2008 Research Experiences for Undergraduates (REU) Participants-Department of Biological Sciences - Carnegie Mellon University

2008 Participants

National Science Foundation
Mentored, Cutting Edge Research Experiences in the Molecular Biosciences
Research Experiences for Undergraduates (REU)

Noel BrownNoël Brown, Muhlenberg College

Mentor: Dr. John Woolford

Pre-rRNA-Protein Interactions in Yeast Using a 3-Hybrid Assay of ITS2 RNA and Nip7 and Ssf1 Proteins

Ribosomes are the organelles in cells that synthesize proteins. They are made up of 4 RNA strands and 79 proteins present in two ribonucleoprotein particles, the 40S and the 60S subunits. The 40S subunit serves as a scaffold to bring messenger RNA and transfer RNA molecules together in order to decode the genetic code. The ribosomal RNA in the center of the 60S subunit serves as a “ribozyme” that catalyzes peptide bond formation. Approximately 180 different proteins are required to assemble rRNAs together with ribosomal proteins to make functional ribosomal subunits. Many of these assembly factors contain amino acid sequence motifs found in proteins that bind RNA. However, none of these potential RNA binding proteins involved in ribosome assembly in eukaryotes has been shown to bind specifically to pre-RNA in vivo. My study focused on one step of ribosome assembly, the endonucleolytic cleavage of the C2 site in the ITS2 sequence, in which the binding of proteins to pre-rRNA is thought to play a key role. Both the Nip7 and Ssf1 proteins are involved in this step in pre-rRNA processing and ribosome assembly. Nip7 is required for this step to occur; it recruits a complex of 5S rRNA and four proteins into the assembling ribosome. Ssf1 is suspected of postponing this cleavage step until just the correct point in the assembly pathway. In order to determine if Nip7 or Ssf1 bind to ITS2 of yeast pre-rRNA, I inserted the genes that code for these proteins into a plasmid, pACT II AD, which carries the LEU2 marker. Separately, I inserted the gene that encodes the ITS2 RNA sequence into a different plasmid, pIII a/MS2-2, which carries the MS2 sequence. Yeast transformed with these plasmids express the hybrid RNA or hybrid proteins under selective conditions. Due to repeated transformation attempts to obtain gap repair, instead of recircularization without the insert, my experiments stopped at this step. Because I used PCR, which can cause mutations, to obtain my insertable sequences, the next step is to electrotransform these plasmids into E.coli. From there, the DNA will be checked for mutations. If no mutations are present, the DNA can be shuttled back into yeast and used to perform a 3-hybrid assay as an in vivo assay for RNA-protein interactions.

Shannon Ellis

Shannon Ellis, King's College

Mentor: Dr. Jonathan Minden

Characterizing the Role of Heat Shock Protein 27 in Cell Death

Apoptosis, or programmed cell death, is the orderly removal of extraneous cells from an organism. This phenomenon eliminates unneeded cells and prevents the formation of tumors. Proteomic studies to identify additional Drosophila proteins involved in apoptosis revealed that an isoform of heat shock protein 27 (Hsp27) is upregulated with increased cell death. Furthermore, RNAi-mediated knockdown of Hsp27 virtually eliminates the naturally occurring epithelial cell death in Drosophila embryos. Hsp27 knockdown yields an additional phenotype, believed to be linked to altered levels of cell death, in which macrophages aberrantly escape into the space between the embryo and the intervitelline membrane that surrounds the embryo. This robust phenotype was used to screen potential loss-of-function (LOF) Hsp27 mutant alleles to obtain a null allele for future LOF analyses. Several mutant lines were identified that exhibited this macrophage phenotype. Hsp27 transcript levels of these mutant lines were examined by RT-PCR and qRT-PCR and showed a pronounced decrease or total absence of Hsp27 mRNA. In addition, two of these mutant lines demonstrating the macrophage phenotype also exhibit decreased levels of epidermal cell death as assayed by staining with the vital dye Acridine Orange. Further, to complement the LOF analyses, a misexpression paradigm is being employed to ask if overexpression of Hsp27 directly triggers cell death. Since overexpression of the master cell death regulators (hid, reaper, or grim) in the fly eye and wing reduces of these structures due to increased cell death, one would expect that if Hsp27 is capable of triggering cell death, it would produce phenotypes that are similar to hid, reaper, and grim overexpression. In summary, the above LOF analyses support the necessity of Hsp27 in cell death, while the ongoing GOF experiments are poised to provide additional insights into the mechanistic function of Hsp27 in this important biological process.

