Participant Abstracts | AGEP SRI Mentors
Northeastern University (Boston, MA)
Bernadette Henares, Elizabeth Boon
Department of Chemistry
Nitric oxide and biofilm production in Shewanella woodyi
Bacterial biofilms are surface-associated bacterial communities. Biofilming bacteria are characterized by the excretion of exopolysaccharides (EPS) - high molecular weight sugars and other molecules. EPS protect the biofilm community from the environment, and concentrate nutrients, among other roles. Signals that regulate the development of biofilm communities are the focus of our research. Nitric oxide (NO), a well-known signaling molecule in eukaryotic species, can trigger biofilm formation. If NO is also a signaling molecule in bacteria, then there must be a bacterial NO sensor. We hypothesize that NO is sensed in bacteria by a member of the highly conserved family of Heme Nitric Oxide/OXygen Binding family (H-NOX) hemoproteins. In many bacteria, an H-NOX gene is in the same putative operon as a gene for diguanylate cyclase (DGC), which is known to play a role in biofilm production. We hypothesize that NO-bound H-NOX regulates the enzymatic activity of DGC, thus linking NO sensing to biofilm formation. In these studies we investigated the role of NO in biofim formation in the model species Shewanella woodyi.
Earlier work proved that H-NOX regulates the activity of DGC, but the effect of NO on biofilm formation in S. woodyi has not been determined. Here we grew biofilms in various NO concentrations and characterized the extent of biofilm formation, by staining for EPS. EPS formation can be quantified using two stains, crystal violet (CV) and congo red (CR). CV staining is used to quantify the amount of biofilm grown on a solid surface. CR is used to quantify the amount of EPS produced by cells in solution under biofilming conditions. Together, these assays give us a quantitative difference in biofilm production as a function of NO concentration.
The project also used fluorescence microscopy (FM) and confocal laser scanning microscopy (CLSM) to image the effect of NO on biofilm formation. For FM, we used Calcofluor, a dye which stains extracellular matrices. The intensity of the light is a direct indication of the amount of biofilm present. In CLSM, a high-resolution type of microscopy, we can see each individual bacterium after staining with propidium iodide. Using microscopy, we can visibly see the differences in biofilm formation between differing NO concentrations, giving us the qualitative difference in biofilm formation. By both assays and microscopy, we have concluded that NO does indeed increase the production of biofilm in S. woodyi.
Figure 1: Propidium iodide-stained S. woodyi with 50uM NO (R) and no NO (L), viewed under CLSM with a Rhodamine laser at 543nm with a magnification of 100. Regions over 5um are defined as biofilm.
Long Island University (Brooklyn, NY)
Antoine Dufour & Jian Cao
Division of Cancer Prevention
The Role of MT1-MMP in Ovarian Cancer: the "Pearl Necklace" Paradox
Epithelial ovarian cancer is the deadliest gynecological cancer in the Western world. A poor clinical prognosis has been linked to cancer metastasis within the peritoneal cavity and is characterized by a complex series of interrelated cellular events. Among the multitude of proteins orchestrating the neoplastic progression and metastatic cascade, matrix metalloproteinases (MMPs) have been demonstrated to play an important role in ovarian carcinogenesis. MMPs are proteases that remodel extracellular matrix proteins; a conserved zinc-binding region within their catalytic domain defines their activity. One subset of this family includes six membrane-associated MMPs (MT-MMPs). They have been demonstrated to impact biological processes by increasing cell migration, invasion, and metastasis. This study examines the role of MT1-MMP in a stably transfected ovarian cancer cell line, SK-OV-3.
Endogenous expression of MT1-MMP in transfected cells caused a unique phenotype in which rings of cells were observed in 2D and 3D cultures. MT1-MMP-transfected SK-OV-3 cells also showed increased migration in a Transwell Migration assay, and increased invasion in a 3D Type I collagen matrix. An increase in cell proliferation was also observed in MT1-MMP/GFP stable cells as compared to GFP-transfected and wild type cells. Using a deletion and mutational analysis approach, SK-OV-3 cells were transiently transfected with different chimeras of MT1-MMP domains, to identify domains involved in the enhancement of cell migration.
