Johnstone IL (1999) "Molecular biology."

Johnstone IL

[1999]

Johnston WL et al. (2012) Genesis "The eggshell in the C. elegans oocyte-to-embryo transition."

Dennis JW, Johnston WL

[Genesis, 2012]

In egg-laying animals, embryonic development takes place within the highly specialized environment provided by the eggshell and its underlying extracellular matrix. Far from being simply a passive physical support, the eggshell is a key player in many early developmental events. Herein, we review current understanding of eggshell structure, biosynthesis, and function in zygotic development of the nematode, C. elegans. Beginning at sperm contact or entry, eggshell layers are produced sequentially. The earlier outer layers are required for secretion or organization of inner layers, and layers differ in composition and function. Developmental events that depend on the eggshell include polyspermy barrier generation, high fidelity meiotic chromosome segregation, osmotic barrier synthesis, polar body extrusion, anterior-posterior polarization, and organization of membrane and cortical proteins. The C. elegans eggshell is proving to be an excellent, tractable system to study the molecular cues of the extracellular matrix that instruct cell polarity and early development.

Johnston RJ et al. (2010) Cell "Preview. A penetrating look at stochasticity in development."

Johnston RJ, Desplan C

[Cell, 2010]

In recent work published in Nature, Raj et al. (2010) use single mRNA molecule quantification to show that variation in gene expression in Caenorhabditis elegans increases in mutants displaying incomplete penetrance. They find that a bimodal response is triggered when noisy expression of an upstream regulator crosses a critical threshold.

Johnston AD et al. (2012) J Toxicol "The Redox System in C. elegans, a Phylogenetic Approach."

Johnston AD, Ebert PR

[J Toxicol, 2012]

Oxidative stress is a toxic state caused by an imbalance between the production and elimination of reactive oxygen species (ROS). ROS cause oxidative damage to cellular components such as proteins, lipids, and nucleic acids. While the role of ROS in cellular damage is frequently all that is noted, ROS are also important in redox signalling. The "Redox Hypothesis" has been proposed to emphasize a dual role of ROS. This hypothesis suggests that the primary effect of changes to the redox state is modified cellular signalling rather than simply oxidative damage. In extreme cases, alteration of redox signalling can contribute to the toxicity of ROS, as well as to ageing and age-related diseases. The nematode species Caenorhabditis elegans provides an excellent model for the study of oxidative stress and redox signalling in animals. We use protein sequences from central redox systems in Homo sapiens, Drosophila melanogaster, and Saccharomyces cerevisiae to query Genbank for homologous proteins in C. elegans. We then use maximum likelihood phylogenetic analysis to compare protein families between C. elegans and the other organisms to facilitate future research into the genetics of redox biology.

St Johnston D et al. (2010) Cell "Cell polarity in eggs and epithelia: parallels and diversity."

St Johnston D, Ahringer J

[Cell, 2010]

Cell polarity, the generation of cellular asymmetries, is necessary for diverse processes in animal cells, such as cell migration, asymmetric cell division, epithelial barrier function, and morphogenesis. Common mechanisms generate and transduce cell polarity in different cells, but cell type-specific processes are equally important. In this review, we highlight the similarities and differences between the polarity mechanisms in eggs and epithelia. We also highlight the prospects for future studies on how cortical polarity interfaces with other cellular processes, such as morphogenesis, exocytosis, and lipid signaling, and how defects in polarity contribute to tumor formation.

Johnston RJ et al. (2003) Nature "A microRNA controlling left/right neuronal asymmetry in Caenorhabditis elegans."

