2008 Summer Scholar Participants-HHMI Undergraduate Program - Carnegie Mellon University

2008 HHMI Summer Scholar Participants

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Danielle Eytan, Carnegie Mellon University
Mentor: Dr. Chien Ho

19F MRI Detection of Acute Cardiac Allograft Rejection Using In SituPerfluorocarbon Labeling of Immune Cells

Organ transplantation is the preferred treatment of end-stage organ failure, however the transplant recipient faces a lifetime of immunosuppressive therapy and the threat of losing the organ due to immunological rejection.  The current gold standard for diagnosing cardiac rejection following transplantation is to obtain a myocardial biopsy sample to detect the infiltration of immune cells to the graft.  Biopsy is not only an invasive procedure, but it is also prone to sampling errors. Therefore, a more sensitive and non-invasive technique to monitor organ rejection and provide personalized patient care is needed.  We are developing MRI techniques using a heterotopic cardiac transplantation model in rats to non-invasively detect and stage organ rejection.  One approach is to label immune cells with MRI contrast agents and detect the accumulation of labeled cells by in vivo MRI.  In this study we are evaluating a novel formulation of a fluorine-based cellular contrast agent.  Transplant recipients are given a direct i.v. injection of the fluorine contrast agent.  Immune cells, such as macrophages, take up the contrast agent in situ and the accumulation of labeled cells can be detected in vivo by 19F MRI.  A conventional 1HMR image provides anatomical context.  The results show that this technique is sensitive to the labeled cells, and histopathology confirmed that the particles had been taken up by macrophages infiltrating the sites of rejection. Correlating the 19F signal with the number of immune cells at the site of rejection may provide a sensitive method for detecting and staging rejection, and potentially may lead to clinical application for personalizing immunosuppressive therapy following cardiac transplantation.

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Justine Harkness, Carnegie Mellon University
Mentor: Dr. Peter Berget

Evaluation of a Disulfide Bond Free scFv as an Intracellular Biosensor Platform

Single chain variable fragments (scFvs) are two-domain proteins created from the variable regions of heavy and light chains of human antibodies. Each scFv domain has one disulfide bond that covalently links two cysteine residues, holding the scFv in its native confirmation.  Single domain scFvs are of particular interest since they are small and can be used as biosensor platforms. MG1 is a single domain heavy chain that binds malachite green (MG) and causes it to fluoresce. Like other scFvs, the wild type MG1 (MG1WT) has two cysteine residues that form a disulfide bond. It was hypothesized that the reducing environment of the cytoplasm would prevent the formation of this disulfide bond in the cytoplasm, thus limiting its usefulness as an intracellular biosensor. A disulfide bond free MG1 would be a viable alternative. A disulfide bond free mutant was created and then affinity matured to regain dye-binding activity (MG1AFM). To test if MG1AFM was more active in the cytoplasm than MG1WT, the genes for MG1 and Venus, a Green Fluorescent Protein derivative, were cloned into a vector that expresses these gene segments as a synthetically constructed trans-membrane fusion protein. Four constructs were made, two which express MG1WT or MG1AFM on the extracellular side, and two which expressed MG1WT or MG1AFM on the cytoplasmic side.  Venus was always expressed on the opposite side of the membrane from the MG proteins and served as a marker of protein expression (a control for the presence of MG1 protein.)  NIH 3T3 cells (mouse fibroblasts) were transduced with retroviruses containing these synthetic gene constructs.  Cells resulting from these infections were analyzed using two different approaches: flow cytometry and fluorescence microscopy. Since MG1 and Venus are expressed as a single protein, a 1:1 ratio of different colored fluorescence was expected.  In flow cytometry this would be indicated by a linear relationship between the intensity of Venus fluorescence vs. MG dye fluorescence.  This pattern was observed in all cases except for Venus-MG1WT, when the MGWT protein was expressed in the cytoplasm. To visually confirm these findings, fluorescence microscopy was performed on each of the cell lines using both a cell permeable and cell impermeable MG dye. Venus signal was present in all cell lines. MG signal was absent in the Venus-MG1WT when the MG1WT protein was located on the cytoplasmic side of the membrane.  However MG signal was present in the Venus-MG1AFM cells when the MG1AFM protein was located on the cytoplasmic side of the membrane.  MG signal was present when the MG1 proteins were expressed on the extracellular side of the membrane.  These findings support the hypothesis that a disulfide free MG1 will remain properly folded in the cytoplasm where as MG1WT is inactive.

