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An aeroponic culture system for the study of root herbivory on Arabidopsis thaliana
Vaughan, Martha M.; Tholl, Dorothea; Tokuhisa, James G. (Biomed Central, 2011-03-10)
Background Plant defense against herbivory has been studied primarily in aerial tissues. However, complex defense mechanisms have evolved in all parts of the plant to combat herbivore attack and these mechanisms are likely to differ in the aerial and subterranean environment. Research investigating defense responses belowground has been hindered by experimental difficulties associated with the accessibility and quality of root tissue and the lack of bioassays using model plants with altered defense profiles. Results We have developed an aeroponic culture system based on a calcined clay substrate that allows insect herbivores to feed on plant roots while providing easy recovery of the root tissue. The culture method was validated by a root-herbivore system developed for Arabidopsis thaliana and the herbivore Bradysia spp. (fungus gnat). Arabidopsis root mass obtained from aeroponically grown plants was comparable to that from other culture systems, and the plants were morphologically normal. Bradysia larvae caused considerable root damage resulting in reduced root biomass and water absorption. After feeding on the aeroponically grown root tissue, the larvae pupated and emerged as adults. Root damage of mature plants cultivated in aeroponic substrate was compared to that of Arabidopsis seedlings grown in potting mix. Seedlings were notably more susceptible to Bradysia feeding than mature plants and showed decreased overall growth and survival rates. Conclusions A root-herbivore system consisting of Arabidopsis thaliana and larvae of the opportunistic herbivore Bradysia spp. has been established that mimics herbivory in the rhizosphere. Bradysia infestation of Arabidopsis grown in this culture system significantly affects plant performance. The culture method will allow simple profiling and in vivo functional analysis of root defenses such as chemical defense metabolites that are released in response to belowground insect attack.
Analysis of T-DNA alleles of flavonoid biosynthesis genes in Arabidopsis ecotype Columbia
Bowerman, Peter A.; Ramirez, Melissa V.; Price, Michelle B.; Helm, Richard F.; Winkel, Brenda S. J. (2012-09-04)
BACKGROUND: The flavonoid pathway is a long-standing and important tool for plant genetics, biochemistry, and molecular biology. Numerous flavonoid mutants have been identified in Arabidopsis over the past several decades in a variety of ecotypes. Here we present an analysis of Arabidopsis lines of ecotype Columbia carrying T-DNA insertions in genes encoding enzymes of the central flavonoid pathway. We also provide a comprehensive summary of various mutant alleles for these structural genes that have been described in the literature to date in a wide variety of ecotypes. FINDINGS: The confirmed knockout lines present easily-scorable phenotypes due to altered pigmentation of the seed coat (or testa). Knockouts for seven alleles for six flavonoid biosynthetic genes were confirmed by PCR and characterized by UPLC for altered flavonol content. CONCLUSION: Seven mutant lines for six genes of the central flavonoid pathway were characterized in ecotype, Columbia. These lines represent a useful resource for integrating biochemical and physiological studies with genomic, transcriptomic, and proteomic data, much of which has been, and continues to be, generated in the Columbia background.
Another turn for p53
Tyson, John J. (Nature Publishing Group, 2006-01-01)
Attracting and Retaining Minorities in the Biomedical Sciences Including Genomics: a Community-based Approach
Smith, Edward J. (2014-08-21)
Bifurcation analysis of a model of the budding yeast cell cycle
Battogtokh, D.; Tyson, John J. (American Institute of Physics, 2004-09-01)
We study the bifurcations of a set of nine nonlinear ordinary differential equations that describe regulation of the cyclin-dependent kinase that triggers DNA synthesis and mitosis in the budding yeast, Saccharomyces cerevisiae. We show that Clb2-dependent kinase exhibits bistability (stable steady states of high or low kinase activity). The transition from low to high Clb2-dependent kinase activity is driven by transient activation of Cln2-dependent kinase, and the reverse transition is driven by transient activation of the Clb2 degradation machinery. We show that a four-variable model retains the main features of the nine-variable model. In a three-variable model exhibiting birhythmicity (two stable oscillatory states), we explore possible effects of extrinsic fluctuations on cell cycle progression. (C) 2004 American Institute of Physics.
