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

2012 Participants

National Science Foundation Research Experiences for Undergraduates (REU)

Undergraduate Research Experiences in Cellular and Molecular Biosciences

Evan CesanekEvan Cesanek, Vassar College

Mentor: Robert Murphy and Devin Sullivan

Learning Structure-Localization Relationships of Fluorescent Bioimaging Probes

In this work, we aimed to develop a method to bridge the gap between cheminformatic descriptions of molecular structure and image-based subcellular localization of styryl probe molecules. We used machine learning to analyze how structural elements of small molecules determine their distribution within cells. This work was based on a large image dataset of HeLa cell samples incubated with 1344 structurally diverse fluorescent styryl molecules. First, we computed a weighted linear mixture of images obtained from independent filter channels to account for variability in the molecules’ emission spectra, yielding a single image for each probe molecule. To quantify variation in the appearance of these images, we computed 173 image features and manually assigned 3 classes. A naïve Bayesian classifier with kernel smoothing was used to predict image class from a set of 210 structural features describing each styryl molecule. We achieved an average classification accuracy of 70% with 10-fold cross-validation. We also predicted intra-class localization using multiple linear regression with 70 structural features as predictors of the first principle component of the image features for each class. Using 10-fold cross-validation, we obtained average Pearson correlation coefficients (r) of .42, .36, and .37.

Elise Croteau-Chonka

Elise Croteau-Chonka, Mount Holyoke College

Mentor: Nathan Urban

Computational Modeling of Coupling-Induced Neuronal Synchrony in Heterogeneous Networks

Oscillatory activity is observed throughout the brain’s expanse of neural networks and is believed to be a key contributor to information processing and signal propagation. For example, synchronized oscillations among neurons can increase the efficiency of postsynaptic depolarization and subsequent spike propagation. Mechanisms capable of generating synchronized oscillations include correlated noisy inputs and specific forms of synaptic connectivity. These mechanisms may help explain olfactory bulb mitral cell activity observed in vivo. Previously, we analyzed neuronal responses to correlated input to investigate the influence of cell-to-cell variation on noise-induced synchrony. This heterogeneity is encompassed in differences in phase-resetting curves (PRC), which characterize the response of an oscillator to weak perturbations. Here, we investigated how PRC heterogeneity influences coupling-induced oscillatory synchrony. We hypothesized that 1) cell-to-cell PRC differences would disrupt synchronization, and 2) synaptic weights could be adjusted to compensate for PRC heterogeneity and facilitate synchronization, optimally balancing synaptic and cellular properties. We began by modeling neurons as phase and noisy-phase oscillators, replicating intrinsic properties and periodic activity observed in olfactory bulb slices. Subsequent experiments focused on the coupling mechanism and optimization of synaptic weights in heterogeneous networks. To quantify synchrony, we performed coherence calculations and other spectral analyses. Others have demonstrated that heterogeneity in neural firing rates or external driving currents can destabilize coupling-induced synchrony. Consistent with these results, we now report that PRC heterogeneity can also destabilize coupling-induced synchrony. Further, we find that synaptic weights can be tractably varied to compensate for heterogeneity. Specifically, as a neuron’s PRC amplitude decreases, increasing the average weight of synapses impinging on that neuron from other neurons in the network can promote oscillatory synchrony. The results of this research have thus illuminated one mechanism by which real, physiologically diverse neurons can achieve oscillatory synchrony and may help explain in vivo mitral cell activity.