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Samuel Kim, Carnegie Mellon University
Mentor: Dr. Gordon Rule

Isolation and Purification of MG1.5 Single Chain Variable Fragment for Structural Determination by Nuclear Magnetic Resonance (NMR) Spectroscopy

Biosensors are used to quantify intracellular reactions and identify their locations. The MG1.5 single chain variable fragment (scFv) is a biosensor platform called a Fluorogen Activating Protein (FAP). This protein binds to the fluorogenic dye, malachite green causing it to fluoresce. By determining the protein-dye structural interactions, we can modify the platform with new amino acid substitutions for increased specificity, sensitivity, and detection capabilities. To determine the structure using NMR spectroscopy, we need to generate a large amount of protein under conditions compatible with heavy isotope labeling. I am using an E.coli expression system to secrete MG1.5 directly into the periplasm space. The MG1.5 is secreted with a His6-tag, allowing for purification by a Ni+2-NTA column. The proton NMR spectrum showed that the scFv did not aggregate indicating a nitrogen spectrum could be taken for further structural information. I generated enough 15N-labeled MG1.5 in minimal media for preliminary NMR analysis this summer; the spectrum indicated the protein is a beta sheet protein that can bind malachite green dye. In the upcoming semester, I plan to label the protein with 13Carbon because carbon labeling provides much more structural information. The NMR analysis can be combined with future x-ray crystallography data for a comprehensive structural determination of MG1.5 and any derivatives made using this platform.

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Megan Martik, Carnegie Mellon University
Mentor: Dr. Veronica Hinman

Mechanisms Involved in the Regulation of the Echinoderm Supra-Anal Territory

Gene regulatory network (GRN) models show the connections between regulatory genes and regulatory DNA. The process of the development of animal body plans is illustrated primarily by GRNs. They describe how the embryo is spatially divided into different domains during development. Each domain is fated to form a particular cell type or body part. Since GRNs describe development of body plans, divergences in the architecture of GRNs underlie evolution in the development of different domains in different animals. I have been studying the expression patterns of two genes, gbx and nk1 in the sea urchin, Strongylocentrotus purpuratus, and the sea star, Asterina miniata. The distinct territory within which they are expressed, I’ve named the “supra-anal territory” due to its location in a ring of ectoderm around the larval anus in both embryos. I have developed a protocol for double fluorescence in situ hybridization (DFISH) to be used in sea stars. This new DFISH procedure allows me to distinguish between overlaps in gene expression of a variety of other transcription factors with gbx and nk1 and whether the supra-anal territory derives from a purely ectodermal or endodermal domain. This summer, I have found that there is a distinct boundary in expression between AmNk1 and AmOtx, but there is an overlap in expression between AmNk1 and AmFoxJ1 as well as AmNk1 and AmFoxA which were previously thought to be distinct expression patterns. Also I am currently working with a morpholino anti-sense oligonucleotide (MASO) designed to block the translation of the nk1 transcript in the sea star. Micro-injection of this MASO will allow me to determine the phenotypic and gene expression differences observed when normal nk1 functionis perturbed, thereby determining the regulatory role that this gene has in sea star development. I am especially interested to see if nk1, gbx, and otx genes are cross-regulated as they are known to be in vertebrate embryos and also because of the cogent DFISH results. This would demonstrate that the GRN subcircuit involving these genes has been conserved for 100s of millions of years.

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Henry Nguyen, Carnegie Mellon University
Mentor: Dr. Subha Das

Synthesis of a Nucleotide Analogue for RNA Structural Analysis

New paradigms for the roles of RNA are emerging and it is clear that RNA plays a much larger role than previously identified. These discoveries are fueled through greater understandings of RNA structure and function relationships. We aim to understand better the contributions of specific structural features, such as the 2'-hydroxl group, to RNA function through atomic substitutions to RNA. We aim to test the hypothesis that RNA containing a 2'-deoxy-2'-chloro (2'-Cl) analogue is unable to be cleaved by self-cleaving ribozymes, yet still retains the inherent conformational flexibility of the natural ribo-sugar phosphate backbone of RNA. Starting with uridine (U), we synthesized in four steps the protected form of 2'-Cl-U required for solid phase RNA synthesis. The stem and loop region of the U1-RNA sequence will be synthesized for co-crystallization with the U1A protein. The U1A protein was obtained by fusion to a (His)6-GST tag containing a TEV cleavage site for purification. It was expressed in E.coli and purified by Ni+2 chromatography, followed by cleavage of the fusion protein using TEV. In future work, the synthetic U1-RNA containing 2'-Cl-U will be co-crystallized with the U1A protein and the structure solved for comparison with the known structures of the natural U1-RNA and protein co-complex. This will determine how well the 2'-Cl substituents mimic the natural ribo 2'-OH residues in U1-RNA. This may also help to determine what structural features of the RNA backbone conformation are important for RNA-protein recognition.

