippo pathway link is conserved in mammals, as activation of EGFR or RAS activates the Yorkie homolog YAP, and EGFR-RAS-MAPK signaling promotes phosphorylation of the Ajuba family protein WTIP and also enhances WTIP binding to the Warts and Salvador homologs LATS and WW45. Our observations implicate the Hippo pathway in EGFR-mediated tumorigenesis and identify a molecular link between these pathways.
Holder, AA, Taylor P, Magnusen AR, Moffett ET, Meyer K, Hong Y, Ramsdale SE, Gordon M, Stubbs J, Seymour LA et al..  2013.  Preliminary anti-cancer photodynamic therapeutic in vitro studies with mixed-metal binuclear ruthenium(II)-vanadium(IV) complexes.. Dalton transactions (Cambridge, England : 2003). 42(33):11881-99. Abstract
We report the synthesis and characterisation of mixed-metal binuclear ruthenium(II)-vanadium(IV) complexes, which were used as potential photodynamic therapeutic agents for melanoma cell growth inhibition. The novel complexes, [Ru(pbt)2(phen2DTT)](PF6)2·1.5H2O 1 (where phen2DTT = 1,4-bis(1,10-phenanthrolin-5-ylsulfanyl)butane-2,3-diol and pbt = 2-(2'-pyridyl)benzothiazole) and [Ru(pbt)2(tpphz)](PF6)2·3H2O 2 (where tpphz = tetrapyrido[3,2-a:2',3'-c:3'',2''-h:2''',3'''-j]phenazine) were synthesised and characterised. Compound 1 was reacted with [VO(sal-L-tryp)(H2O)] (where sal-L-tryp = N-salicylidene-L-tryptophanate) to produce [Ru(pbt)2(phen2DTT)VO(sal-L-tryp)](PF6)2·5H2O 4; while [VO(sal-L-tryp)(H2O)] was reacted with compound 2 to produce [Ru(pbt)2(tpphz)VO(sal-L-tryp)](PF6)2·6H2O 3. All complexes were characterised by elemental analysis, HRMS, ESI MS, UV-visible absorption, ESR spectroscopy, and cyclic voltammetry, where appropriate. In vitro cell toxicity studies (with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay) via dark and light reaction conditions were carried out with sodium diaqua-4,4',4'',4''' tetrasulfophthalocyaninecobaltate(II) (Na4[Co(tspc)(H2O)2]), [VO(sal-L-tryp)(phen)]·H2O, and the chloride salts of complexes 3 and 4. Such studies involved A431, human epidermoid carcinoma cells; human amelanotic malignant melanoma cells; and HFF, non-cancerous human skin fibroblast cells. Both chloride salts of complexes 3 and 4 were found to be more toxic to melanoma cells than to non-cancerous fibroblast cells, and preferentially led to apoptosis of the melanoma cells over non-cancerous skin cells. The anti-cancer property of the chloride salts of complexes 3 and 4 was further enhanced when treated cells were exposed to light, while no such effect was observed on non-cancerous skin fibroblast cells. ESR and (51)V NMR spectroscopic studies were also used to assess the stability of the chloride salts of complexes 3 and 4 in aqueous media at pH 7.19. This research illustrates the potential for using mixed-metal binuclear ruthenium(II)-vanadium(IV) complexes to fight skin cancer.
Khorobrykh, A, Dasgupta J, Kolling DRJ, Terentyev V, Klimov VV, Dismukes CG.  2013.  Evolutionary origins of the photosynthetic water oxidation cluster: bicarbonate permits Mn(2+) photo-oxidation by anoxygenic bacterial reaction centers.. Chembiochem : a European journal of chemical biology. 14(14):1725-31. Abstract
The enzyme that catalyzes water oxidation in oxygenic photosynthesis contains an inorganic cluster (Mn4 CaO5 ) that is universally conserved in all photosystem II (PSII) protein complexes. Its hypothesized precursor is an anoxygenic photobacterium containing a type 2 reaction center as photo-oxidant (bRC2, iron-quinone type). Here we provide the first experimental evidence that a native bRC2 complex can catalyze the photo-oxidation of Mn(2+) to Mn(3+) , but only in the presence of bicarbonate concentrations that allows the formation of (bRC2)Mn(2+) (bicarbonate)1-2 complexes. Parallel-mode EPR spectroscopy was used to characterize the photoproduct, (bRC2)Mn(3+) (CO3 (2-) ), based on the g tensor and (55) Mn hyperfine splitting. (Bi)carbonate coordination extends the lifetime of the Mn(3+) photoproduct by slowing charge recombination. Prior electrochemical measurements show that carbonate complexation thermodynamically stabilizes the Mn(3+) product by 0.9-1 V relative to water ligands. A model for the origin of the water oxidation catalyst is presented that proposes chemically feasible steps in the evolution of oxygenic PSIIs, and is supported by literature results on the photoassembly of contemporary PSIIs.
