2007 Summer Scholar Participants-HHMI Undergraduate Program - Carnegie Mellon University

2007 HHMI Summer Scholar Participants

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Sheena Dohar, Carnegie Mellon University
Mentor: Dr. Elizabeth Jones

Creating a Novel Biosensor by Improving the Binding of TO1-2P Dye Molecules to ScFV1 Antibodies

The intent of this project is to create a novel biosensor with increased fluorescence capabilities that allows for the detection of cellular movement. The method employed facilitates the visualization of biological molecules by improving upon existing immunofluorescence techniques.  Specifically, this experiment utilizes antibodies expressed on the surface of yeast cells.  Fluorescent dye molecules can bind to these antibodies extracellularly, emitting fluorescence.  Intracellularly, however, the same binding cannot occur because of the internal reducing environment of the cell.  In order to compensate for this problem, seventeen constructions were designed that incorporate various combinations of two different classes of mutations in the sequence of single chain variable fragment (ScFv) antibody DNA.  These constructions were created by the gap repair method.  Thus, by combining two different classes of mutations in DNA in this way, it is our hope that the binding of TO1-2P dye molecules to the scFv1 of an antibody in a reducing environment where disulfide bridges cannot form will be enhanced.  We have successfully created three constructs, and soon expect to confirm the successful completion of the remaining fourteen as they are all currently in the final sequencing stage of development. Our ultimate goal is to create a construct that contains all of the mutations of both classes.  Fluorescence activated cell sorting (FACS) will then be utilized to separate yeast cells whose displayed antibody binding is increased as a result of various combinations of mutations in the scFv DNA.   The creation of a biosensor that would work intracellularly could be widely used in many types of research dealing with molecular binding of two molecules.  For example, it can be utilized in pharmacological research to direct drug molecules to cells by obtaining information about the location of cells in the body. In addition, this research could be vastly extended and applied to other types of molecular and biomedical experimentation.

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Laurel Farmer, Carnegie Mellon University
Mentor: Dr. Eric Ahrens

Magnetic Resonance Microscopy to Investigate Phenotypic Variation in Dnmt1o Deficient Mouse Embryos

The mechanisms for maintaining monoallelic gene expression, important for normal embryo development, can be altered such that biallelic expression occurs. A recent study found that the loss of maintenance methylation by the Dnmt1o protein in early-gestation mouse embryos resulted in mosaic embryos displaying a wide array of anatomical abnormalities and developmental delays. The next step we have taken is to create clone lines from cells in the mosaic embryo. We expect to find that clones in the same line have identical defects and delays. Magnetic Resonance Microscopy (MRM) is well suited to study these unusual developmental phenotypes because the resulting data are non-invasive, near-cellular resolution, and three dimensional (3D). We have acquired data in a series of staged Dnmt1o defective embryos, optimizing the MRM pulse sequences to highlight anatomical features of interest. Images were analyzed using 3D reconstruction software, and the developing embryo features annotated and correlated with known histology. Preliminary results support our identical-defect hypothesis. The results of this work have important implications for the detailed characterization of the mutant phenotypes and the use of MRI for mammalian fetal developmental studies.

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Rebecca Martz , Carnegie Mellon University
Mentor: Dr. Brooke McCartney

The Role of an Adenomatous Polyposis Coli 2 Complex in Actin Organization

Adenomatous polyposis coli (APC) is mutated in colon cancer and normally functions to negatively regulate the Wnt pathway and to organize microtubules (MT) and actin. The mechanisms by which APC proteins affect the cytoskeleton are poorly understood. Drosophila APC2 and the formin Diaphanous (Dia) function together to organize syncytial actin. APC2 and Dia may affect actin directly or may influence actin through MT stability. To understand how this complex influences actin we took two approaches. First, We examined actin and plasma membrane organization in different APC2 mutant syncytial embryos. Unexpectedly, we observed allele specific changes that may suggest the importance of the Arm repeats of APC2. In addition, the defects we observed in plasma membrane organization did not always correlate with the actin defects, suggesting that different mechanisms are involved in their organization. Because EB1 interacts with the APC2/Dia complex to affect MT stability in mammalian cells, we visualized actin during syncytial development in live EB1 mutants. We found that in EB1 mutant embryos there were severe defects in actin organization that appeared to originate from furrow defects. Because MT defects develop prior to the actin defects in EB1 mutants, we predict that correct MT organization impacts actin furrow formation, and that EB1 facilitates MT stability and/or organization primarily. This data contributes to our understanding of the mechanisms by which APC2 affects the cytoskeleton, and will thus allow us to gain a better understanding of why mutations in human APC promote colon cancer.

