2011 Research Experiences for Undergraduates (REU) Participants-Department of Biological Sciences - Carnegie Mellon University

2011 Participants

National Science Foundation Research Experiences for Undergraduates (REU)

Undergraduate Research Experiences in Cellular and Molecular Biosciences

Nate BraunNathanael Braun, Cedarville University

Mentor: Brooke McCartney

The Role of GSK3-beta and Akt in APC2 Regulation of Actin Furrow Formation in Drosophila Syncytial Embryos

A common mutation in colon cancer is that of the tumor suppressor Adenomatous Polyposis Coli (APC). APC proteins are most well known for their role as negative regulators of Wnt signaling, but they are also regulators of the microtubule and actin cytoskeletons. In order to study how APC proteins regulate the actin cytoskeleton, we are using Drosophila syncytial embryos as a model system. Our lab has shown that APC2 can form a complex with the formin Diaphanous (Dia) to regulate the formation and extension of actin-based pseudocleavage furrows in these embryos. How the APC2-Dia complex is regulated is still unclear. Phosphorylation of APC proteins regulates their activity in other contexts, and we hypothesize that it may also play a role here. Glycogen synthase kinase 3 β (GSK3β) is a kinase that regulates APC2 function in Wnt signaling, and preliminary data suggests that it may regulate APC2 function in actin furrow extension. Akt is a kinase which negatively regulates GSK3β. These data suggest that APC2’s effect on actin may be regulated by the Akt-GSK3β pathway. I genetically manipulated GSK3β and Akt and assayed for effects on the extension of actin furrows. The increase or decrease of function of GSK3β and Akt disrupted the furrow extension. This provides evidence to suggest that the APC2-Dia complex may regulated by the Akt-GSK3β pathway and prompts further examination of this pathway in the regulation of actin.

Susana Chan

Susana Chan, Barry University

Mentor: Eric Ahrens

Determining the Fate of Perfluoropolyether Labeling Agent after Cells are Lysed or Undergo Apoptosis

Cellular therapy introduces pharmacologically manipulated cells into the body to battle different pathologies. Difficulty arises in quantifying the number of therapeutic cells and establishing whether they are present in the targeted area. Ahrens’ lab has developed a technique that can trace therapeutic cells by labeling them with perfluoropolyether (PFPE), a fluorine-rich labeling agent and imaging them using 19F MRI. 19F MRI has high sensitivity to labeled cells thus providing a good marker for cell tracking with no background signals from the host tissues. To prepare for 19F MRI, a nanoemulsion is formulated using polyethyleneimine (PEI) and PFPE. PEI is used to aid the entry of PFPE into cells since PFPE is both hydrophobic and lipophobic.  The PEI portion facilitates cell membrane entry. However, little is known of the whereabouts of PFPE after the therapeutic cells complete the task and undergo cell death. In this study, we tested the hypothesis that PEI will not aid any further entry of the nanoemulsion into cells upon therapeutic cell death. We used 9L and Jurkat cells as model therapeutic cells. Both cells lines were labeled using the nanoemulsion. The labeled 9L cells were lysed using a lysis buffer and the labeled Jurkat cells were lysed through an apoptotic pathway with the addition of an anti-Fas antibody. The lysed cells were then re-suspended in media and added to unlabeled cells. The unlabeled cells were collected to determine the fluorine uptake using 19F NMR. The purpose of lysing labeled cells and then re-suspending them in media was to release the intracellular PFPE. The signal for 19F atoms detected from the unlabeled cells suggests that intracellular PFPE was able re-enter cells upon co-incubation. However, the uptake of 19F atoms per cell was less than when the cells were initially labeled with the nanoemulsion.

Schnaude DorizanSchnaude Dorizan, University of Maryland, Baltimore County

Mentor: Nathan Urban

The Analysis of Mouse Behavior Based on Sound

Mice are a common model system for studying biological processes, behavior and the connection between.  Compared to other mammalian model systems, mice are easy to breed and can be manipulated genetically. To better make use of mice as model systems for study of brain disease and behavior, we must develop methods for efficient and reproducible analysis of mouse behavior. Current methods of behavioral analysis in rodents are too labor or cost-intensive to be applied on the scale of hundreds or thousands of mice. We are developing an approach to analysis of mouse behavior based solely on multi-site sound recordings from the animal’s home cage. The short term goals of this project were to collect data sets that would allow us to determine the feasibility of this approach. Specifically we acquired sound recordings using two microphones placed in the mouse cage while also performing video recordings of mouse behavior.  We sought to optimize the sound recordings and then to determine whether we could use the sound recordings to identify when the mouse was engaging in one of six behaviors that we identified from the video recordings.