A Bistable Switch Mechanism for Stem Cell Domain Nucleation in the Shoot Apical Meristem
Battogtokh, D.; Tyson, John J. (Frontiers, 2016-05-23)
Cell Cycle Control by a Minimal Cdk Network
Gerard, Claude; Tyson, John J.; Coudreuse, Damien; Novak, Bela (PLOS, 2015-02-01)
In present-day eukaryotes, the cell division cycle is controlled by a complex network of interacting proteins, including members of the cyclin and cyclin-dependent protein kinase (Cdk) families, and the Anaphase Promoting Complex (APC). Successful progression through the cell cycle depends on precise, temporally ordered regulation of the functions of these proteins. In light of this complexity, it is surprising that in fission yeast, a minimal Cdk network consisting of a single cyclin-Cdk fusion protein can control DNA synthesis and mitosis in a manner that is indistinguishable from wild type. To improve our understanding of the cell cycle regulatory network, we built and analysed a mathematical model of the molecular interactions controlling the G1/S and G2/M transitions in these minimal cells. The model accounts for all observed properties of yeast strains operating with the fusion protein. Importantly, coupling the model’s predictions with experimental analysis of alternative minimal cells, we uncover an explanation for the unexpected fact that elimination of inhibitory phosphorylation of Cdk is benign in these strains while it strongly affects normal cells. Furthermore, in the strain without inhibitory phosphorylation of the fusion protein, the distribution of cell size at division is unusually broad, an observation that is accounted for by stochastic simulations of the model. Our approach provides novel insights into the organization and quantitative regulation of wild type cell cycle progression. In particular, it leads us to propose a new mechanistic model for the phenomenon of mitotic catastrophe, relying on a combination of unregulated, multi-cyclin-dependent Cdk activities.
Cell cycle regulation by feed-forward loops coupling transcription and phosphorylation
Csikasz-Nagy, Attila; Kapuy, Orsolya; Toth, Attila; Pal, Csaba; Jensen, Lars Juhl; Uhlmann, Frank; Tyson, John J.; Novak, Bela (Nature Publishing Group, 2009-01-01)
The eukaryotic cell cycle requires precise temporal coordination of the activities of hundreds of ‘executor’ proteins (EPs) involved in cell growth and division. Cyclin-dependent protein kinases (Cdks) play central roles in regulating the production, activation, inactivation and destruction of these EPs. From genome-scale data sets of budding yeast, we identify 126 EPs that are regulated by Cdk1 both through direct phosphorylation of the EP and through phosphorylation of the transcription factors that control expression of the EP, so that each of these EPs is regulated by a feed-forward loop (FFL) from Cdk1. By mathematical modelling, we show that such FFLs can activate EPs at different phases of the cell cycle depending of the effective signs (+ or -) of the regulatory steps of the FFL.We provide several case studies of EPs that are controlled by FFLs exactly as our models predict. The signal-transduction properties of FFLs allow one (or a few) Cdk signal(s) to drive a host of cell cycle responses in correct temporal sequence.
Comparison of Domain Nucleation Mechanisms in a Minimal Model of Shoot Apical Meristem
Battogtokh, D.; Tyson, John J. (2016-04-20)
Existing mathematical models of the shoot apical meristem (SAM) explain nucleation and confinement of a stem cell domain by Turing's mechanism, assuming that the diffusion coefficients of the activator (WUSCHEL) and inhibitor (CLAVATA) are significantly different. As there is no evidence for this assumption of differential diffusivity, we recently proposed a new mechanism based on a bistable switch model of the SAM. Here we study the bistable-switch mechanism in detail, demonstrating that it can be understood as localized switches of WUSHEL activity in individual cells driven by a non-uniform field of a peptide hormone. By comparing domain formation by Turing and bistable-switch mechanisms on a cell network, we show that the latter does not require the assumptions needed by the former, which are not supported by biological evidences.
