2007 REU Participants-Department of Biological Sciences - Carnegie Mellon University

2007 Participants

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

Nancy Anoruo

Nancy Anoruo, University of Florida

Mentor: Dr. Brooke McCartney

The Effects of Activated Diaphanous on Cytoskeletal Organization and Wing Morphology in Drosophila

Many genes mutated in human cancers are crucial components of signal transduction pathways and are vital to normal cell function. The tumor suppressor Adenomatous polypopsis coli (APC) is mutated in most colon cancers and also plays a critical role in several cell processes, including signal transduction and cytoskeletal organization. The precise mechanisms by which APC protein affects the cytoskeleton are poorly understood. Our lab has shown that Drosophila APC2 interacts with the formin Diaphanous to affect actin organization in the early embryo. To understand how the interaction between APC2 and Diaphanous affects actin, we examined the cellular consequences of expression of a constitutively active form of Diaphanous (DiaCA). Because formins can nucleate actin and have been implicated in microtubule stability, we predicted that expression of DiaCA may have effects on both cytoskeletal systems. We expressed DiaCA in larval wing imaginal discs using the GAL4/UAS system for expression throughout the disc and using the FLIP-out system to restrict expression to small patches of cells called clones. When DiaCA was expressed throughout the wing disc, it resulted in high levels of actin accumulation and morphological disruption. Surprisingly, DiaCA clones did not display significantly increased levels of actin. However, some of the clone cells were more rounded than the control cells and tended to scatter throughout the disc. Because Drosophila wing bristles and hairs are composed of actin and microtubules, we examined these structures in adult flies expressing DiaCA throughout the wing disc and observed significant structural defects. Flies with DiaCA clones did not appear to survive to adulthood, precluding the analysis of wing morphology under these conditions. These results will be used to further our understanding of the mechanisms by which APC mutations affect cells, ultimately resulting in colon cancer.

Alys CheatleAlys Cheatle, The Catholic University of America

Mentor: Dr. Charles Ettensohn

Hormone-Inducible Expression of Alx1 in the Lytechinus variegatus Embryo

Alx1 is a beta catenin dependent homeodomain protein; its expression is necessary for skeletogenesis and ingression of primary mesenchyme cells (PMCs) in Lytechinus variegates (Lv). Previously, Alx1’s role in these processes was determined by blocking its expression using morpholino antisense oligonucleotides, which prevented both ingression of PMCs and skeletogenesis. However, this approach can only be used to determine Alx1’s function during early development because these oligonucleotides are injected at fertilization resulting in persistent repression during development. Our goal is to obtain a greater understanding of Alx1’s function throughout development by temporally controlling its expression. We generated a DNA construct encoding LvAlx1 fused to green fluorescent protein (GFP), and the glucocorticoid receptor (GR) ligand binding domain. Addition of the GR ligand binding domain allows Alx1 localization to be regulated.  In the absence of the hormone ligand dexamethasone, the ligand binding domain will trap the fusion protein in the cytoplasm by causing it to bind to a cytoplasmic heat shock protein (HSP). Upon addition of dexamethasone, the fusion protein will dissociate from the HSP and enter the nucleus where Alx1 can activate the transcription of other skeletogenic genes. The green fluorescent protein will allow any migration of Alx1 to be observed. We synthesized mRNA of the LvAlx1 fusion for injection into fertilized eggs. Dexamethasone will be added to injected eggs at various embryological stages to assess Alx1’s function at different points in development. Optimization of this hormone-inducible system using LvAlx1 will allow its future use in the study of additional skeletogenic genes.