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Rohit Ramnath, Carnegie Mellon University
Mentor: Dr. Tina Lee

Structure-Function Analysis of Sec13 in the COPII Coat

The secretory pathway, which is the pathway for cell secretion, is used for communication, excretion and many other functions in cells. In the pathway, proteins move from the Endoplasmic Reticulum(ER) in coated vesicles. There are 3 kinds of coats, and the coat mediating the movement from the ER to the Golgi is called the COPII coat. This coat is made up of the Sar1 GTPase, the Sec23/24 adaptor complex and an outer cage heterodimer composed of Sec13 and Sec31. All of these proteins have been shown to be minimal essential components of the COPII coat in vitro. Although the crystal structure of each of the COPII coat subunits has been solved, the interaction interfaces between subunits that are critical to the function of the coat have not been studied in detail.To determine the critical interaction interfaces in Sec13, I would first deplete cells of Sec13 using siRNA transfections and look for phenotypic changes. I will then attempt to rescue the phenotypic changes using wild-type Sec13, and then test a series of site-directed mutants in a structure-function analysis. Preliminary studies have shown that a sequential double knockdown is necessary to deplete the cells of Sec13 to a high degree. However, the loss of Sec13 does not cause changes in the Golgi apparatus, which is a measure of ER export. This challenges the assumption that Sec13 is essential for ER export. A wild-type rescue construct has been made to rescue any phenotypic changes that we see.

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Lauren Thorpe, Carnegie Mellon University
Mentor: Dr. Brooke McCartney

Assessing the Cellular Consequences of Loss of APC Function in Drosophila Wing Imaginal Discs Using Mitotic Recombination Techniques

The colon cancer tumor suppressor adenomatous polyposis coli (APC) functions in both negative regulation of Wnt/Wingless (Wg) signaling and in cytoskeletal organization. Both Drosophila and humans have two APC proteins, APC1 and APC2, which have redundant function. Therefore, to study the consequences of the loss of APC function in Drosophila, both APC1 and APC2 must be mutant. APC2 APC1 double mutant flies are not viable, but mitotic recombination can be used to induce patches of APC2 APC1 double mutant tissue. APC2g10APC1Q8 (double null) clones in adult wings exhibit cell fate transformations consistent with induction of the Wg pathway. In wing imaginal discs, APC2g10APC1Q8 clones exhibit a phenotype reminiscent of colon polyps that is characterized by outpocketing of the mutant cells from the rest of the epithelium. While clones are numerous throughout larval discs, significantly fewer are seen in the adult wings. Most of these surviving clones are seen in the wing blade, with relatively few in the other adult structures which derive from the wing disc. To determine the fate of the APC2g10APC1Q8 clones we examined their number, location, and phenotype at stages of development between puparium formation and eclosion. Preliminary data suggest that clones near the hinge may pinch off and are lost, consistent with their absence in the adult wing, while some clones in the blade invaginate between the two epithelial layers of the wing. In addition, the outpocketing phenotype is observed in areas of the pupal disc where it is not seen in larval stages, suggesting that clones in different areas of the wing disc take on that phenotype at different times. Exploration of these results may contribute to further understanding of the basis of colon cancer development.

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Cody Vild, Carnegie Mellon University
Mentor: Dr. Jonathan Minden

Identifying Mass Changes in the Pros35 Proteasome Subunit During Drosophila Embryonic Development by Mass Spectrometry