Hobert O, Johnston RJ

[Nature, 2003]

How left/right functional asymmetry is layered on top of an anatomically symmetrical nervous system is poorly understood. In the nematode Caenorhabditis elegans, two morphologically bilateral taste receptor neurons, ASE left (ASEL) and ASE right (ASER), display a left/right asymmetrical expression pattern of putative chemoreceptor genes that correlates with a diversification of chemosensory specificities(1,2). Here we show that a previously undefined microRNA termed lsy-6 controls this neuronal left/right asymmetry of chemosensory receptor expression. lsy-6 mutants that we retrieved from a genetic screen for defects in neuronal left/right asymmetry display a loss of the ASEL-specific chemoreceptor expression profile with a concomitant gain of the ASER-specific profile. Alsy-6 reporter gene construct is expressed in less than ten neurons including ASEL, but not ASER. lsy-6 exerts its effects on ASEL through repression of cog-1, an Nkx-type homeobox gene, which contains a lsy-6 complementary site in its 30 untranslated region and that has been shown to control ASE-specific chemoreceptor expression profiles(3). lsy-6 is the first microRNA to our knowledge with a role in neuronal patterning, providing new insights into left/right axis formation.

Wendy Johnston et al. (2008) Development & Evolution Meeting "Chitin and its Binding Partners in Oocyte and Early Embryo Development"

Wendy Johnston, James Dennis, Aldis Krizus

[Development & Evolution Meeting, 2008]

In C. elegans, one of the earliest events at fertilization is the secretion of a multilayered chitinous eggshell. Our lab has shown that the hexosamine pathway, chitin and the chitin-binding proteins, CEJ-1 and/or CPG-2, are developmentally essential at the one-cell stage for multiple actin-dependent events, including high fidelity meiosis, polar body extrusion and embryonic polarization. (Johnston et al., 2006, BMC Biology, 4:35), Consistent with these embryonic roles, chitin, CEJ-1::mCherry and CPG-2::Venus are localized to the eggshell and the extraembryonic matrix between the developing embryo and the eggshell. A third chitin-binding protein, H02I12.1, is also essential for embryonic development. However, distinct from chitin, CEJ-1:mCherry and CPG-2:Venus, H02I12.1::GFP is localized at or near the surface of developing oocytes. This localization suggests that in addition to, or instead of, an embryonic role, H02I12.1 in the extracellular matrix may be required for oocyte development. The proximal germline of H02I12.1(RNAi) hermaphrodites had an increased number of oocytes expressing phosphohistone H3. Furthermore, the number of oocytes in the proximal germline was increased and oocytes displayed significant physical overlapping. Additionally, embryos were often pinched in two, resulting in the production of anucleate cytoplasts. Together, these results suggest that H02I12.1 in the oocyte extracellular matrix inhibits oocyte maturation and may play a role in oocyte/sheath or oocyte/oocyte interaction. H02I12.1(RNAi) oocytes also failed to bind a number of GalNAc-recognising lectins, including SBA, MPA and GS-I, indicating that one or more transmembrane or extracellular matrix glycoconjugates are absent or defective. Our results show that the glycopolymer, chitin, and the chitin-binding proteins, CEJ-1, CPG-2 and H02I12.1, are required for multiple developmental events just preceding and just following fertilization. We are currently testing the hypothesis that chitin and its binding partners function as a scaffold that organizes specific transmembrane glycoproteins for intracellular signaling and polarity in the maturing oocyte and the newly fertilized embryo.

Wendy L. Johnston et al. (2007) International Worm Meeting "Chitin-binding domain proteins required for germline and embryonic development."

Wendy L. Johnston, Aldis Krizus, James W. Dennis

[International Worm Meeting, 2007]

The C. elegans genome encodes an 11-member family of non-chitinase, chitin binding domain-containing proteins (CBDs) with distinct expression profiles. Our lab is investigating the roles of chitin and CBDs in development. We have shown that chitin and two CBD family members, CEJ-1 and CPG-2, are required in the single-cell embryo for high fidelity segregation of meiotic chromosomes, for polar body extrusion and for polarization (Johnston et al., 2006). Interestingly, while CEJ-1 and CPG-2 are functionally redundant for embryonic development, localization of CEJ-1::mCherry and CPG-2::Venus is largely distinct. CEJ-1::mCherry has a punctate cortical localization in oocytes, and is non-punctate and gradually enriched in the eggshell. Conversely, CPG-2::Venus is detected in the zone between the eggshell and the embryo. These findings imply that meiotic chromosome segregation, polar body extrusion and polarization require a very small amount of CEJ-1 or CPG-2. We are also investigating the developmental role of another CBD family member, H02I12.1, which is embryonic lethal by RNAi. H02I12.1::GFP is expressed at boundaries between developing germline nuclei. Chitin is not detected in the germline, suggesting that H02I12.1 may bind to non-chitin glycoconjugates. H02I12.1(RNAi) embryos have chitin, but are fragile, and often pinch in two as they enter the uterus, suggesting somatic gonad contraction/dilation may be defective. Furthermore, the germline is also defective, with an extended zone of oocyte phosphohistone H3, and a Pro phenotype in which there is a zone of germline proliferation proximal to the region of gametogenesis. The Pro phenotype has previously been reported for glp-1 alleles with mutations in the extracellular domain, and for mutant alleles of genes involved in ribosome biogenesis in the somatic gonad. Taken together, these results suggest that H02I12.1 interacting with non-chitin glycoconjugates may modulate signaling between germ cells and the somatic gonad.