J. Michael HendersonJ. Michael Henderson, University of Central Oklahoma

Mentor: Dr. Gordon Rule

Investigations Into Organic Dye Binding Antibody Single Chain Variable Fragments Through Bacterial Expression Systems

Antibodies have emerged as a model system for the experimental study of ligand-protein interactions. Antibody single-chain variable fragments (scFvs) are recombinant antibody fragments consisting of only variable light chain (VL) and variable heavy chain (VH) domains covalently linked by means of a polypeptide chain. Antibody single-chain variable fragments that bind common organic dyes such as fluorescein and thiazole orange (TO1) have been explored as potential biosensors. When these organic dyes bind to the antibody fragments, the complex experiences either an increase or decrease in the intensity of fluorescence. Research this summer has focused on the expression of organic dye binding scFvs through E. coli rather than S. cerevisiae as previously documented. A bacteria based expression system will ultimately lead to a higher production of functional antibody fragments since E. coli growth is rapid and inexpensive, and the ease with which the bacteria can be genetically manipulated. An affinity matured gene sequence for the TO1 binding scFv (AM2-2) was amplified and inserted in a bacterial plasmid by TOPO Cloning. Once the TOPO vector had been synthesized and isolated, SfiI restriction enzymes cleaved the scFv encoding region. To confirm the presence of the scFv fragment following SfiI digestion, a series of double restriction enzyme digestions (EcoRI and BsmBI) were performed. The DNA fragment was then ligated into the pAK400 plasmid; consequently, the plasmid encoded for a periplasmically secreted scFv that binds TO1. Isolation and purification of the scFv was conducted by metal-chelation chromatography. NMR spectroscopy was utilized in determining the structure of the antibody fragment bound to TO1. With the construction of a new bacterial expression vector, future investigations can be conducted into enhancing ligand affinity by manipulating the primary amino acid sequence of the polypeptide linker and the variable domains of the scFvs.

Daniel LeesDaniel Lees, Amherst College

Mentor: Dr. Russell Schwartz

Discovering Transitions Between Pathways for Self-Assembly

Icosehedral models for self-assembly provide a simple, but realistic framework for modeling viral capsid assembly. Experimental data shows that different organisms use different pathways to assemble similar geometric structures. Computer simulations may provide information on how and why these differences exist. Certain assembly conditions are believed to provide certain pathways.  Our simulations between these known data points show that the pathways are not necessarily mutually exclusive. By running the simulation with five times the detail as previous tests, a more accurate map of these transition regions was created. Instead of a sharp boundary between pathways, there is a transition area in the parameter-space where assembly utilizes multiple pathways. This result suggests that assembly may be more complicated that previously believed. Extending this result to non-simulated systems could imply that in vitro models for self-assembly are inadequate to model in vivo systems. Since in vivo systems have much higher concentrations of protein subunits, these systems may utilize different pathways than the lower concentration in vitro systems that most of the current viral self-assembly theory is based upon.  More research will need to be conducted to better understand how the existence of boundary regions affects our theory of self-assembly.  This is important since discovering the nature of these boundaries could aid scientists in creating inhibitors for viral reproduction for use in treating viruses. Understanding the boundaries of self-assembly could also help the development of nanotechnology that mimics the assembly techniques of natural organisms.

Daniel MedranoDaniel Medrano, Columbia University

Mentor: Dr. David Hackney

Evaluating Reverse Constant Rates of Phosphate and Its Effects on Kinesin Motility

Kinesin is a heterodimer motor protein that transports cargo throughout the cell using a hand-over-hand mechanism directed toward the positive end of microtubules. This movement is driven by the free energy arising from the hydrolysis of ATP to ADP, which causes the kinesin motor to move an 8nm distance along the tubulin filament. The effectiveness of kinesin motility, however, cannot be measured directly and requires knowing the reverse rate reaction of phosphate, which can inhibit or promote the reaction. In this study, we examine Drosophilia kinesin motility by creating an in vivo assay containing biotinilated bovine serum albumin (BSA), which adheres to the glass wall. Tetrameric avadin is then used to bridge this with biotin site of the enzyme of study, one containing the motor domain of the protein. To create this enzyme, a contrasfection of the motor head domain and the BirA gene, encoding for the biotin site was made using glycerol-washed cell lines. The vector encoding for the BirA contained a gene resistant to chloramphenicol, while a second vector containing the Drosophilia head domain was made resistant to ampcillin. Once the cotransfection took place, the resultant vector was held resistant to both. The resulting enzyme was then bound to the avadin in the glass well assay, and axoneme from sea urchin sperm tails were used to replicate kinesine movement along microtubule filaments. Preliminary results successfully show Biotin inhibiting avadin binding to the Nte, thus preventing axonemes from binding to the glass surface. Once an adequate control assay is created that will enable for adequate measurement of the axionome velocity along the assay, phosphorous concentrations will be varied to further examine the effectiveness of kinesine motility.