Li, Y, Harris L, Dooner HK.  2013.  TED, an autonomous and rare maize transposon of the mutator superfamily with a high gametophytic excision frequency.. The Plant cell. 25(9):3251-65. Abstract
Mutator (Mu) elements, one of the most diverse superfamilies of DNA transposons, are found in all eukaryotic kingdoms, but are particularly numerous in plants. Most of the present knowledge on the transposition behavior of this superfamily comes from studies of the maize (Zea mays) Mu elements, whose transposition is mediated by the autonomous Mutator-Don Robertson (MuDR) element. Here, we describe the maize element TED (for Transposon Ellen Dempsey), an autonomous cousin that differs significantly from MuDR. Element excision and reinsertion appear to require both proteins encoded by MuDR, but only the single protein encoded by TED. Germinal excisions, rare with MuDR, are common with TED, but arise in one of the mitotic divisions of the gametophyte, rather than at meiosis. Instead, transposition-deficient elements arise at meiosis, suggesting that the double-strand breaks produced by element excision are repaired differently in mitosis and meiosis. Unlike MuDR, TED is a very low-copy transposon whose number and activity do not undergo dramatic changes upon inbreeding or outcrossing. Like MuDR, TED transposes mostly to unlinked sites and can form circular transposition products. Sequences closer to TED than to MuDR were detected only in the grasses, suggesting a rather recent evolutionary split from a common ancestor.
Robinson, DM, Go Y B, Mui M, Gardner G, Zhang Z, Mastrogiovanni D, Garfunkel E, Li J, Greenblatt M, Dismukes CG.  2013.  Photochemical water oxidation by crystalline polymorphs of manganese oxides: structural requirements for catalysis.. Journal of the American Chemical Society. 135(9):3494-501. Abstract
Manganese oxides occur naturally as minerals in at least 30 different crystal structures, providing a rigorous test system to explore the significance of atomic positions on the catalytic efficiency of water oxidation. In this study, we chose to systematically compare eight synthetic oxide structures containing Mn(III) and Mn(IV) only, with particular emphasis on the five known structural polymorphs of MnO2. We have adapted literature synthesis methods to obtain pure polymorphs and validated their homogeneity and crystallinity by powder X-ray diffraction and both transmission and scanning electron microscopies. Measurement of water oxidation rate by oxygen evolution in aqueous solution was conducted with dispersed nanoparticulate manganese oxides and a standard ruthenium dye photo-oxidant system. No Ru was absorbed on the catalyst surface as observed by XPS and EDX. The post reaction atomic structure was completely preserved with no amorphization, as observed by HRTEM. Catalytic activities, normalized to surface area (BET), decrease in the series Mn2O3 > Mn3O4 ≫ λ-MnO2, where the latter is derived from spinel LiMn2O4 following partial Li(+) removal. No catalytic activity is observed from LiMn2O4 and four of the MnO2 polymorphs, in contrast to some literature reports with polydispersed manganese oxides and electro-deposited films. Catalytic activity within the eight examined Mn oxides was found exclusively for (distorted) cubic phases, Mn2O3 (bixbyite), Mn3O4 (hausmannite), and λ-MnO2 (spinel), all containing Mn(III) possessing longer Mn-O bonds between edge-sharing MnO6 octahedra. Electronically degenerate Mn(III) has antibonding electronic configuration e(g)(1) which imparts lattice distortions due to the Jahn-Teller effect that are hypothesized to contribute to structural flexibility important for catalytic turnover in water oxidation at the surface.