Johnstone IL et al. (2000) European Worm Meeting "Assembly of cuticle collagens"

Muriel JM, Johnstone IL, McMahon LM

[European Worm Meeting, 2000]

The cuticle of Caenorhabditis elegans is an extra-cellular matrix consisting predominantly of small collagens which are encoded by a dispearsed multi gene family of approximately 150 members. During cuticle synthesis, the collagen genes are expressed in a specific temporal series, the earliest being expressed approximately four hours prior to secretion and polymerisation of the matrix, the latest concurrent with secretion. We have shown that the correct timing of expression is important for assembly of individual cuticle collagens, and by epitiope tagging various collagen genes, we have demonstrated that different collagens are located in distinct cuticle sub-structures. We have shown that several "early" expressed collagens, including DPY-2, DPY-10 and DPY-7, are located in what appears to be a single cuticle sub-structure. As expected, mutations in the dpy-7 gene cause the absence of DPY-7 protein from the structure, as assayed by a specific mono-clonal antibody. However, mutations other early class collagen genes, including dpy-2 and dpy-10, which have similar dpy loss of function phenotypes, also cause the absence of DPY-7. Mutations in dpy-13, which is expressed later than this class, does not affect DPY-7 location. Our interpretation is that several early class collagens interact to form a single cuticle sub-structure and that absence (by mutation) of any one collagen causes absence of the entire structure. This is supported by the phenotypes of various double mutants. Finally, altering the timing of expression of dpy-7 to slightly later during cuticle synthesis than wild type, causes a disrupted location of the DPY-7 protein into the cuticle. A similar alteration of timing of dpy-2 or dpy-10 causes the same altered location of DPY-7. We suggest this proves interaction between these proteins.

Johnston T et al. (2018) Sci Rep "Propyl-5-hydroxy-3-methyl-1-phenyl-1H-pyrazole-4-carbodithioate (HMPC): a new bacteriostatic agent against methicillin-resistant Staphylococcus aureus."

Kwon B, Kelso MJ, Gilmore MS, Mylonakis E, Johnston T, Van Tyne D, Fawzi NL, Chen RF

[Sci Rep, 2018]

The emergence of Staphylococcus aureus strains resistant to 'last resort' antibiotics compels the development of new antimicrobials against this important human pathogen. We found that propyl 5-hydroxy-3-methyl-1-phenyl-1H-pyrazole-4-carbodithioate (HMPC) shows bacteriostatic activity against S. aureus (MIC=4ug/ml) and rescues Caenorhabditis elegans from S. aureus infection. Whole-genome sequencing of S. aureus mutants resistant to the compound, along with screening of a S. aureus promoter-lux reporter array, were used to explore possible mechanisms of action. All mutants resistant to HMPC acquired missense mutations at distinct codon positions in the global transcriptional regulator mgrA, followed by secondary mutations in the phosphatidylglycerol lysyltransferase fmtC/mprF. The S. aureus promoter-lux array treated with HMPC displayed a luminescence profile that was unique but showed similarity to DNA-damaging agents and/or DNA replication inhibitors. Overall, HMPC is a new anti-staphylococcal compound that appears to act via an unknown mechanism linked to the global transcriptional regulator MgrA.