Amy Kerin

Amy Kerin, Washington & Jefferson College

Mentor: Dr. Veronica Hinman

Characterization of Cell Types in Neurogenic Domains of the Sea Star Embryo

Developmental gene regulatory networks (GRNs) are models of the interactions among transcription factors and regulatory DNA that describe how a fertilized egg develops into a complex embryo. An embryo has many unique territories that are defined by different cell types. These cell types are the output of different GRNs. The Hinman laboratory is working to determine the GRNs for two neuronal territories in the sea star, the apical plate and the ciliary bands. The apical plate and ciliary bands are found in many animals. The apical plate is located at the anterior end of the embryo, is usually associated with serotonergic neurons, and is thought to play a sensory function. The ciliary bands are found around the mouth and are used for feeding and locomotion. The goal of this project is to characterize cell types in these territories in the sea star by using immunohistochemistry and whole mount in situ hybridization (WMISH). Sea star embryos were cultured and fixed at two through five days using three different fixing methods. Immunostaining revealed serotonergic neurons in the apical plate and synaptotagmic positive cells in the ciliary bands. This is in accordance to previously published results. Acetylcholinergic and dopaminergic neurons are scattered throughout the front oral hood. WMISH was performed using probes for genes expressed in ciliated cells, which are known to be present in the apical plate of the sea urchins. These genes are located in the apical plate as well as the ciliary bands of the sea star embryo. Embryos will then be injected with morpholino antisense oligonucleotides to disrupt the function of a gene of interest. Immunostaining and WMISH will be performed on these injected embryos. These expression patterns will be compared with the ones done on normal embryos to establish how the disrupted gene has affected different types of cells. Ultimately, these experiments as well as future ones will help determine the GRNs for the apical plate and ciliary bands of the sea star embryo.

Olivia Molinar

Olivia Molinar, University of Texas at El Paso

Mentor: Dr. Brooke McCartney

Dissecting the Molecular Pathway Connecting Wnt Signaling to Morphological Changes in Drosophila

The colon cancer tumor suppressor Adenomatous polyposis coli (APC) is a multifunctional protein that plays a role in cytoskeletal organization and acts as a negative regulator of the Wnt signaling pathway. In the Wnt pathway, APC functions in the destruction complex, a multiprotein complex that targets Armadillo (Arm) for proteolytic destruction. When cells receive Wnt signals, the destruction complex is inhibited and the levels of Arm rise. As a consequence of Arm accumulation in the cytoplasm, the protein enters the nucleus where it acts in a transcription factor complex to activate Wnt target genes. Wnt target genes are known to influence cell fate decisions during development, but much less is known about the role of Wnt targets in morphogenesis. Our laboratory has shown that when APC is removed from patches (clones) of the Drosophila wing imaginal epithelium, Wnt signaling is inappropriately activated, resulting in improper apical constriction and basal extrusion of mutant tissue. The main goal of this project is to dissect the molecular pathway connecting Wnt signaling to morphological changes. We predicted that reduction of the Wnt effector Arm in APC mutant clones would suppress the Wnt induced morphological changes. However, we found that reduction of arm did not result in gross suppression of either the larval or adult phenotypes. Currently, we are analyzing both larval and adult tissues to determine if subtle suppression occurred. Lack of strong suppression suggests that a dose reduction of arm is insufficient to significantly reduce signaling through the pathway. The next step is to determine if suppression of the aberrant morphology will occur by completely removing the Wnt effector Arm from the APC mutant tissue. Proof of this kind of suppression will indicate that other components of the pathway connecting Wnt signaling to morphogenesis can be identified as suppressors of the morphological changes in APC mutant clones.