Xu, Y, Guerra TL, Li Z, Ludwig M, Dismukes CG, Bryant DA.  2013.  Altered carbohydrate metabolism in glycogen synthase mutants of Synechococcus sp. strain PCC 7002: Cell factories for soluble sugars.. Metabolic engineering. 16:56-67. Abstract
Glycogen and compatible solutes are the major polymeric and soluble carbohydrates in cyanobacteria and function as energy reserves and osmoprotectants, respectively. Glycogen synthase null mutants (glgA-I glgA-II) were constructed in the cyanobacterium Synechococcus sp. strain PCC 7002. Under standard conditions the double mutant produced no glycogen and more soluble sugars. When grown under hypersaline conditions, the glgA-I glgA-II mutant accumulated 1.8-fold more soluble sugars (sucrose and glucosylglycer-(ol/ate)) than WT, and these cells spontaneously excreted soluble sugars into the medium at high levels without the need for additional transporters. An average of 27% more soluble sugars was released from the glgA-I glgA-II mutant than WT by hypo-osmotic shock. Extracellular vesicles budding from the outer membrane were observed by transmission electron microscopy in glgA-I glgA-II cells grown under hypersaline conditions. The glgA-I glgA-II mutant serves as a starting point for developing cell factories for photosynthetic production and excretion of sugars.
Vinyard, DJ, Xu Y, Bennette N, McNeely K, Bryant DA, Dismukes CG.  2013.  Natural osmolytes are much less effective substrates than glycogen for catabolic energy production in the marine cyanobacterium Synechococcus sp. strain PCC 7002.. Journal of biotechnology. 166(3):65-75. Abstract
ADP-glucose pyrophosphorylase, encoded by glgC, catalyzes the first step of glycogen and glucosylglycer(ol/ate) biosynthesis. Here we report the construction of the first glgC null mutant of a marine cyanobacterium (Synechococcus sp. PCC 7002) and investigate its impact on dark anoxic metabolism (autofermentation). The glgC mutant had 98% lower ADP-glucose, synthesized no glycogen and produced appreciably more soluble sugars (mainly sucrose) than wild type (WT). Some glucosylglycerol was still observed, which suggests that the mutant has another, inefficient ADP-glucose synthesis pathway. In contrast, hypersaline conditions (1M NaCl) were lethal to the mutant strain, indicating that, unlike other strains, the elevated sucrose does not compensate for the reduced GG as osmolyte. In contrast to WT, nitrate limitation did not cause bleaching of N-containing pigments or carbohydrate accumulation in the glgC mutant, indicating impaired recycling of nitrogen stores. Despite the 2-fold increase in osmolytes, both the respiration and autofermentation rates of the glgC mutant were appreciably slower (2-4-fold) and correlated quantitatively with the lower fraction of insoluble carbohydrates relative to WT (85% vs. 12%). However, the remaining insoluble carbohydrates still accounted for a high fraction of the carbohydrate catabolized (38%), indicating that insoluble carbohydrates rather than osmolytes were the preferred substrate for autofermentation.
Vinyard, DJ, Zachary CE, Ananyev GM, Dismukes CG.  2013.  Thermodynamically accurate modeling of the catalytic cycle of photosynthetic oxygen evolution: a mathematical solution to asymmetric Markov chains.. Biochimica et biophysica acta. 1827(7):861-8. Abstract
Forty-three years ago, Kok and coworkers introduced a phenomenological model describing period-four oscillations in O2 flash yields during photosynthetic water oxidation (WOC), which had been first reported by Joliot and coworkers. The original two-parameter Kok model was subsequently extended in its level of complexity to better simulate diverse data sets, including intact cells and isolated PSII-WOCs, but at the expense of introducing physically unrealistic assumptions necessary to enable numerical solutions. To date, analytical solutions have been found only for symmetric Kok models (inefficiencies are equally probable for all intermediates, called "S-states"). However, it is widely accepted that S-state reaction steps are not identical and some are not reversible (by thermodynamic restraints) thereby causing asymmetric cycles. We have developed a mathematically more rigorous foundation that eliminates unphysical assumptions known to be in conflict with experiments and adopts a new experimental constraint on solutions. This new algorithm termed STEAMM for S-state Transition Eigenvalues of Asymmetric Markov Models enables solutions to models having fewer adjustable parameters and uses automated fitting to experimental data sets, yielding higher accuracy and precision than the classic Kok or extended Kok models. This new tool provides a general mathematical framework for analyzing damped oscillations arising from any cycle period using any appropriate Markov model, regardless of symmetry. We illustrate applications of STEAMM that better describe the intrinsic inefficiencies for photon-to-charge conversion within PSII-WOCs that are responsible for damped period-four and period-two oscillations of flash O2 yields across diverse species, while using simpler Markov models free from unrealistic assumptions.