Etoroabasi EkpeEtoroabasi Ekpe, Yale University

Mentor: Chuck Ettensohn

Role of VEGF Signaling in Cell Specification during Transfating

I am studying how the genome encodes sea urchin skeletal development. Unlike many echinoderms, including starfish and sea cucumbers, sea urchins develop an elaborate endoskeleton during the embryonic stage. Skeletogenesis is the process whereby primary mesenchyme cells (PMCs) secrete calcite and proteins to form the endoskeleton. Various genes interact in a gene regulatory network (GRN) to control this process in PMCs, and the vascular endothelial growth factor receptor (vegfr) gene is one of them. The role of vegfr is also evident in transfating, the process by which secondary mesenchyme cells, in the absence of PMCs, become PMCs and develop an endoskeleton. However, when embryos are treated with axitinib, a VEGFR inhibitor, transfating and skeletogenesis do not occur. By determining which genes in the PMC-GRN are expressed in the presence of the inhibitor, we can identify the step at which transfating is blocked. I used whole mount in situ hybridization (WMISH) to analyze the expression of several genes in the PMC-GRN by first testing probes complementary to these genes and finding their working concentrations and color reaction times. In the second part of my project I used these probes to examine gene expression during transfating in the presence and absence of axitinib, an inhibitor of VEGF/VEGFR signaling. Transfating was induced by removing the PMCs surgically from embryos. Using the probes alx1, ets1, and tbr, we found higher gene expression in the control embryos compared to axitinib-treated embryos. This showed that expression of these genes during transfating is dependent upon VEGF signaling in contrast to normal development, in which the expression of VEGFR is dependent on alx1, ets1, and tbr. This indicates that the skeletogenic GRN is wired differently in the transfating cells and PMCs:  VEGF signaling acts at the very top of the network in transfating cells but near the bottom in normal development.

Ashley Fidler

Ashley Fidler, The College of William and Mary

Mentor: David Hackney

The Role of Unc-76 in the Induction of Kinesin-1 Auto-Inhibition Release

Kinesin-1 is a heterotetrameric protein fundamental to the movement of cargoes throughout the cell. Composed of two heavy chains (KHCs) and two light chains (KLCs), kinesin’s KHC contains a tail domain that binds cellular cargoes and a head domain that instigates motion along a microtubule through a cycle of ATP hydrolysis. In the absence of proper molecular signals, one of kinesin’s dimeric C-terminal tail domains binds to the N-terminal head domains, impairing their motive ability by inhibiting ADP release. Previous work has shown that the Drosophila protein Unc-76 has the capacity to relieve the kinesin molecule’s auto-inhibition, restoring its motor function. In this project, I attempted to elucidate the mechanism of this reaction between Unc-76 and kinesin using several biochemical techniques, including fluorescence resonance energy transfer (FRET). Initially, our FRET studies were complicated by the formation of insoluble kinesin tail-Unc-76 complexes in buffers with low salt concentrations. When populations of kinesin tails and Unc-76 were mixed, the solution became visibly more turbid and experienced a significant amount of light scatter. After centrifuging the samples, SDS-PAGE experiments revealed that considerable amounts of protein were removed from solution and that the kinesin tail concentrations were more greatly affected than those of Unc-76. However, at higher salt concentrations, we observed a decrease in the donor Cy3 peak without a significant corresponding increase in the acceptor Cy5 peak to indicate the existence of FRET. Therefore, to apply FRET to this system, we must develop a methodology that averts the formation of these insoluble complexes in solution while maintaining favorable conditions for kinesin-Unc-76 binding. By developing an effective FRET methodology, we hope to not only to clarify the role of Unc-76, a protein crucial for axonal growth, but also to provide a basis for the study of other kinesin-interacting proteins.

Alexander Hurley

Alexander Hurley, Centre College

Mentor: Jon Minden

Detecting Unique Proteins in Rheumatoid Arthritis Patients with Interstitial Lung Disease

Rheumatoid Arthritis (RA) patients can be affected by a deadly disease complication known as Interstitial Lung Disease (ILD). A research team at the University of Miami believe that this complication is caused by the presence of unique citrullinated proteins in lung tissue. Confirming this prediction is valuable because these specific proteins could be used to produce a bioassay that could identify the early onset of ILD. To determine the validity of this hypothesis, citrullinated and uncitrullinated protein profiles from RA patients both with and without ILD were prepared and then compared in our lab using Two-Dimensional Difference Gel Electrophoresis and prototype gel imaging technology. Our results did not suggest that the RA-ILD patients possessed citrullinated proteins wholly unique from the RA-No ILD patients. However, this project did represent one of the initial attempts to test this hypothesis with the aforementioned techniques, and so several reiterations of the experiment must be run with minor alterations before the hypothesis can be rejected with certainty.