Ariel Le

Ariel Le, Harriet L. Wilkes Honors College at Florida Atlantic University

Mentor: David Hackney

Kinesin-Fluorogen Activating Protein Constructs for Motility Assays

Kinesin is a motor protein that functions in intracellular cargo transport. The movement of kinesin has been seen using green fluorescent protein (GFP) and Quantum Dots (Q dots). However, GFP’s intrinsic nonsensitivity and photobleaching and Q dots’ large size allows kinesin’s movement to be viewed for a short amount of time. It is hypothesized that such drawbacks can be overcome by fusing kinesin with a fluorogen activating protein (FAP) coupled with a fused dye called a dyedron. A molecule of FAP is a portion of an antibody that contains a single chain variable fragment (scFV) where a dye such as malachite green was selected to bind. The malachite green is attached to four Cy3 molecules, which proportionally amplify the intensity of malachite green through fluorescence resonance energy transfer (FRET). The objective of my project was to create kinesin-FAP constructs to be coupled with dyedrons for future use in motility assays. The FAP sequence was amplified using PCR, where restriction sites were included in the primers. Utilizing these restriction sites, the FAP was digested then ligated to both the K412 motor domain of a kinesin1 vector and the TC910-952 tail domain fused to a thioredoxin region. These ligated constructs were then transformed into E. coli cells. The TC910-952-FAP construct containing a His-tag, was efficiently purified using phosphocellulose and a nitrilotriacetic acid (NTA) column. The K412-FAP construct was also cloned, but difficult to purify. Using a spectrophotometer, malachite green showed a red spectral shift from 617 nm to 656 nm upon binding to TC-FAP. The spectral shift was quantified and exhibited strong binding between FAP and the kinesin tail, showing it can serve as a reliable control for future motility assays that could lead to further understanding kinesin’s stepping mechanism.

Travis Lear

Travis Lear, Saint Mary's College of Maryland

Mentor: Jon Minden

Increasing the Sensitivity of Protein Identification using Immobilized Trypsin

Advances in two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS) have enabled unprecedented progress in proteomic research. Proteins found at lower concentrations such as transcription factors and cell signaling molecules are often responsible for critical cell events. Since physiological proteins exist in concentrations over nine orders of magnitude, researchers require methods with high dynamic range to identify and quantify these rare proteins while capturing the complete proteome of a cell. Researchers typically use 2DE to separate proteins in a sample by isoelectric point then by molecular weight. Proteins of interest need to be digested with a trypsin solution, typically for 16 hours, to be sequenced using MS. However, we observed that soluble trypsin exhibits autolysis, resulting in trypsin fragments that can hinder the identification of rare proteins in complex samples. We incubated trypsin to exhaust autolysis and assayed trypsin activity at multiple time points in a 24-hour period. The assay was accomplished by allowing trypsin to digest a fluorescent protein substrate whose breakdown products were quantified on SDS-PAGE at each time point. This workflow was applied to both soluble trypsin and trypsin immobilized on agarose beads. Soluble trypsin exhibits loss of activity after 24 hours while trypsin beads showed retention of activity after 20 hours, due to decreased autolysis. We were unable to conclusively identify our target protein through MS, due to sample contamination and high background.

Lauren MscibrodaLauren Mascibroda, Cedar Crest College

Mentor: Alan Waggoner

Floppy Fluorescein: A Novel Fluorogen Exhibiting pH Sensitivity

The Waggoner lab works with fluorogens: non-intrinsically fluorescent molecules which fluoresce when bound and held in a planar conformation by specific fluorogen activating proteins (FAPs). These fluorogens can be engineered to be sensitive enough to show changes in and around a cell, such as pH, depending on the FAP it binds. My intention was to find single chain variable fragments (scFvs) of human antibodies expressed on the cell surface of yeast that will bind a novel candidate fluorogen called floppy fluorescein, which is structurally similar to the dye fluorescein. Yeast expressing scFvs were sorted using flow cytometry to enrich the population for true FAP binding of the fluorogen. Floppy fluorescein was found to specifically bind and activate on certain yeast cells, being excited at and emitting different wavelengths of light depending on the FAP and its environment. We determined that floppy fluorescein is pH sensitive based on its spectral properties and behavior at differing pH levels.

