Roh-Johnson M et al. (2012) Science "Triggering a cell shape change by exploiting preexisting actomyosin contractions."

McClellan JH, Werts AD, Goldstein B, Betzig E, Tulu US, Higgins CD, Kiehart DP, Roh-Johnson M, Gao L, Shemer G

[Science, 2012]

Apical constriction changes cell shapes, driving critical morphogenetic events, including gastrulation in diverse organisms and neural tube closure in vertebrates. Apical constriction is thought to be triggered by contraction of apical actomyosin networks. We found that apical actomyosin contractions began before cell shape changes in both Caenorhabitis elegans and Drosophila. In C. elegans, actomyosin networks were initially dynamic, contracting and generating cortical tension without substantial shrinking of apical surfaces. Apical cell-cell contact zones and actomyosin only later moved increasingly in concert, with no detectable change in actomyosin dynamics or cortical tension. Thus, apical constriction appears to be triggered not by a change in cortical tension, but by dynamic linking of apical cell-cell contact zones to an already contractile apical cortex.

Johnson, M. et al. (2017) International Worm Meeting "A model for assessing the impact of complex environmental exposures on neurodegeneration in Caenorhabditis elegans."

Miller, G., Johnson, M., Jones, D., Niedzwiecki, M., Walker, D., Kalia , V., Bradner, J.

[International Worm Meeting, 2017]

Neurodegeneration is a complex molecular process that involves a combination of genetic, environmental, and age-related risk factors. Little is known about how environmental exposures interact with genetic factors and aging to induce neurodegeneration. Here, we describe a platform to assess the impact of complex environmental exposures in Caenorhabditis elegans (C.elegans). Work from our laboratory and others has previously shown that disruption of the vesicular monoamine transporter 2 (VMAT2; SLC18A2) confers vulnerability to dopamine toxicity in mice and Parkinson's disease in humans. The mutant cat-1 lacks the worm ortholog to VMAT2. We evaluated the effect of cat-1 deletion on lifespan using the hands-free, high-throughput Lifespan Machine, as developed by Stroustrup et al. (2013, 2016). We found that cat-1 mutants have an altered lifespan as compared to wild-type controls. In order to study the impact of exposures to complex chemical mixtures, we needed to develop a high-throughput method to measure hundreds of exogenously applied chemical and subsequent metabolic products in the worms. Using three different high-resolution metabolomics platforms, the Agilent 6560 Ion Mobility Q-TOF LC/MS, the Thermo Q-Exactive HF LC/MS, and the Thermo Q Exactive GC hybrid quadrupole-Orbitrap GC-MS/MS, we are able to detect and quantify low levels of a wide variety of environmental chemicals (>350) representative of realistic exposures. These include polychlorinated biphenyls, brominated flame-retardants, organochlorine pesticides, phosphoester chemicals, herbicides, insecticides and pesticide synergists. Moreover, the combination of liquid chromatography (LC) and gas chromatography (GC) metabolomic platforms allows us additional measures of polar metabolites from environmental chemicals and endogenous metabolites (>5000). This combined analytical strategy thus provides a means to capture differential toxic effects of chemical metabolic products and the biological response to chemical exposures through metabolome-wide association study (MWAS). The high-resolution metabolomics platform can provide comprehensive coverage on as few as 100-1000 worms. The combination of genetically vulnerable mutants, the automated analysis of lifespan, and the high-resolution metabolomics analysis of endogenous and exogenous chemicals creates an ideal platform for studying gene-environment interactions in models of neurodegeneration and aging in C. elegans.

Roh-Johnson M et al. (2009) J Cell Sci "In vivo roles for Arp2/3 in cortical actin organization during C. elegans gastrulation."

Goldstein B, Roh-Johnson M

[J Cell Sci, 2009]

The Arp2/3 complex is important for morphogenesis in various developmental systems, but specific in vivo roles for this complex in cells that move during morphogenesis are not well understood. We have examined cellular roles for Arp2/3 in the Caenorhabditis elegans embryo. In C. elegans, the first morphogenetic movement, gastrulation, is initiated by the internalization of two endodermal precursor cells. These cells undergo a myosin-dependent apical constriction, pulling a ring of six neighboring cells into a gap left behind on the ventral surface of the embryo. In agreement with a previous report, we found that in Arp2/3-depleted C. elegans embryos, membrane blebs form and the endodermal precursor cells fail to fully internalize. We show that these cells are normal with respect to several key requirements for gastrulation: cell cycle timing, cell fate, apicobasal cell polarity and apical accumulation and activation of myosin-II. To further understand the function of Arp2/3 in gastrulation, we examined F-actin dynamics in wild-type embryos. We found that three of the six neighboring cells extend short, dynamic F-actin-rich processes at their apical borders with the internalizing cells. These processes failed to form in embryos that were depleted of Arp2/3 or the apical protein PAR-3. Our results identify an in vivo role for Arp2/3 in the formation of subcellular structures during morphogenesis. The results also suggest a new layer to the model of C. elegans gastrulation: in addition to apical constriction, internalization of the endoderm might involve dynamic Arp2/3-dependent F-actin-rich extensions on one side of a ring of cells.