Alexandria MelendezAlexandria Melendez, University of Texas at El Paso

Mentor: Dr. Eric Ahrens

Quantification of Primary Amines in Nanoemulsions to Improve Cellular Uptake

Recently, cell tracking methods have been developed that utilize perfluorocarbon labeling of cells in vitro followed by in vivo 19F MRI of cells after transfer. Perfluoropolyether (PFPE) was emulsified by microfluidization to create nanoemulsions with droplet sizes ranging from 100-200 nm. Polyethyleneimine (PEI) was incorporated during the emulsification process. Resulting nanoemulsion droplets contain primary, secondary and tertiary amines. We propose that there is a relationship between the number of primary amines on the nanoemulsion droplet surface and the cellular uptake of these nanoemulsions. By this logic, the more PEI that is incorporated into a nanoemulsion, the greater the cellular uptake of that emulsion will be unless, there exists a finite number of primary amines; a number at which cellular uptake will began to level off and then decrease. To test this hypothesis we utilized the Kaiser test; an assay that utilizes ninhydrin which reacts with free, primary amines to produce Ruhemann’s blue and a blue colored solution. The degree of saturation of this colored reaction product may be quantified by spectrophotometry. Four emulsions with different amounts of surface primary amines were then used to label Jurkat cells. The uptake was meausred by 19F NMR. Presence of primary amines on nanoemulsion droplet surface significantly increased the cellular uptake of PFPE and did not induce any cell toxicity. Furthermore, we observed that increase of the primary amines presence beyond certain amounts induced decrease in uptake and cellular toxicity. We also examined the stability of these nanoemulsions in extreme environments by exposing them to pH levels ranging from 1- >12. The emulsions were incubated at a various number of hours in 37°C and then Dynamic Light Scattering was performed on each to test the nanoparticle size in each emulsion. pH produced no drastic changes in the nanoparticle diameters.  In summary, the optimal amount of primary amines on nanoemulsion droplet surface was established. Furthermore, these results indicate a sufficient degree of PFPE nanoemulsion stability, and suggest that the nanoemulsions likely remain stable when exposed to acidic, in vivo conditions.