Concept inventory development reveals common student misconceptions about microbiology
Briggs, Amy G.; Hughes, Lee E.; Brennan, Robert E.; Buchner, John; Horak, Rachel E. A.; Katz Amburn, D. Sue; McDonald, Ann H.; Primm, Todd P.; Smith, Ann C.; Stevens, Ann M.; Yung, Sunny B.; Paustian, Timothy D. (American Society for Microbiology, 2017-10)
Correction: Mitotic-Chromosome-Based Physical Mapping of the Culex quinquefasciatus Genome.
Naumenko, Anastasia N.; Timoshevskiy, Vladimir A.; Kinney, Nicholas A.; Kokhanenko, Alina A.; deBruyn, Becky S.; Lovin, Diane D.; Stegniy, Vladimir N.; Severson, David W.; Sharakhov, Igor V.; Sharakhova, Maria V. (2015)
Correction
Development, validation and application of the Microbiology Concept Inventory
Paustian, T. D.; Briggs, A. G.; Brennan, R. E.; Boury, N.; Buchner, J.; Harris, S.; Horak, R. E. A.; Hughes, Lee E.; Katz Amburn, D. Sue; Massimelli, M. J.; McDonald, A. H.; Primm, T. P.; Smith, A. C.; Stevens, Ann M.; Yung, S. B. (American Society for Microbiology, 2017-10)
Diversity and Abundance of Ice Nucleating Strains of Pseudomonas syringae in a Freshwater Lake in Virginia, USA
Pietsch, Renee B.; Vinatzer, Boris A.; Schmale, David G. III (Frontiers, 2017-03-09)
Dynamic Modeling of the Interaction Between Autophagy and Apoptosis in Mammalian Cells
Tavassoly, I.; Parmar, J.; Shajahan-Haq, A. N.; Clarke, R.; Baumann, William T.; Tyson, John J. (2015-04)
Autophagy is a conserved biological stress response in mammalian cells that is responsible for clearing damaged proteins and organelles from the cytoplasm and recycling their contents via the lysosomal pathway. In cases of mild stress, autophagy acts as a survival mechanism, while in cases of severe stress cells may switch to programmed cell death. Understanding the decision process that moves a cell from autophagy to apoptosis is important since abnormal regulation of autophagy occurs in many diseases, including cancer. To integrate existing knowledge about this decision process into a rigorous, analytical framework, we built a mathematical model of cell fate decisions mediated by autophagy. Our dynamical model is consistent with existing quantitative measurements of autophagy and apoptosis in rat kidney proximal tubular cells responding to cisplatin-induced stress.
The dynamic response of the Arabidopsis root metabolome to auxin and ethylene is not predicted by changes in the transcriptome
Hildreth, Sherry B.; Foley, Evan E.; Muday, Gloria K.; Helm, Richard F.; Winkel, Brenda S. J. (Nature Research, 2020-01-20)
While the effects of phytohormones on plant gene expression have been well characterized, comparatively little is known about how hormones influence metabolite profiles. This study examined the effects of elevated auxin and ethylene on the metabolome of Arabidopsis roots using a high-resolution 24 h time course, conducted in parallel to time-matched transcriptomic analyses. Mass spectrometry using orthogonal UPLC separation strategies (reversed phase and HILIC) in both positive and negative ionization modes was used to maximize identification of metabolites with altered levels. The findings show that the root metabolome responds rapidly to hormone stimulus and that compounds belonging to the same class of metabolites exhibit similar changes. The responses were dominated by changes in phenylpropanoid, glucosinolate, and fatty acid metabolism, although the nature and timing of the response was unique for each hormone. These alterations in the metabolome were not directly predicted by the corresponding transcriptome data, suggesting that post-transcriptional events such as changes in enzyme activity and/or transport processes drove the observed changes in the metabolome. These findings underscore the need to better understand the biochemical mechanisms underlying the temporal reconfiguration of plant metabolism, especially in relation to the hormone-metabolome interface and its subsequent physiological and morphological effects.