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Rohit Ramnath, Carnegie Mellon University
Mentor: Dr. Nathan Urban

The Development of Connectivity in the Mouse Accessory Olfactory Bulb

The accessory olfactory bulb is responsible for the recognition and discrimination of non-volatile compounds and small molecules, such as pheromones. Sensory neurons from the vomeronasal organ (VNO) project to the accessory olfactory bulb (AOB), where the axons from these neurons form condensed structures of neuropil called glomeruli. Synapses between the axons of sensory neurons from the VNO and the dendrites of the principal neurons of the AOB, the mitral cells, occur within the glomeruli. Thus, the glomeruli are important synaptic sites for communication between the sensory periphery and the brain. Changes in structure of the glomeruli and the connectivity between neurons of the VNO and mitral cells throughout development can be used as a model to study the morphological changes that occur in neurons in the developing brain. We have studied the development of glomeruli in the AOB in genetically modified mice which express the green fluorescent protein (GFP) in cells that express a specific vomeronasal receptor type, the V2R1b receptor. Our data shows that the glomeruli appear to undergo axonal coalescence between postnatal-day 0 (p0) and postnatal-day 4 (p4). The structures begin as diffuse axonal projections, which condense to form structures resembling highly structured, stereotypical adult glomeruli. The AOB itself increases in volume between p0 and p30, while the volume of the glomerular layer relative to the AOB remains constant. The changes we have observed, point to the first few post-natal days as critical for the emergence and development of glomeruli in the AOB. Future experiments will examine both mitral cell morphology and synapse formation during this critical period of development in this system.

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Jonathan Sitrin, Carnegie Mellon University
Mentor: Dr. Jonathan Minden

Toward Understanding the Ventral-Specific Changes of Proteasome Subunit Pros35 and its Role in DrosophilaVentral Furrow Formation

Drosophila gastrulation involves four stages of morphogenetic development, beginning with ventral furrow formation (VFF). VFF leads to the formation of the mesoderm tissue layer, producing muscles and the immune system and when improperly developed, in humans, can lead to spina bifida. A 2D-DIGE (two dimensional difference gel electrophoresis) proteomics analysis of ventralized versus lateralized embryos revealed a difference in three proteasome subunits (Pros35, Pros25 and CG17331), implicating the proteasome in the developmental pathway determining ventral cell fate. Mass spectrometry showed a seven amino acid cleavage at the C-terminus of Pros35, revealing a PDZ domain ligand sequence. We show that this cleavage event can be inhibited in vitro by two different protease inhibitors, illuminating the possibility of a signaling event involving two proteases. We propose a model in which the revealed PDZ domain ligand targets the proteasome to the apical surface of ventral cells, allowing the proteasome to degrade an unidentified substrate to promote VFF. Several candidate binding proteins containing PDZ domains are enriched on the apical surface of ventral cells during VFF. This model, however, has been confounded by the recent discovery that the cleavage event is masked or prevented in fresh embryos when using an identical assay to that previously used with frozen embryos. It is possible that the cleavage may be an artifact of the freezing process during embryo collection as opposed to a natural signaling event. However, embryo injection of the protease inhibitors causes defective VFF in 50% of the embryos, supporting our original model. These contradictory results lead us to proposing a different approach to show that the in vitro cleavage event during proteasome collection matches an in vivo event during VFF. This will be tested using a 2D-DIGE analysis comparing fresh ventralized and lateralized embryos. This experiment should reveal the state of Pros35 during VFF and support or disprove the model presented.

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Natalie Straight, Carnegie Mellon University
Mentor: Dr. Peter Berget

Development of Protein Kinase A (PKA) Biosensor Based on scFv-based Fluorogen Activating Proteins (FAPs)

The single-chain variable fragment (scFv) MG1.5 binds malachite green dye causing it to fluoresce upon binding. It was hypothesized that this protein could be used as a Protein Kinase A (PKA) activation biosensor. Previous work inserted amino acid sequences into MG1.5 that carry a serine residue that is phosphorylated by PKA. This prototype PKA biosensor causes brighter fluorescence of bound malachite green dye after PKA phosphorylation compared to samples not treated with PKA. Currently, thirteen additional potential MG1.5 PKA-phosphorylation biosensor constructs have been created with the amino acid sequence insertion LLRRASLGP. Over the course of the summer, twelve analogues to these constructs were created. However, they differ by one amino acid (replacing the serine residue with aspartate). These are known as “phosphomimic” sequences. The phosphomimic amino acid sequences were inserted by cutting the thirteen plasmids open at unique PmeI restriction enzyme sites and then ligating in the DNA coding for the phosphomimic sequence. Colony PCR and sequence analysis were used to screen for cells with the proper insertion. These plasmids were then transformed into Saccharomyces cerevisiae and the modified FAPs were displayed on the surface of these yeast cells. Flow cytometric analysis was utilized to compare cells displaying serine and aspartate MG1.5 inserts. Current analysis shows potential for one or more of the constructs to be used as a PKA biosensor.