Vinyard, DJ, Gimpel J, Ananyev GM, Cornejo MA, Golden SS, Mayfield SP, Dismukes CG.  2013.  Natural variants of photosystem II subunit D1 tune photochemical fitness to solar intensity.. The Journal of biological chemistry. 288(8):5451-62. Abstract
Photosystem II (PSII) is composed of six core polypeptides that make up the minimal unit capable of performing the primary photochemistry of light-driven charge separation and water oxidation in all oxygenic phototrophs. The D1 subunit of this complex contains most of the ligating amino acid residues for the Mn(4)CaO(5) core of the water-oxidizing complex (WOC). Most cyanobacteria have 3-5 copies of the psbA gene coding for at least two isoforms of D1, whereas algae and plants have only one isoform. Synechococcus elongatus PCC 7942 contains two D1 isoforms; D1:1 is expressed under low light conditions, and D1:2 is up-regulated in high light or stress conditions. Using a heterologous psbA expression system in the green alga Chlamydomonas reinhardtii, we have measured growth rate, WOC cycle efficiency, and O(2) yield as a function of D1:1, D1:2, or the native algal D1 isoform. D1:1-PSII cells outcompete D1:2-PSII cells and accumulate more biomass in light-limiting conditions. However, D1:2-PSII cells easily outcompete D1:1-PSII cells at high light intensities. The native C. reinhardtii-PSII WOC cycles less efficiently at all light intensities and produces less O(2) than either cyanobacterial D1 isoform. D1:2-PSII makes more O(2) per saturating flash than D1:1-PSII, but it exhibits lower WOC cycling efficiency at low light intensities due to a 40% faster charge recombination rate in the S(3) state. These functional advantages of D1:1-PSII and D1:2-PSII at low and high light regimes, respectively, can be explained by differences in predicted redox potentials of PSII electron acceptors that control kinetic performance.
Barr, MM, Androwski RJ, Rashid A, Lee H, Lee J, Barr MM.  2013.  Dauer-specific dendrite arborization in C. elegans is regulated by KPC-1/Furin.. Current biology : CB. 23(16):1527-35. Abstract
Dendrites often display remarkably complex and diverse morphologies that are influenced by developmental and environmental cues. Neuroplasticity in response to adverse environmental conditions entails both hypertrophy and resorption of dendrites. How dendrites rapidly alter morphology in response to unfavorable environmental conditions is unclear. The nematode Caenorhabditis elegans enters into a stress-resistant dauer larval stage in response to an adverse environment.
Xiong, W, He L, Li Y, Dooner HK, Du C.  2013.  InsertionMapper: a pipeline tool for the identification of targeted sequences from multidimensional high throughput sequencing data.. BMC genomics. 14:679. Abstract
The advent of next-generation high-throughput technologies has revolutionized whole genome sequencing, yet some experiments require sequencing only of targeted regions of the genome from a very large number of samples. These regions can be amplified by PCR and sequenced by next-generation methods using a multidimensional pooling strategy. However, there is at present no available generalized tool for the computational analysis of target-enriched NGS data from multidimensional pools.
Vinyard, DJ, Ananyev GM, Dismukes CG.  2013.  Photosystem II: the reaction center of oxygenic photosynthesis.. Annual review of biochemistry. 82:577-606. Abstract
Photosystem II (PSII) uses light energy to split water into chemical products that power the planet. The stripped protons contribute to a membrane electrochemical potential before combining with the stripped electrons to make chemical bonds and releasing O2 for powering respiratory metabolisms. In this review, we provide an overview of the kinetics and thermodynamics of water oxidation that highlights the conserved performance of PSIIs across species. We discuss recent advances in our understanding of the site of water oxidation based upon the improved (1.9-Å resolution) atomic structure of the Mn4CaO5 water-oxidizing complex (WOC) within cyanobacterial PSII. We combine these insights with recent knowledge gained from studies of the biogenesis and assembly of the WOC (called photoassembly) to arrive at a proposed chemical mechanism for water oxidation.