Johnson, Lindsay M. et al. (2019) International Worm Meeting "Spontaneous mutational variation in metabolic network enzyme activity in C. elegans."

Smith, Olivia, Johnson, Lindsay M., Baer, Charles F., Hahn, Daniel

[International Worm Meeting, 2019]

Human metabolic diseases are becoming increasingly common and understanding the role genetic mutation plays in these diseases is an important first step in treatment. Large GWAS studies conducted on the human metabolome have shown that only a small fraction of the heritability of metabolic traits can be explained by cumulative mQTL. The remainder of the heritability indicates that variation is either due to a few, relatively new, highly deleterious variants, which is unlikely because they would be quickly removed by natural selection, or by many small variants that are not likely to be removed via natural selection. Regardless of how many genes are causing this heritability, it implies that spontaneous mutations are playing a role. Here we will employ C. elegans to analyze the relationship between the cumulative effects of spontaneous mutation on adenosine metabolism and on the function of the associated enzymes. The goals of this project are (1) to associate the effects of spontaneous mutations with variation in the metabolome and (2) quantify the covariance between the activity and concentration of metabolic enzymes in the adenosine pathway and metabolic phenotypes. We will also attempt to correlate mutational and network statistics for those metabolites in the adenosine pathway. Pilot data from four mutation accumulation (MA) lines and their common ancestor (N2) show significant variance for adenosine deaminase activity and concentration, adenosine kinase activity and concentration, as well as concentration of several metabolites in the adenosine pathway.

Johnson, Lindsay M. et al. (2017) International Worm Meeting "Network architecture and the cumulative effects of spontaneous mutations on the C. elegans metabolome."

Chandler, Luke, Johnson, Lindsay M., Baer, Charles F.

[International Worm Meeting, 2017]

A functional metabolic network is key for an organism's survival, and thus for its fitness. Spontaneous mutations reduce fitness, so it stands to reason they affect metabolism in some way. However, not all metabolites are equally important in maintaining organismal function, and the relationship between mutation and an organism's metabolic network has rarely been investigated. By investigating a set of mutation accumulation(MA) lines of Caenorhabditis elegans, we aim to better understand the effect mutations have on the metabolic network. Using a set of 43 MA lines and their ancestor, a pool of 29 metabolites was measured for changes in mean metabolite concentration (?M) and mutational variance (VM). We constructed a metabolic network map of these metabolites for C. elegans from the KEGG database, with the aim of characterizing the relationship between VM, ?M, and heritability found for these 29 metabolites and features of the metabolic network. Initial analysis shows a positive association between the largest k-core grouping of a metabolite and ?M and VM.

Casonya M Johnson (2001) International C. elegans Meeting "THERE'S A HUMP IN YOUR BACK!! RNA INTERFERENCE IN THE CLASSROOM"

Casonya M Johnson

[International C. elegans Meeting, 2001]

Undergraduate students often have difficulty understanding the relationship between nucleotides, genes, and the regulation of developmental processes. In an undergraduate sophomore level laboratory exercise, I used RNA interference assays to reinforce concepts in gene expression. Over four laboratory periods, students were able to (1) build a three-dimensional model of a eukaryotic gene, (2) use the BLAST and OMIM databases to identify genes in C. elegans with significant homology to selected human genes, (3) perform RNA interference assays by soaking, and (4) statistically evaluate the data obtained from RNAi.

Steven M Johnson et al. (2001) International C. elegans Meeting "Temporal Regulation of the let-7 stRNA"

Steven M Johnson, Shin-Yi Lin, Frank J Slack

[International C. elegans Meeting, 2001]

The heterochronic gene pathway in C. elegans controls the timing of developmental events. Within this pathway of at least ten genes, there are two genes, lin-4 and let-7 , which instead of coding for proteins encode short RNA products. lin-4 and let-7 RNAs accumulate during the first and fourth larval stages respectively and act as timing switches that initiate a cascade of developmental changes in the somatic cells between the first and second larval stages and the fourth larval and adult stages respectively. These are therefore referred to as small temporal RNAs (stRNA). The let-7 stRNA is also temporally regulated during development of other animals including Drosophila and zebrafish. Since in C. elegans , the appearance of the stRNAs correlates with temporal transitions in cell fates after molting, we can distill the timing of temporal transitions down to the timing of appearance of the stRNAs. We are therefore interested in understanding how let-7 is regulated. Since both lin-4 and let-7 are first transcribed as ~70 nt pre-RNAs that are processed to the mature 21-22 nt transcripts, there are multiple potential points where regulation could take place, i.e., processing, transcription, RNA stability. To test the simple hypothesis that the let-7 RNA is temporally regulated at the level of transcription, we fused the let-7 promoter to GFP and saw that the temporal expression of LET-7 recapitulates that observed in developmental Northern blots. Therefore let-7 appears to be temporally regulated at the transcriptional level. Preliminary studies with functional knockouts of ama-1 support the hypothesis that RNA polymerase II transcribes LET-7. Additionally, Northern blot analysis indicates that many of the heterochronic genes upstream of let-7 seem to play a role in the regulation of the let-7 stRNA product. Further studies are underway to confirm these initial findings and also to elucidate other cis- and trans -acting elements involved in this temporal and spatial regulation. This study may provide information pertinent to regulation of stRNAs in higher animals.