Grace SoloffGrace Soloff, Ursinus College

Mentor: David Hackney

Ionic Interactions between Kinesin Head and Tail Domains Due to Phosphorylation

Kinesin-1 is an ATP-dependent motor protein that transports cargo to (+) ends of microtubules. Its heavy-chain dimer is composed of an N-terminal motor domain (head), neck region, and tail responsible for carrying cargo. Kinesin can bend, forming a head-tail complex, causing autoinhibition by preventing ADP release. Previous research from other labs indicates polyQ expansion in Huntingin decreases fast axonal transport (FAT). PolyQ expansion activates JNK3 kinase, which phosphorylates kinesin and other proteins. It was also shown that mutating serine residue 176 to glutamate in human kinesin as a phosphorylation mimic decreases FAT. Our previous research shows mutating Ser182 (Drosophila melanogaster homolog) to glutamate as a phosphorylation mimic increases head-tail affinity. We aim to extend the phosphorylation process to the mechanism of tighter tail binding in kinesin autoinhibition. We hypothesized a charge effect causes greater head-tail affinity changes in S182E mutants than wild type kinesin heads. This study used stopped-flow fluorometry to detect fluorescence in tryptophan/dansyl chloride fluorescence resonance energy transfer (FRET). Observation of tail association and dissociation rates at varying salt concentrations for S182E heads showed affinity of tails for heads is dependent on ionic strength, indicated by a larger slope of a line comparing log[Kd] to log[ionic strength] in S182E heads than that of wild type. For wild type and S182E heads, affinity decreased with higher salt concentrations. In full-length constructs of kinesin, the tryptophan/dansyl chloride FRET pair is not viable due to tryptophan in kinesin cargoes. We investigated alternative labeling kinesin heads and tails with FRET pairs. Cy3-labeled heads/Cy5-labeled tails showed strong FRET signals. Kinesin heads were also tetramethylrhodamine (TMR)-labeled, and TMR dimer splitting due to tails binding heads was possible, but not optimal. In the future, we hope to express full-length kinesin and study the effects of direct Ser182 phosphorylation on tail binding affinity and cargo binding.

Kayla Topper

Kayla Topper, Bridgewater College

Mentor: Brooke McCartney

Understanding Changes in Tissue Morphology in Response to Wnt Siganling

Wnt signaling is necessary for normal development due to its significant functions in cell proliferation and differentiation. Inappropriate activation of Wnt signaling is linked to human diseases, such as colon cancer. Previously, our lab demonstrated a novel role for Wnt signaling in the regulation of epithelial cell shape in the Drosophila wing imaginal disc. Over activation of the Wnt pathway caused apical constriction and basal extrusion in flat portions of the disc (blade region), while in highly folded regions (dorsal hinge) apical extrusion occurs. Apical constriction coupled with basal extrusion causes a bulge of cells to become trapped beneath the normal layer of cells. Because a similar phenotype appears to initiate polyps that can progress to colon cancer, the Drosophila imaginal discs are a good model for understanding the cellular changes that promote polyposis. This summer I attempted to understand why some cells apically constrict while others apically extrude. Our hypothesis suggested that the differing structure of the disc regions (folded or flat) causes the cells to have different mechanical properties. These properties may be responsible for the opposing effects in the different areas of the discs. I examined larval haltere and leg imaginal discs to determine whether the folded regions apically extruded and flat regions apically constricted, as in the wing discs. Activation of the Wnt pathway was stimulated in two ways: by loss of function of APC1 and APC2, and with a stabilized form of Armadillo. Preliminarily, the structure of the disc does appear to influence morphology in cells activating Wnt signaling, which is consistent with the hypothesis. Surprisingly, opposing phenotypes were observed between the two genotypes in some disc regions, suggesting that the level of Wnt activation may also play a role in cell and tissue morphology.

Nicole Traphagen

Nicole Traphagen, Clarkson University

Mentor: Veronica Hinman

Dissecting Novelty: Examining the Role of Delta in Mesoderm Specification in Sea Cucumbers

Throughout development, multiple-potential territories split into separate cell types.  Echinoderms are a good model system for studying the way these separations evolve. During the development of echinoderms, the mesoderm segregates into discrete cell types. In sea stars, the mesoderm gives rise to the coelom and mesenchyme cells. In sea urchins the coelom, early mesenchyme, late mesenchyme, and skeletogenic mesenchyme are all formed from the mesoderm. Sea cucumbers also have coelom, early mesenchyme, late mesenchyme, and skeletogenic mesenchyme cells that arise from the mesoderm. However, the early mesenchyme in sea urchins is similar to the late mesenchyme in sea cucumbers. The Delta-Notch pathway is involved in distinguishing different cell fates in the mesoderm of both sea urchins and sea stars. In order to form a model for how mesoderm separation occurs in sea cucumbers, the genes involved in the Delta-Notch signaling pathway were cloned. The expression patterns of these genes were examined in the sea cucumber Paristochopus parvimensis using whole mount in situ hybridization. These expression patterns indicate that the early mesenchyme separates from the mesoderm without Delta-Notch signaling, while the late mesenchyme and the coelom segregate later and Delta-Notch signaling is involved in this process.