Robb, NC, Cordes T, Hwang L C, Gryte K, Duchi D, Craggs TD, Santoso Y, Weiss S, Ebright RH, Kapanidis AN.  2013.  The transcription bubble of the RNA polymerase-promoter open complex exhibits conformational heterogeneity and millisecond-scale dynamics: implications for transcription start-site selection.. Journal of molecular biology. 425:875-885. Abstract
Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts ~14 base-pairs around the transcription start site, and forms a single-stranded "transcription bubble" within a catalytically active RNAP-DNA open complex (RP(o)). There is significant flexibility in the transcription start site, which causes variable spacing between the promoter elements and the start site; this in turn causes differences in the length and sequence at the 5' end of RNA transcripts, and can be important for gene regulation. The start-site variability also implies the presence of some flexibility in the positioning of the DNA relative to the RNAP active site in RP(o). The flexibility may occur in the positioning of the transcription bubble prior to RNA synthesis and may reflect bubble expansion ("scrunching") or bubble contraction ("unscrunching"). Here, we assess the presence of dynamic flexibility in RP(o) with single-molecule Förster Resonance Energy Transfer. We obtain experimental evidence for dynamic flexibility in RP(o) using different FRET rulers and labelling positions. An analysis of FRET distributions of RP(o) using burst variance analysis reveals conformational fluctuations in RP(o) in the millisecond timescale. Further experiments using subsets of nucleotides and DNA mutations allowed us to reprogram the transcription start sites, in a way that can be described by repositioning of the single-stranded transcription bubble relative to the RNAP active site within RP(o). Our study marks the first experimental observation of conformational dynamics in the transcription bubble of RP(o) and indicates that DNA dynamics within the bubble affect the search for transcription start sites.
Mathieu, J, Cauvin C, Moch C, Radford SJ, Sampaio P, Perdigoto CN, Schweisguth F, Bardin AJ, Sunkel CE, McKim K et al..  2013.  Aurora B and cyclin B have opposite effects on the timing of cytokinesis abscission in Drosophila germ cells and in vertebrate somatic cells. Dev Cell. 26(3):250-65.Website
Calvino, M., Messing J.  2013.  Discovery of MicroRNA169 gene copies in genomes of flowering plants through positional information. Genome Biol Evol. 5:402-17. AbstractWebsite
Expansion and contraction of microRNA (miRNA) families can be studied in sequenced plant genomes through sequence alignments. Here, we focused on miR169 in sorghum because of its implications in drought tolerance and stem-sugar content. We were able to discover many miR169 copies that have escaped standard genome annotation methods. A new miR169 cluster was found on sorghum chromosome 1. This cluster is composed of the previously annotated sbi-MIR169o together with two newly found MIR169 copies, named sbi-MIR169t and sbi-MIR169u. We also found that a miR169 cluster on sorghum chr7 consisting of sbi-MIR169l, sbi-MIR169m, and sbi-MIR169n is contained within a chromosomal inversion of at least 500 kb that occurred in sorghum relative to Brachypodium, rice, foxtail millet, and maize. Surprisingly, synteny of chromosomal segments containing MIR169 copies with linked bHLH and CONSTANS-LIKE genes extended from Brachypodium to dictotyledonous species such as grapevine, soybean, and cassava, indicating a strong conservation of linkages of certain flowering and/or plant height genes and microRNAs, which may explain linkage drag of drought and flowering traits and would have consequences for breeding new varieties. Furthermore, alignment of rice and sorghum orthologous regions revealed the presence of two additional miR169 gene copies (miR169r and miR169s) on sorghum chr7 that formed an antisense miRNA gene pair. Both copies are expressed and target different set of genes. Synteny-based analysis of microRNAs among different plant species should lead to the discovery of new microRNAs in general and contribute to our understanding of their evolution.
Messing, J, Holding D.  2013.  Evolution, Structure, and Function of Prolamin Storage Proteins.. Seed Genomics. :139-158.
Li, Y., Segal, G., Wang, Q., Dooner HK.  2013.  Gene tagging with engineered Ds elements in maize. Methods in Molecular Biology: Plant Transposable Elements. :83-99.
Klimuk, E, Akulenko N, Makarova KS, Ceyssens P-J, Lavigne R, Severinov K.  2013.  Host RNA polymerase inhibitors encoded by φKMV-like phages of Pseudomonas. Virology. 436:67-74.