MT1-MMP also induces an Epithelial to Mesenchymal Transition (EMT). Changes in expression of E-cadherin, a cell-cell adhesion protein, indicate the phenotypic plasticity that occurs in ovarian cancer progression. The Snail transcription family is thought to repress E-cadherin expression, leading to EMT. EMT markers were analyzed by RT-PCR and it was demonstrated that the transcriptional factor Snail was upregulated in the MT1-MMP/GFP stable SK-OV-3 cells. Decreased expression in E-cadherin was demonstrated by RT-PCR, Western Blot and Immunohistochemistry. In conclusion, it was demonstrated that MT1-MMP increases migration, invasion, and proliferation of ovarian cancer cells and leads to an Epithelial to Mesenchymal Transition by repressing E-cadherin, thus promoting aggressiveness of these cells.
Figure 1. A stable SK-OV-3 cell line expressing a GFP and MT1-GFP chimera. Morphologic differences were observed between GFP and MT1-GFP under two-dimensional culture. Scale bar, 20 uM.
Karla S. Dixon
CUNY Brooklyn College (Brooklyn, NY)
Marjolijn Mertz, Lorna Role, David Talmage
Department of Pharmacology, Department of Neurobiology & Behavior; Center for Nervous System Disorders
Type III Neuregulin 1 and the development of cholinergic neurons in basal forebrain nuclei
The chemical compound acetylcholine (ACh) is a neurotransmitter in both the peripheral nervous system and central nervous system in many organisms including humans. In the brain, ACh signaling is important for attention, motivation and memory; disruption in ACh signaling has been reported in brain disorders as diverse as schizophrenia and Alzheimer's disease.
In the spinal cord and brain stem, Type III Neuregulin 1 (NRG 1) is necessary for the survival of acetylcholine-producing, or cholinergic, neurons. At present we do not know if NRG1 is required for the survival and/or function of cholinergic neurons in the forebrain. If it is, then a genotypic disruption of NRG1 should disrupt cholinergic neurons. Some anticipated disruptions include: a decrease in the number of cholinergic neurons, a decrease in the length of cholinergic neurons, and a change in the density of cholinergic neurons.
To address the role of NRG1 signaling in the proper development and function of forebrain cholinergic neurons, we have used a line of mice, the ChAT-eGFP; Nrg1tm1Lwr line in which green fluorescent protein is expressed in all ACh synthesizing neurons, and one copy of the Type III Neuregulin 1 gene is disrupted. A GFP antibody has been used to detect cholinergic neurons and their projections in brain sections. The cholinergic neurons are now being counted and the numbers will be compared to WT mice.
This project has implications in the pharmaceutical industry for treating schizophrenia. Schizophrenia is a mental disorder that distorts one's ability to think logically. Some symptoms of the disease include auditory hallucinations, disruption of sensory gating and flat affect. NRG 1 is a susceptibility factor in schizophrenia. Since NRG 1 is also known to code for ACh it is hoped that the schizophrenic NRG 1 malfunction is related to ACh.
CUNY Brooklyn College (Brooklyn, NY)
Shamoon Naseem, James Konopka
Department of Molecular Genetics and Microbiology
Identifying novel signal pathways activated by the sugar N-acetylglucosamine (GlcNAc)
GlcNAc (N-Acetylglucosamine) signaling contributes to several human diseases, including cancer. However, the full range of GlcNAc signaling activities is not well understood. We are using genetic approaches in fungi to identify novel signaling pathways that are likely to be conserved in humans. GlcNAc signaling has not been studied previously in fungi because the commonly studied yeasts, S. cerevisiae and S. pombe, lack the genes required to grow on GlcNAc and do not respond to this sugar. In contrast, most other fungi are able to catabolize GlcNAc: some members of the Candida genus are also induced by GlcNAc to switch from budding to a filamentous pattern of growth, often known as hyphal growth. The pathways by which fungi respond to GlcNAc are not known, therefore the haploid yeast Candida lusitaniae is being targeted to define two novel signal pathways; the pathway that induces the expression of the genes needed to catabolize GlcNAc and the pathway that induces morphological changes. The purpose of this project was to further explore GlcNAc catabolism, which is relevant to further exploration and understanding of the GlcNAc hyphal induction pathway.