Oluwatoyin OrunjaOluwatoyin Orunja, University of Maryland Baltimore County

Mentor: Dr. Justin Crowley

Quantum Dots as Retrograde Tracers

Quantum Dots (QDots) are relatively new fluorescent crystals that may offer several advantages in the study of neural circuit organization. The goal of this project was to analyze the efficiency of QDots as neural tracers, specifically as retrograde tracers. QDots may eventually be more effective than conventional fluorophores because they are highly resistant to photobleaching. In addition, QDots have a high absorbency and high quantum yield. Conventional fluorophores have emission bands that are broad and their excitation spectra are non-overlapping, so different excitation spectra are needed to produce different fluorescence channels. By contrast, QDots have narrow fluorescence emission bands, increasing the number of discrete fluorescence channels that can be used in a single experiment with a single excitation wavelength. This makes higher order multiplexing possible because several different QDot species can be injected in different areas of the brain, with the goal of separating them by their fluorescence emissions. Thus, retrograde neural labeling studies with QDots could identify the afferent populations for brain regions of interest at a higher resolution than has been possible previously. Retrograde labeling is when neurons transport a label from axon terminations back to the cell bodies instead of away from them down axons (antereograde). This type of experiment identifies the input population to the injection site. We injected primary visual cortex with various preparations of quantum dots. In these experiments, we expected to find retrograde label in the visual cortex and in the lateral geniculate nucleus (LGN). Through surgical injection procedures, QDots were injected into layer four of the visual cortex of a ferret. Approximately 48 hours later, the ferret was killed, and then perfused with saline (0.9%), paraformaldehyde (4% in 0.1M PB), and then paraformaldehyde and sucrose (30%) solutions. Labeled brain structures were cut on a sliding microtome and mounted on glass slides for microscopic analysis. The cortex and LGN were viewed under a fluorescent microscope and analyzed for retrograde labeling. We injected the primary visual cortex with various preparations of QDots. Before the QDots were used, they were subjected to purification through gel filtration chromatography and gel electrophoresis. The QDots were loaded onto a prepacted Superose 6 column, and then chromatographed using an FPLC system. Then electrophoresis was conducted in 1% agarose, TAE buffer at a pH of 8; the gel ran for about one hour.  After analyzing the LGN of ferrets injected with QDots, the extent of retrograde labeling was analyzed. The QDots species that were injected into these ferrets were QDots 525, 565, 605, and 655 all coated with carboxyl amphiphilic. We observed labeling on the LGN with all of these QDots species except for 565. We also used QDots 605 and 655 coated in polyethylene glycol 5K. We observed labeling from QDot 605 PEG. Although labeling in the LGN was observed, the labeling was sparse on some slides, and scattered on others. Based on our experience with other retrograde tracers, we expected to see retrograde labeling that was concentrated with specific QDots in distinct areas. This unusual pattern suggests an abnormal pattern or mechanism of QDot uptake. When the slides exhibiting labeling in the LGN were viewed several weeks later, they were no longer fluorescence. This could be due to the instability of the quantum dots, so more experiments will be performed to stabilize the quantum dots in the future.

Davalyn PowellDavalyn Powell, Dickinson College

Mentor: Dr. Alison Barth

Fluorescence Activated Cell Sorting of Neocortical FosGFP Positive Neurons in Transgenic Mice

The segregation and characterization of different types of neuronal cells in various regions of the brain is central to the understanding of its operation as a whole. To view differing types of neural activity, transgenic mice were developed that express the green fluorescent protein (GFP) upon stimulation of the activity-dependent gene c-fos. The fosGFP transgenic mouse allows for direct visualization of GFP in living cells based upon activation of fos expression. When the fosGFP mouse is subjected to one of many sensory stimulants that activates the c-fos gene, many neurons within the cerebral cortex will appear green under fluorescent microscopy. However, other neurons in the same regions of the brain do not exhibit c-fos activation and GFP fluorescence. To determine what molecular variations cause some neurons to express the transgene while others do not, we are interested in being able to physically separate the two classes of cells that are usually interspersed within the cortex. We have developed a protocol for fluorescent activated cell sorting (FACS) of fosGFP+ cells for future microarray analysis. We performed tissue dissociation using the enzyme papain on acute brain slices from layer 2/3 of the fosGFP mouse neocortex after in vivo stimulation. To reduce dissociation-induced expression of fosGFP, the transcription inhibitor, α-amanitin, and translation inhibitor, anisomycin, were used. Their presence ensured that the GFP-expressing cells had been activated by in vivo experience. The FACS sorter then separated the heterogeneous collection of cells based on fluorescent activity into homogeneous populations of cells that exhibited elevated fosGFP expression. Once the samples of cells have been purified in this manner, it may be possible to characterize differences in gene expression using microarray analysis. The distinct characteristics that these neurons possess will molecularly define this specific subset of neurons in the neocortex and possibly their role in greater neural function.