Wu, Y, Yuan L, Guo X, Holding DR, Messing J.  2013.  Mutation in the seed storage protein kafirin creates a high-value food trait in sorghum. Nat Commun. 4:2217. AbstractWebsite
Sustainable food production for the earth's fast-growing population is a major challenge for breeding new high-yielding crops, but enhancing the nutritional quality of staple crops can potentially offset limitations associated with yield increases. Sorghum has immense value as a staple food item for humans in Africa, but it is poorly digested. Although a mutant exhibiting high-protein digestibility and lysine content has market potential, the molecular nature of the mutation is previously unknown. Here, building on knowledge from maize mutants, we take a direct approach and find that the high-digestible sorghum phenotype is tightly linked to a single-point mutation, rendering the signal peptide of a seed storage protein kafirin resistant to processing, indirectly reducing lysine-poor kafirins and thereby increasing lysine-rich proteins in the seeds. These findings indicate that a molecular marker can be used to accelerate introduction of this high nutrition and digestibility trait into different sorghum varieties.
Guerra, LT, Xu Y, Bennette N, McNeely K, Bryant DA, Dismukes GC.  2013.  Natural osmolytes are much less effective substrates than glycogen for catabolic energy production in the marine cyanobacterium Synechococcus sp. strain PCC 7002 .. J. Biotechnol.. 166:65-75. Abstract
ADP-glucose pyrophosphorylase, encoded by glgC, catalyzes the first step of glycogen and glucosylglycer(ol/ate) biosynthesis. Here we report the construction of the first glgC null mutant of a marine cyanobacterium (Synechococcus sp. PCC 7002) and investigate its impact on dark anoxic metabolism (autofermentation). The glgC mutant had 98% lower ADP-glucose, synthesized no glycogen and produced appreciably more soluble sugars (mainly sucrose) than wild type (WT). Some glucosylglycerol was still observed, which suggests that the mutant has another, inefficient ADP-glucose synthesis pathway. In contrast, hypersaline conditions (1M NaCl) were lethal to the mutant strain, indicating that, unlike other strains, the elevated sucrose does not compensate for the reduced GG as osmolyte. In contrast to WT, nitrate limitation did not cause bleaching of N-containing pigments or carbohydrate accumulation in the glgC mutant, indicating impaired recycling of nitrogen stores. Despite the 2-fold increase in osmolytes, both the respiration and autofermentation rates of the glgC mutant were appreciably slower (2-4-fold) and correlated quantitatively with the lower fraction of insoluble carbohydrates relative to WT (85% vs. 12%). However, the remaining insoluble carbohydrates still accounted for a high fraction of the carbohydrate catabolized (38%), indicating that insoluble carbohydrates rather than osmolytes were the preferred substrate for autofermentation.
Barbosa, N, Minakhina S, Medina DJ, Balsara B, Greenwood S, Huzzy L, Rabson AB, Steward R, Schaar DG.  2013.  PDCD2 functions in cancer cell proliferation and predicts relapsed leukemia.. Cancer biology & therapy. 14(6):546-555. AbstractWebsite
PDCD2 is an evolutionarily conserved eukaryotic protein with unknown function. The Drosophlia PDCD2 ortholog Zfrp8 has an essential function in fly hematopoiesis. Zfrp8 mutants exhibit marked lymph gland hyperplasia that results from increased proliferation of partially differentiated hemocytes, suggesting Zfrp8 may participate in cell growth. Based on the above observations we have focused on the role of PDCD2 in human cancer cell proliferation and hypothesized that aberrant PDCD2 expression may be characteristic of human malignancies. We report that PDCD2 is highly expressed in human acute leukemia cells as well as in normal hematopoietic progenitors. PDCD2 knockdown in cancer cells impairs their proliferation, but not viability relative to parental cells, supporting the notion that PDCD2 overexpression facilitates cancer cell growth. Prospective analysis of PDCD2 in acute leukemia patients indicates PDCD2 RNA expression correlates with disease status and is a significant predictor of clinical relapse. PDCD2's role in cell proliferation and its high expression in human malignancies make it an attractive, novel potential molecular target for new anti-cancer therapies.