Strains of Candida lusitaniae, a haploid species which changes to hyphal morphology when GlcNAc is introduced, were used as models in our small-scale pilot study. The data indicate that cells can be readily screened by replica-plating transformants onto GlcNAc medium to assess their growth and ability to switch to filamentous growth.
A targeted approach is also being developed to examine specific genes involved in GlcNAc breakdown by deletion mutagenesis. Transforming C. lusitaniae with special polymerase chain reaction (PCR) product mixtures having random insertional nucleotide bases at each end and the selectable marker URA3 results in transformants with a random gene knocked out. Afterwards, we plated the cells onto selective media to obtain mutant "knockouts". The essential goal was to plate the mutants onto plates with GlcNAc as the sole carbon source to screen for GlcNAc catabolism-related gene deletion mutants. Genetic sequencing techniques using the known sequence of the "knockout" PCR product would allow us to discover which gene was mutated and identify it as a new GlcNAc catabolic gene. The same technique is being applied to a species, Yarrowia lipolytica. Together, these methods will be used to identify novel GlcNAc signaling components in fungi that can be assessed for roles in human cancer.
Figure 1. Hyphal formation of Candida albicans, one of three morphologies in yeast, can be caused by catabolizing GlcNAc, leading to the GlcNAc hyphal induction pathway.
Stony Brook University (Stony Brook, NY)
Department of Pharmacology
Role of MTERF1 residue F322 in dsDNA base flipping
The mitochondrial genome encodes 13 proteins essential for ATP production. Regulation of mitochondrial gene expression depends on proteins that are encoded by the nuclear DNA and transported into the mitochondria. Proper regulation is critical to couple the synthesis of electron transport chain proteins with the energetic needs of the cell.
Defects in mitochondrial gene expression are associated with various myopathies and neurological disorders. By understanding the regulatory mechanisms controlling mitochondrial gene expression we can better understand the relationship between transcription regulation and mitochondrial disease.
MTERF (Mitochondrial Termination Factors) are a family of proteins involved in regulating transcription. There are four members of the family, one of which is MTERF1. The structure of MTERF1 has recently been solved (Yakubovskaya et al., 2010) and it has been found to bind dsDNA in such a way that it is able to stop transcription. When MTERF1 recognizes the transcription termination sequence, it binds to dsDNA and adopts a unique conformation in which it unwinds dsDNA and promotes base flipping. Base flipping is one of the essential events necessary for transcription termination by further stabilizing the MTERF1/DNA complex. Stable binding of MTERF1 to DNA effectively interferes with RNA polymerase elongation, ceasing transcription.
There are many protein residues that play a role in the behavior of MTERF1, but the specific function of each of these residues is unknown. We are focusing on phenylalanine 322 (F322) which may play a role in promoting DNA base flipping. We hypothesize that F322 in MTERF1 may act as a wedge to intercalate stacked bases of dsDNA and cause them to be flipped outward. In order to study the effect of F322 on base flipping, we mutated the phenylalanine at position 322 to alanine by site directed mutagenesis. We further purified and crystallized the MTERF1F322A protein and determined the structure of the mutant in complex with the termination sequence by x-ray crystallography. To determine if F322 plays a role in base flipping, we superimposed the structure of MTERF1F322A and wild type MTERF1 in order to compare the positions of the bases in dsDNA.
Figure 1. MTERF1F322A/DNA complex superimposed on MTERF1/DNA complex. Dull blue and green represent the position of dsDNA when WT MTERF1 binds to it while cyan and yellow represent the position of DNA when MTERF1F322A binds. Positions of the residues in the mutant protein are shown green while WT residues are shown in red and pink.
Reference: Yakubovskaya, E., Edison, M., Byrnes, J., Hambardjiva, E., and Garcia-Diaz, M. (2010). Helix Unwinding and Base Flipping Enable Human MTERF1 to Terminate Mitochondrial Transcription. Cell. 5.14: 1-12.
Ph.D. student in Biopsychology
Mentor to Karla Dixon
Ph.D. student in Molecular and Cellular Pharmacology
Pictured with mentee Jennifer DeLeon
Ph.D. Student in Ecology and Evolution
Mentor to Leah Norona
Natalie St. Fleur
Ph.D. student in Chemistry
Pictured with mentee Alexander Andia
Ph.D. student in Molecular Genetics and Microbiology
Pictured with mentee Diana Faustin