John Oxford

John Oxford, Georgia Southern University

Mentor: Dr. Jonathan Jarvik

Development of a Fluorogen Activating Protein HRV-3C Protease Biosensor

Single Chain Fragment Variable (ScFv) antibodies have been designed to be displayed on the surface of yeast and mammalian cells, bind to varying antibodies, and then visualized via flow confocal fluorescence microscopy. A method of testing protease activity on cellular membranes with ScFvs could serve as a model for developing anti-viral drugs that inhibit protease activity. The fluorogen activating protein (FAP) used in our construct is malachite green (MG) 13-16, which consists of a heavy chain and a light chain. When the heavy chain is positioned directly next to the light chain, the MG fluorgen binding site is blocked, thus fluorogen may only bind when these chains are physically separated. The overall goal of this project is to replicate this previous design and test HRV-3C on fibroblast NIH-3T3 cells. This was accomplished by first designing a plasmid DNA vector that consisted of the MG13-16 gene whose protein is expressed on the cell membrane's outer surface, a transmembrane (TM) gene that spans the membrane, and finally a green fluorescing protein (GFP) gene that serves as a known marker of tagged proteins which will be located on membrane's inner surface. The site between the heavy and light chains was then cleaved with the restriction enzyme BamHI which allowed for the insertion of a HRV-3C oligonucleotide that codes for the protease cleavage site. Once this was accomplished, the construct was transfected into NIH-3T3 cells. Four 6-well plates were filled with serum-free media where two plates contained the vector with the normal HRV-3C sequence and two plates contained the sequence in reverse. We hypothesized that the cells containing the forward sequence would have their FAP cleaved which would allow MG fluorogen to bind, while the cells with the reverse sequence would not be cleaved and therefore would not allow fluorogen to bind. Fluorescence was visualized via confocal fluorescence microscopy. Our data for the forward sequence indicated that, in the presence of the protease, the FAP was cleaved and fluorogen was allowed to bind. When no protease was added, no fluorogen binding was observed except for an expected 5% that is always observed. However, it appeared that the fluorogen could bind to the FAP when the HRV-3C site was in reverse, with and without the presence of the protease. Still, this model will hopefully be used further studies with membrane protein biosensors.

Alexis Peterson

Alexis Peterson, St. John's University

Mentor: Dr. Alison Barth

Mapping Sensory Representations by fos-Green Floursecent Protein and junB during the Stimulation of Selected Whiskers of Freely Moving Mice

Immediate early genes (IEGs), such as c-fos and junB, are a class of genes that are rapidly synthesized in neurons following neuronal stimulation. In this study IEGs served as activity markers that detected change in frequency, duration, and amplitude of somatosensory (whisker) stimulation. The aim of this study was to determine whether neuronal ensembles activated by whisker stimulation are consistent over time and within same time frames. Typically, both junB and c-fos proteins will peak 30 to 60 minutes after neuronal activation. However, in a fos-GFP transgenic mouse, where GFP is driven by activation of the fos promoter, there is a relatively long (2 to 4 hour) time lag between neuronal stimulation and GFP fluorescence. The time difference between peak junB expression and GFP fluorescence enabled us to use these two proteins as distinct temporal markers for the activation of specific neuronal ensembles. These experiments determined the relative overlap of neocortical neurons activated by two stimulus trials separated in time, in which the two IEGs, fos-GFP and junB, each had protein expression that varied in representation. The stimulus was whisker displacement that was both repetitive and variable in orientation and generated by metal shavings glued to multiple whiskers on the facial whiskerpad. This project offered an insight into the stability of cellular representations of sensory input in the barrel cortex of freely moving mice. This study will add to current knowledge in the field of neuroscience because this magnetic device allowed for the controlled stimulus delivery to drive fos-GFP and junB expression to be monitored across time.

Alex Ritter

Alex Taylor Ritter, Concordia College

Mentor: Dr. Adam Linstedt

SNARE-Mediated Homotypic Fusion in Golgi Ribbon Formation

The ribbon-like structure of the Golgi apparatus is thought to be the end result of homotypic fusion of cisternal membranes of ministacks, which are pre-Golgi intermediate structures. Organization of ministack compartments for fusion is facilitated in part by the protein GRASP65 that links homotypic compartments of the ministacks and brings them into close contact. This process is called tethering. However, the Grasp65 protein alone is not sufficient to facilitate membrane fusion. Once the membranous compartments of the ministacks are in close proximity, a different set of proteins is believed to fuse the membranes into one continuous, ribbon-like structure. The identity of these proteins is currently unknown. SNARE proteins mediate membrane fusion in eukaryotic cells. A number of different SNARE proteins have been identified in Golgi membranes including Syntaxin 5, hBet1, Sec22, and Membrin. We believe that these four SNARE proteins along with GRASP 65 are sufficient to drive homotypic membrane fusion, possibly bringing about the fusion step that is required for Golgi ribbon formation. To test this hypothesis, I utilized a non-Golgi membranous structure to model the activity of the proteins. Mitochondrial outer membranes provide an accessible model membrane to use in this case. I constructed plasmids that target the suspected Golgi SNAREs to the mitochondrial outer membrane. I then expressed these plasmids in HeLa cells along with GRASP 65 and assayed for membrane fusion using Fluorescence Recovery After Photobleaching (FRAP).  As a result of my efforts, we are well on our way to determining the role of Golgi SNAREs in membrane fusion.