Guerra, TL, Levitan O, Frada MJ, Sun JS, Falkowski PG, Dismukes GC.  2013.  Regulatory branch points affecting protein and lipid biosynthesis in the diatom Phaeodactylum tricornutum. Biomass and Bioenergy. 59:306-315. AbstractWebsite
It is widely established that nutritional nitrogen deprivation increases lipid accumulation but severely decreases growth rate in microalgae. To understand the regulatory branch points that determine the partitioning of carbon among its potential sinks, we analyzed metabolite and transcript levels of central carbon metabolic pathways and determined the average fluxes and quantum requirements for the synthesis of protein, carbohydrates and fatty acid in the diatom Phaeodactylum tricornutum. Under nitrate-starved conditions, the carbon fluxes into all major sinks decrease sharply; the largest decrease was into proteins and smallest was into lipids. This reduction of carbon flux into lipids together with a significantly lower growth rate is responsible for lower overall FA productivities implying that nitrogen starvation is not a bioenergetically feasible strategy for increasing biodiesel production. The reduction in these fluxes was accompanied by an 18-fold increase in α-ketoglutarate (AKG), 3-fold increase in NADPH/NADP+, and sharp decreases in glutamate (GLU) and glutamine (GLN) levels. Additionally, the mRNA level of acetyl-CoA carboxylase and two type II diacylglycerol-acyltransferases were increased. Partial suppression of nitrate reductase by tungstate resulted in similar trends at lower levels as for nitrate starvation. These results reveal that the GS/GOGAT pathway is the main regulation site for nitrate dependent control of carbon partitioning between protein and lipid biosynthesis, while the AKG/GL(N/U) metabolite ratio is a transcriptional signal, possibly related to redox poise of intermediates in the photosynthetic electron transport system.
Kumaraswamy, GK, Guerra T, Qian X, Zhang S, Bryant DA, Dismukes GC.  2013.  Reprogramming the glycolytic pathway for increased hydrogen production in cyanobacteria: metabolic engineering of NAD+-dependent GAPDH. Energy Environ. Sci.. 6:3722-3731. AbstractWebsite
Catabolism of glycogen stored by cyanobacteria occurs during anaerobic auto-fermentation and produces a range of C1–C3 fermentation products and hydrogen via hydrogenase. We investigated both augmenting and rerouting this carbon catabolism by engineering the glycolysis pathway at the NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH-1), its major regulation site at the nexus of two pathways (Oxidative Pentose Phosphate pathway, OPP, and glycolysis/gluconeogenesis). Null (gap1::aphII) and overexpression (gap1+) strains of Synechococcus sp. strain PCC 7002 were constructed in order to produce more NADPH (via rerouting carbon through OPP) and more NADH (via opening the glycolytic bottleneck), respectively. For gap1::aphII quantitative analyses after four days of dark auto-fermentation showed undiminished glycogen catabolism rate, significant increases of intracellular metabolites in both OPP and upper-glycolysis, decrease in lower-glycolysis intermediates, 5.7-fold increase in NADPH, 2.3-fold increase in hydrogen and 1.25-fold increase in CO2vs. wild type (WT). These changes demonstrate the expected outcome of redirection of carbon catabolism through the OPP pathway with significant stimulation of OPP product yields. The gap1+ strain exhibits a large 17% increase in accumulation of glycogen during the prior photoautotrophic growth stage (gluconeogenesis), in parallel with a 2-fold increase in the total [NAD+ + NADH] pool, foreshadowing an increased catabolic capacity. Indeed, the rate of glycogen catabolism during subsequent dark auto-fermentation increased significantly (58%) vs. WT, resulting in increases in both NADH (4.0-fold) and NADPH (2.9-fold) pools, and terminal fermentation products, hydrogen (3.0-fold) D-lactate (2.3-fold) and acetate (1.4-fold). The overall energy conversion yield over four days from catabolized glycogen to hydrogen increased from 0.6 mole of hydrogen per mole of glucose (WT) to 1.4 (gap1::aphII) and 1.1 (gap1+) under headspace accumulation conditions (without hydrogen milking). These findings demonstrate the significant potential of metabolic engineering for redirecting carbon pathways for carbohydrate catabolism and hydrogen production in cyanobacteria.
Gallavotti, A.  2013.  The role of auxin in shaping shoot architecture. Journal of Experimental Botany. 64(9):2593-2608. AbstractWebsite
The variety of plant architectures observed in nature is predominantly determined by vegetative and reproductive branching patterns, the positioning of lateral organs, and differential stem elongation. Branches, lateral organs, and stems are the final products of the activity of meristems, groups of stem cells whose function is genetically deter- mined and environmentally influenced. Several decades of studies in different plant species have shed light on the essential role of the hormone auxin in plant growth and development. Auxin influences stem elongation and regulates the formation, activity, and fate of meristems, and has therefore been recognized as a major hormone shaping plant architecture. Increasing our knowledge of the molecular mechanisms that regulate auxin function is necessary to understand how different plant species integrate a genetically determined developmental programme, the establish- ment of a body plan, with constant inputs from the surrounding environment. This information will allow us to develop the molecular tools needed to modify plant architecture in several crop species and in rapidly changing environments.