Casonya M Johnson (2005) International Worm Meeting "Demonstrating the interdisciplinary nature of biology to students entering high school"

Casonya M Johnson

[International Worm Meeting, 2005]

High school students and freshmen biology majors often have two things in common - a strong aversion to mathematics and the unshakeable belief that chemistry has nothing to do with biology. The goal of this project is to introduce students to the interdisciplinary nature of biology, and of science in general, before they form such negative associations with chemistry or mathematics. Toward this goal, through a competitive process, two 8th graders from Baltimore City private or public schools were selected for summer internship positions based on (1) strength of recommendations from 8th grade science, English and math teachers; (2) overall strength of math, foreign language, and science scores from 6th, 7th, and 8th grade; and (3) the ability to write a well-written paragraph that explains why they were interested in the program. We also selected two students that are currently in high school (9th, 10th or 11th grade) to participate in this program. These students will work in pairs under the direct supervision of two undergraduate students to determine whether selected C. elegans strains are homozygous or heterozygous for a recessive gene. This summer internship will run for eight weeks during the summer of 2005. At the end of the summer, each pair of students will prepare a poster presentation of their research activities. They will be asked to participate in the Annual High School Science Fairs hosted by Morgan State University and Towson State University Baltimore, Maryland. All four students will also be able to take a copy of their posters with them to their high school in the Fall of 2005. Here we report on the 8-week schedule for the internship program, samples of our introduction to mathematics and chemistry as it is used in the biological research laboratory, methods and protocols used, and our mid-project evaluation. We have also prepared more detailed information on our web site at http://jewel.morgan.edu/~cjohnso9. This work was supported by NSF grant number MCB0451745, which is a supplement to research grant number MCB0212336.

Steven M Johnson et al. (2005) International Worm Meeting "Roles for nucleosome position and structure in germline gene function"

Heather L McCullough, Steven M Johnson, Daniel P Riordan, Andrew Z Fire

[International Worm Meeting, 2005]

The nucleosome is the underlying feature of the chromosome and its positioning on eukaryotic DNA is a crucial element in transcriptional regulation. With striking genomic sequence phasing of putative positioning signals, C. elegans provide a very interesting system in which to study the global question of nucleosomal positioning. We are interested in following the in vivo positioning of nucleosomes in the very unusual chromatin environment of the C. elegans germline by addressing three questions: first, on a global level, how uniform are nucleosome positions on a given gene, second, to what degree is nucleosome position variable as a correlate of cell type and physiology, and third, can specific nucleosome positioning signals alter a gene's expression pattern? Our strategy is to use the high-throughput techniques of modified SAGE analysis and tiling arrays to answer these questions by effectively elucidating and defining the "nucleosome position-ome" in C. elegans. In a set of pilot experiments, we are optimizing the isolation of precise nucleosome core fragments of DNA in their physiological locations. Current efforts are underway to define the molecular nature and specifically the ends of the nucleosome core DNAs we isolate in order to precisely determine positions and optimize our procedure for the high-throughput techniques we will use to define the "nucleosome position-ome." We hope that analysis of the DNA sequences occupied by the nucleosomes in vivo on a genome-wide scale will reveal the underlying rules of nucleosome positioning and chromatin organization, which in turn could translate into the ability to understand and engineer finely and reproducibly regulated genetic systems.

Steven M Johnson et al. (2003) International Worm Meeting "Expression and functional analysis of mir-84, a let-7 microRNA family member"

Steven M Johnson, Kristy L Reinert, Frank J Slack

[International Worm Meeting, 2003]

The mir-84 gene encodes a microRNA with high homology to the stRNA let-7. Like let-7, mir-84 is temporally regulated. The similarity between these two paralogous genes led us to ask if this homology extends to their expression patterns and their functions.To address the question of spatial expression we fused 2.2 kb of genomic sequence immediately upstream of the coding sequence for the miR-84 product to gfp. Injection of this fusion construct into wild type worms resulted in temporally regulated gfp expression in cells that give rise to the vulva and uterus, but not in the hypodermal seam cells where let-7 RNA is thought to be expressed. In addition, developmental Northern blots reveal that mir-84 is expressed beginning in the L2 stage, one stage earlier than let-7. Our preliminary data suggest that the spatial and temporal expression patterns of mir-84 are not homologous to those of let-7.To address the question of homologous function we made transgenic worms harboring an overexpression construct containing 3.0 kb of genomic sequence spanning the mir-84 gene starting 2.2 kb upstream and ending 0.8 kb downstream of the coding sequence. These transgenic worms displayed phenotypes resulting in abnormal vulval and/or uterine morphologies that would seem to be distinct from the precocious heterochronic seam cell phenotype seen in overexpression of let-7. Nevertheless, preliminary data from our ongoing analysis suggest that there could be distinct heterochronic phenes associated with overexpression of miR-84.