Dynamical Localization of DivL and PleC in the Asymmetric Division Cycle of Caulobacter crescentus: A Theoretical Investigation of Alternative Models
Subramanian, Kartik; Paul, Mark R.; Tyson, John J. (PLOS, 2015-07-01)
Cell-fate asymmetry in the predivisional cell of Caulobacter crescentus requires that the regulatory protein DivL localizes to the new pole of the cell where it up-regulates CckA kinase, resulting in a gradient of CtrA~P across the cell. In the preceding stage of the cell cycle (the “stalked” cell), DivL is localized uniformly along the cell membrane and maintained in an inactive form by DivK~P. It is unclear how DivL overcomes inhibition by DivK~P in the predivisional cell simply by changing its location to the new pole. It has been suggested that co-localization of DivL with PleC phosphatase at the new pole is essential to DivL’s activity there. However, there are contrasting views on whether the bifunctional enzyme, PleC, acts as a kinase or phosphatase at the new pole. To explore these ambiguities, we formulated a mathematical model of the spatiotemporal distributions of DivL, PleC and associated proteins (DivJ, DivK, CckA, and CtrA) during the asymmetric division cycle of a Caulobacter cell. By varying localization profiles of DivL and PleC in our model, we show how the physiologically observed spatial distributions of these proteins are essential for the transition from a stalked cell to a predivisional cell. Our simulations suggest that PleC is a kinase in predivisional cells, and that, by sequestering DivK~P, the kinase form of PleC enables DivL to be reactivated at the new pole. Hence, co-localization of PleC kinase and DivL is essential to establishing cellular asymmetry. Our simulations reproduce the experimentally observed spatial distribution and phosphorylation status of CtrA in wild-type and mutant cells. Based on the model, we explore novel combinations of mutant alleles, making predictions that can be tested experimentally.
Dynamical modeling of syncytial mitotic cycles in Drosophila embryos
Calzone, Laurence; Thieffry, Denis; Tyson, John J.; Novak, Bela (Nature Publishing Group, 2007-07-01)
Immediately following fertilization, the fruit fly embryo undergoes 13 rapid, synchronous, syncytial nuclear division cycles driven by maternal genes and proteins. During these mitotic cycles, there are barely detectable oscillations in the total level of B-type cyclins. In this paper, we propose a dynamical model for the molecular events underlying these early nuclear division cycles in Drosophila. The model distinguishes nuclear and cytoplasmic compartments of the embryo and permits exploration of a variety of rules for protein transport between the compartments. Numerical simulations reproduce the main features of wild-type mitotic cycles: patterns of protein accumulation and degradation, lengthening of later cycles, and arrest in interphase 14. The model is consistent with mutations that introduce subtle changes in the number of mitotic cycles before interphase arrest. Bifurcation analysis of the differential equations reveals the dependence of mitotic oscillations on cycle number, and how this dependence is altered by mutations. The model can be used to predict the phenotypes of novel mutations and effective ranges of the unmeasured rate constants and transport coefficients in the proposed mechanism.