Sarah RomereimSarah Romereim, Baker University

Mentor: Dr. Jonathan Minden

Slingshot Phosphatase Induces Apical Constriction During Ventral Furrow Formation in Drosophila melanogaster

During embryogenesis in Drosophila melanogaster, ventral furrow formation is essential for the morphogenesis of mesodermal tissues. A key step during ventral furrow formation is the constriction of the apical membranes of the cells near the ventral midline. This constriction process has been shown to be correlated with elevated levels of Slingshot phosphatase (Ssh) in wild type embryos. Ssh dephosphorylates and activates cofilin, which is involved in actin depolymerization and plays an important role in cytoskeletal rearrangement. The transcription factor Twist (Twi) transcribes a set of genes that is required for proper ventral furrow formation. In twist (twi) mutants, the apical membranes do not constrict, though a narrow, transient furrow is still formed. The goal of this project was to determine whether or not Ssh is one of the components involved in ventral furrow formation by discovering if Ssh is activated by Twi in the ventral region. By examining whole and cross-sectioned embryos stained with anti-Twist and anti-Slingshot antibodies, I was able to confirm that higher levels of Ssh are present during and shortly after ventral furrow formation in the same region in which Twi expression is observed in wild-type embryos. I also observed that twi mutant embryos lack elevated Ssh levels along the ventral midline, but maintain higher Ssh levels in other areas such as the amnioproctodeal invagination, as expected. This indicates that the presence of Twi is required for Ssh expression in the ventral region. The lack of higher levels of Ssh in ventral cells of twi mutants may result in reduced cofilin activity and could explain the failure of these cells to constrict. Thus, our observations suggest that Twist is necessary for Slingshot expression, and Slingshot itself is required for the apical constriction needed for proper ventral furrow formation.

Thomas SuterThomas Suter, William Jewell College

Mentor: Dr. Javier López

Generating Single Chain Variable Fragment Antibodies Against pre-mRNA Splicing: Lariat Product as a Tool for Genome-Scale Splicing Analysis

This project aims at generating single chain variable fragment antibodies (ScFvs) that can bind with high selectivity and affinity to the branch point of the lariat product generated upon the splicing of introns from pre-mRNA. Alternative splicing is the generation of different mRNAs by varying the pattern of pre-mRNA splicing. These alternative arrangements are generated through the splicing of segments of the mRNA, including non-coding inter-exon regions known as introns. Whenever such a splicing event occurs, the pre-mRNA being excised is cleaved through interaction with the spliceosome to wrap around upon itself, thus binding to itself to form a three-way branch point junction and thereby forming a lariat structure. Once the remaining mRNA is free of non-coding introns, it can then go on to be translated into protein. However, the lariat product of splicing remains an important part of the splicing and development of mRNA, and the code of the lariat can provide information about the intron and the surrounding gene from which it's spliced.From multiple hybridoma cell lines that were previously shown by the Lopez lab to secrete antibodies that could bind with high selectivity and affinity to the lariat branch point, four cell lines were cultured from single cells.These four cell lines were again tested for branch site binding activity via ELISA assay and were harvested for mRNA.This mRNA was used for 5’ RLM RACE in order to amplify and clone the variable region of the different cell lines’ antibodies given their known isotyped constant region.These clones, which contained the active site variable regions of the branch point binding antibodies, were then sequenced and will be used to generate ScFvs.These clones will then undergo directional evolution towards higher affinity and specificity for binding to the branch junction of the lariat product via yeast surface display. The directionally evolved ScFvs will then be tested for affinity and specificity against lariats containing branch junctions of a variety of nucleotide compositions. Once the ScFvs have been determined to be able to bind to the breadth of known branch junctions, and therefore presumably the breadth of known lariat products, the ScFvs will be used in various whole cell extracts to extract lariats for sequencing. The characterization of these lariats will provide information on genomic introns, and thus will aid in whole genomic characterization.

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