Kelly Turner

Kelly Turner, University of Maryland Baltimore County

Mentor: Eric Ahrens

Survival of Δccc1 Yeast Expressing Human Ferritin Genes on an Iron Medium

With soft-tissue contrast and high spatial resolution, magnetic resonance imaging (MRI) is ideal for in vivo imaging in research to track cell migration, visualize gene expression, and visualize preclinical therapeutic gene delivery. MRI reporter molecules would be highly beneficial as modest sensitivity currently limits use of MRI techniques in some of these research avenues. Our lab has done significant research in this emerging field by investigating the use of metalloproteins in the ferritin family. Ferritin proteins oligomerize to form shells in which iron is safely sequestered and mineralized. Overexpression of ferritin triggers heightened internalization and storage of iron, allowing for greater contrast in MRI imaging. This project focuses on expression of human ferritin genes in Saccharomyces cerevisiae as a screen for libraries of ferritin mutants useful as MRI reporter molecules. We aimed to transform the Δccc1 yeast strain so that it would express human ferritin genes. This knockout strain lacks the ccc1 gene for iron sequestration within cell vacuoles and should display lethality when grown in an iron-rich medium. Expression of ferritin genes in these cells was hypothesized to rescue them from death by storing iron in the cells in a non-toxic form; however, phenotypic selection proved to be a setback in the project. When plated on varying concentrations of ferric citrate, the Δccc1 strain already grew equal to that of the wild type strain. Further experimentation into the cause of this occurrence is necessary to determine later success of this protocol in creating a library of ferritin mutants.

Eleanor Vane

Eleanor Vane, Clarkson University

Mentor: Alan Waggoner and Bruce Armitage

Using the Promiscuous scFv Protein, R1, to Bind Dyes that Fluoresce in the Visible and Near-IR Range

Fluoromodules, which are composed of a fluorogenic dye that binds to a fluorogen activating protein (FAP), cause fluorescence and are useful in fluorescence microscopy for live cell imaging. The fluorogenic dyes were conceived and synthesized based on rational design and previous literature on fluorescent dyes. These dyes were then used in the process of fluorescence-activated cell sorting (FACS) to screen a library of human single-chain antibodies (scFv) displayed on the surface of yeast in order to select for the scFv-based FAP that has the greatest fluorescence. Two scFvs that have been identified through this process, K7 and R1, have been found to be promiscuous, which is to say, they bind several different fluorogenic dyes. In order to create a versatile fluorescent label that can be used alongside other types of fluorescent labels, our lab is trying to create a sort of “toolkit” of fluorogenic dyes that fluoresce throughout the visible and near-IR range once they are bound. The purpose of this project is to discover if R1 binds to a group of fluorogenic cyanine dyes, and if it does, determine the fluorescence enhancement of the complex, where it fluoresces, and how thermostable it is. The fluorescence spectra of equimolar amounts of each dye and R1 were recorded to determine the fluorescence enhancement and maximum emission wavelength of each complex. The thermostability of the dye-R1 complexes was evaluated by measuring the change in fluorescence intensity for each complex when heated from 10-70C. This research has shown that R1 binds TO, TO3, TOPRO1, TOPRO3, TOPRO5, POPRO1, YOPRO1, and DIR in the nanomolar range, causes large increases in fluorescence intensity, and forms relatively thermostable complexes.

The following students were co-sponsored by the Howard Hughes Medical Institute (HHMI).

Catherine ByrdCatherine Byrd

Mentor: Chien Ho

Role of Woolly Mammoth Central Cavity Hemoglobin Residues in Oxygen Delivery

Semawit GebrehiwotSemawit Gebrehiwot, Carnegie Mellon University

Mentor: Adam Linstedt

Site-directed Mutagenesis of Shiga Toxin

Jessika LouissaintJessika Louissaint

Mentor: Veronica Hinman

Characterization of Function & the Expression of the Hedgehog (HH) Gene during Neurogenesis in a Non-Chordate Deuterostome Patiria miniata

Darlene ReidDarlene Reid

Mentor: Catalina Achim

Charge Transfer Study of Peptide Nucleic Acids linked to Cytochrome c



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