Hillary St. John

Hillary St. John, St. Lawrence University

Mentor: Dr. Bruce Armitage

Fluorescence Labeling of Proteins with DNA Nanotags

Understanding the function and location of biomolecules is vital to the study of complex systems. One way to detect and track these molecules is to attach fluorescent labels. The purpose of this project was to use DNA as a scaffold for both covalently and non-covalently bound fluorophores to create DNA nanotags and apply them by labeling antibodies. In the non-covalently bound system, the DNA nanotag was attached to an antibody using chemical cross-linking methods. Specifically, a heterobifunctional cross-linker combined with both the thiol modified DNA and the amino group on the antibody. Addition of free cyanine dye to the nanotag solution led to the intercalation of the fluorophore into the duplex DNA and subsequently, fluorescence of the nanotag. Fluorescence spectroscopy analysis of polystyrene beads attached to nanotag labeled antibodies was used to show successful attachment of the DNA to the antibody. The covalently bound system consisted of a DNA duplex with a single covalently attached fluorophore. Fluorescence microscopy was used to compare the two nanotag systems. Yeast cells were tagged with either the covalent or non-covalent labeling system. In living yeast cells the two systems were effective and comparable to each other as well as to commercially available antibodies with attached fluorophores. However, in the non-covalent system, free dye was able to penetrate dead cells and produced intense fluorescence. Although both DNA nanotags proved to be effective labels, the covalent system is better suited for complex biological systems.

Dennis Villegas

Dennis Villegas, New Jersey Institute of Technology

Mentor: Dr. Marcel Bruchez

Development of a Model FRET Based Protein Kinase C Biosensor

Molecular biosensors have the potential to further our understanding of the complex signal transduction pathways involved in our cells. Of particular significance are the protein kinases and phosphatases, a family of enzymes that add or remove phosphate groups to other target proteins, which are thought to play a significant role in the many pathways of cell communication.  In order to build a universal biosensor that could detect the addition or removal of a phosphate group, synthesis of a polypeptide chain is required with specificity towards a particular enzyme. This polypeptide backbone will not only contain amino acids, but will also include either terpyridine or hydroxyquinoline, which will serve as ligands. To quantify the effects of protein kinase C on our biosensor, we will examine the Fluorescence Resonance Energy Transfer (FRET), where the energy from the dye attached to the amino terminus of the peptide (Cy3) is transferred to another dye (Cy5) attached to the other end of the polypeptide, which will be made possible by the proximity of the two dyes in the synthesized strand due to the beta-hairpin conformation that the strand adopts in the presence of a divalent cation, such as zinc, a ligand, and a phosphate group. Successful laboratory experiments could ultimately lead to using this biosensor in vivo. By building a biosensor that could detect the phosphorylation or dephosphorylation effects of enzymes such as protein kinase c, we could gain a much deeper understanding of the complex signal transduction pathway in cells.

Saintedym Wills

Saintedym Wills, Binghamton University

Mentor: Dr. Mark Macbeth

Construction and Analysis of D392N Mutation in hADAR2 and its Catalytic Domain

RNA editing by adenosine deaminases that act on RNA (ADARs) convert adenosine to inosine in double-stranded RNA.  ADARs are crucial for proper neuronal function since several RNAs encoding neuronal proteins are edited. The crystal structure of the catalytic domain of human ADAR2 reveals a zinc ion in the active site that suggests how the deamination reaction is catalyzed. The Macbeth lab has identified a molecule, inositol hexakisphosphate, buried within the catalytic domain that is essential for ADAR activity. Two phosphate groups of IP6 interact with amino acid residues including K519, D392 and K483 to create a salt bridge with the zinc ion. A site-directed D392N mutation was constructed in hADAR2 and the catalytic domain truncation. This conservative mutation of a negatively charged aspartic acid to neutral but isometric asparagine may reveal the essential nature of the salt bridge network between IP6 and the active site. The mutant genetic material was transformed into Saccharomyces cerevisiae, which like other eukaryotes contains IP6. Yeast cells harboring the full length ADAR mutant and catalytic domain mutant genes showed protein expression after induction with galactose. After isolation of the mutant protein, editing activity will be compared to the wild type ADAR.  The structure of the mutant catalytic domain will be determined by x-ray crystallography to assess the effect of this mutation on the protein structure.

Please send inquiries about our Research Experiences for Undergraduates program to bio-reu@andrew.cmu.edu.