Effect of Salmonella enterica serovar Typhimurium VNP20009 and VNP20009 with restored chemotaxis on 4T1 mouse mammary carcinoma progression
Coutermarsh-Ott, Sheryl; Broadway, Katherine M.; Scharf, Birgit E.; Allen, Irving C. (Impact Journals, 2017-05-16)
A variety of bacterial strains have been evaluated as bio-therapeutic and immunomodulatory agents to treat cancer. One such strain, Salmonella enterica serovar Typhimurium VNP20009, which is attenuated by a purine auxotrophic mutation and modified lipid A, is characterized in previous models as a safely administered, tumor colonizing agent. However, earlier work tended to use less aggressive cancer cell lines and immunocompromised animal models. Here, we investigated the safety and efficacy of VNP20009 in a highly malignant murine model of human breast cancer. Additionally, as VNP20009 has recently been found to have a defective chemotaxis system, we tested whether restoring chemotaxis would improve anti-cancer properties in this model system. Exposure to VNP20009 had no significant effect on primary mammary tumor size or pulmonary metastasis, and the tumor colonizing process appeared chemotaxis independent. Moreover, tumor-bearing mice exposed to Salmonella exhibited increased morbidity that was associated with significant liver disease. Our results suggest that VNP20009 may not be safe or efficacious when used in aggressive, metastatic breast cancer models utilizing immunocompetent animals.
Enhanced Mucosal Defense and Reduced Tumor Burden in Mice with the Compromised Negative Regulator IRAK-M.
Rothschild, Daniel E.; Zhang, Yao; Diao, Na; Lee, Christina K.; Chen, Keqiang; Caswell, Clayton C.; Slade, Daniel J.; Helm, Richard F.; LeRoith, Tanya; Li, Liwu; Allen, Irving C. (2016-12-03)
Aberrant inflammation is a hallmark of inflammatory bowel disease (IBD) and colorectal cancer. IRAK-M is a critical negative regulator of TLR signaling and overzealous inflammation. Here we utilize data from human studies and Irak-m(-/-) mice to elucidate the role of IRAK-M in the modulation of gastrointestinal immune system homeostasis. In human patients, IRAK-M expression is up-regulated during IBD and colorectal cancer. Further functional studies in mice revealed that Irak-m(-/-) animals are protected against colitis and colitis associated tumorigenesis. Mechanistically, our data revealed that the gastrointestinal immune system of Irak-m(-/-) mice is highly efficient at eliminating microbial translocation following epithelial barrier damage. This attenuation of pathogenesis is associated with expanded areas of gastrointestinal associated lymphoid tissue (GALT), increased neutrophil migration, and enhanced T-cell recruitment. Further evaluation of Irak-m(-/-) mice revealed a splice variant that robustly activates NF-κB signaling. Together, these data identify IRAK-M as a potential target for future therapeutic intervention.
Exogenous Auxin Elicits Changes in the Arabidopsis thaliana Root Proteome in a Time-Dependent Manner.
Slade, William O.; Ray, W. Keith; Hildreth, Sherry B.; Winkel, Brenda S. J.; Helm, Richard F. (MDPI, 2017-07-10)
Auxin is involved in many aspects of root development and physiology, including the formation of lateral roots. Improving our understanding of how the auxin response is mediated at the protein level over time can aid in developing a more complete molecular framework of the process. This study evaluates the effects of exogenous auxin treatment on the Arabidopsis root proteome after exposure of young seedlings to auxin for 8, 12, and 24 h, a timeframe permitting the initiation and full maturation of individual lateral roots. Root protein extracts were processed to peptides, fractionated using off-line strong-cation exchange, and analyzed using ultra-performance liquid chromatography and data independent acquisition-based mass spectrometry. Protein abundances were then tabulated using label-free techniques and evaluated for significant changes. Approximately 2000 proteins were identified during the time course experiment, with the number of differences between the treated and control roots increasing over the 24 h time period, with more proteins found at higher abundance with exposure to auxin than at reduced abundance. Although the proteins identified and changing in levels at each time point represented similar biological processes, each time point represented a distinct snapshot of the response. Auxin coordinately regulates many physiological events in roots and does so by influencing the accumulation and loss of distinct proteins in a time-dependent manner. Data are available via ProteomeXchange with the identifier PXD001400.

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