The proteasome plays an important role in the selective degradation of ubiquitinated proteins. This ensures that a cell will not accumulate potentially harmful amounts of unfolded or obsolete proteins.The proteasome, also known as the 26S proteasome, is comprised of many different components, which are broken down into the 20S and 19S subunits. The 20S contains the catalytic core and the 19S is a regulatory complex that interacts with the ends of the 20S unit. The 20S can be broken down into further subunits — one of particular interest is the Pros35 subunit. It is an α-type subunit of the 20S proteasome. Pros35 undergoes a specific modification in the development of ventral cells, which form the mesoderm (muscles and immune system). When an embryo extract from Drosophila melanogaster was run on a two-dimensional difference gel, some 50 difference spots were seen.  A group of five spots was identified as Pros35 isoforms. Three of these spots had relatively the same molecular weight, but different isoelectric points. Two other spots mimicked the same isoelectric point of one of the ventral-specific difference spots, but had lower molecular weights. I hope to recreate this result in a 2D electrophoresis gel, and then elute these spots from the gel. The protein from these spots can then be further analyzed by mass spectrometry, which will give us further insight in to which post-translational modification Pros35 is undergoing. Hopefully, we can see what mass changes are occurring. To get to this point I must first isolate and purify the Pros35 in sufficient quantity that it can be analyzed. This will be done by purifying the embryo extract on anionic and gel filtration columns. Once we can understand the different post-translation modifications Pros35 is undergoing, then we can explore a possible mechanism for these changes and why they occur.

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Natalie Weir, Carnegie Mellon University
Mentor: Dr. Chien Ho

Roles of Amino Acid Residues in the Distal Heme Pocket on the Structure-Function Relationship in Human Hemoglobin

This study explores the structural and functional effects of amino acid substitutions at helical position E11, which is located 3-6 Å from the oxygen-binding iron atom of each subunit of the hemoglobin (Hb) molecule. The usual amino acid residue at the E11 postion is valine for both the α and ß chains of human normal adult hemoglobin (Hb A). We are investigating how stereochemical changes of the ligand-binding site can affect the oxygen-binding properties and overall functioning of the Hb molecule by changing the amino acid residue at postion E11. Four recombinant hemoglobins (rHbs), rHb (V62L), rHb (aV62I), rHb (ßV67L), and rHb (ßV67I), have been expressed in Escherichia coli and purified. To characterize these rHbs, oxygen (O2) affinity, Bohr effect, cooperativity, auto-oxidation, and proton nuclear magnetic resonance (1H NMR) spectroscopy have been measured for each mutant.  Substistution of valine for leucine or isoleucine in the a-subunit and rHb (ßV67L) do not exhibit significant functional deviations in the O2 affinity, Bohr effect, and cooperativity from those of Hb A. However, rHb (ßV67I) shows a two-fold decrease in O2 affinity while maintaining cooperativity. As expected, these mutations perturb the ligand-binding site of their respective subunits, as evidenced by 1H NMR. In addition, rHb (aV62L) and rHb (aV62I) exhibit a small perturbation of the a1ß2 interface in the deoxy state. Auto-oxidation studies have shown that the mutants in this study undergo faster oxidation than Hb A. In comparison to the other three mutants, rHb (ßV67I) is most resistant to oxidation. Mutations that generate the highest rates of auto-oxidation (aV62I and ßV67L) also produce the greatest structural perturbation in the heme-pocket region. Study of these mutations enhances our understanding of the heme-pocket environment and will facilitate the design of hemoglobins with interesting properties. These studies will improve our understanding of the structure-function relationship of hemoglobin, a model for allosteric proteins.

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Mariela Zeledon, Carnegie Mellon University
Mentor: Dr. Elizabeth Jones

PBN1 Alanine Scanning Project in S. cerevisiae

PBN1 is a vital gene in the yeast S. cerevisiae whose only known function—folding Prb1p—is non-vital. This project aims to discover the primary function of this gene, using a method of site-directed mutagenesis known as alanine scanning to create conditional mutants. All the possible mutations created using this method have been previously designed by other researchers. Thus far, 10 of them have been introduced into the genome of yeast using Polymerase Chain Reaction (PCR) with long forward primers encoding the mutation and a reverse primer located such that the kanMX gene for G418 resistance is included in the PCR product. The PCR product has been purified and transformed into the yeast genome of G418-sensitive diploid yeast. The g418-resistant transformants were sporulated and dissected. Haploid spores were tested for amino acid deficiencies, G418-resistance, for the known PBN1 function (HPA overlay assay) and for temperature sensitivity. Several temperature-sensitive spores have been identified and remain to be further tested by restriction enzyme digestion of the DNA and sequencing in order to confirm that the pbn1 mutation is present in these temperature-sensitive spores. Developing a library of conditional mutants will help us elucidate the vital function of PBN1.