Noah Most

Noah Most, Grinnell College

Mentor: Chuck Ettensohn

Expanding the Gene Regulatory Network in Sea Urchins (Strongylocentrotus purpuratus)

The embryonic development of the sea urchin has been rigorously studied. In fact, many consider our understanding of the morphogenesis of the Primary Mesenchyme Cells (PMCs)—those cells that are responsible for the secretion of the calcitic skeleton of the larval urchin—to be more complete than any other embryonic cell population. PMCs, the progeny of the large micromeres of the 32-cell stage embryo, depart the blastula wall during gastrulation; these skeletogenic cells ingress into the blastocoel and fuse with one another, aided by filopodial protrusions. The fusion of PMC filopodia creates a syncytial network that encloses a sequestered extracellular space. Into this space, PMCs deposit biominerals to produce two spicules, skeletal branches that influence the shape and movement of the pluteus larva. Yet, as detailed as our morphological understanding is, our comprehension of the molecular basis of these changes requires further inquiry. In humans, a number of pathologies stem from the disrupted regulatory control of biomineralization; thus, it is important to develop an in-depth understanding of the genes and proteins involved in the dramatic morphological changes of skeletogenesis. Others’ proteomic analysis of the spicule matrix, an organic mesh of proteins and carbohydrates that surrounds and is occluded within the spicules, has identified several candidates for participation in skeletogenesis. I employed whole-mount in situ hybridization to visualize the expression patterns of these genes to indicate the spatial and temporal properties of gene activity, and I analyzed the protein domains of each gene to elucidate potential gene function. For instance, I found a likely signaling ligand that contains a DSL domain with several EGF repeats and a potential spicule matrix protein with a CLECT domain. Overall, this research mapped the expression patterns of nine new genes, five of which were PMC-specific, and set the stage for more specific functional analysis.

Kedar Perkins

Kedar Perkins, University of Maryland, Baltimore County

Mentor: Phil Campbell

Relating Inflammatory Response to Stem Cell Differentiation

Inflammation is an important part of the wound healing process. However, the interactions between immune cells and progenitor/stem cells are not well established. To study the cross talk between such cells, immune cells were treated with lipopolysaccharide (LPS) or interleukin-10 (IL10) to drive cells towards a pro-inflammatory or anti-inflammatory phenotype, respectively. Four treatment groups consisting of control, LPS, IL10, and a combination of LPS and IL10 were used to generate conditioned media (CM), which consists of signaling molecules secreted by these cells under a pro-inflammatory or anti-inflammatory phenotype. In these experiments, CM from fetal skin dendritic cells (FSDCs; dendritic cells) and J774A.1 cells (macrophages) were cultured with myogenic stem cell lines such as C2C12 myoblasts on coverslips printed with the bone-promoting growth factor, BMP2. In such experiments, C2C12 cells underwent osteoblast differentiation on the printed BMP2 patterns as evidenced by positive staining for the osteoblast marker alkaline phosphatase (ALP). Although differences were observed between control, LPS, IL10 and LPS+IL10, this may have been due to uneven cell seeding densities. Therefore, this experiment needs to be repeated. To determine if LPS and IL10 have a direct effect on muscle derived stem cells (MDSC), cells were seeded into a twenty four well plate where LPS, IL10, and LPS+IL10 were administered with and without BMP2. Due to pipetting issues the experiment will need repeating. To evaluate how growth factors influence stem cell differentiation, dermamatrix strips were printed with growth factors and placed subcutaneously within mice with the goal of forming ectopic muscle, tendon, and bone. Although experiments are ongoing, the first set of histology results appear promising and will require further evaluation. These experiments will enable the better understanding of the inflammatory response and wound healing process may allow for better treatment and restoration of musculoskeletal injuries.

Teal Russell

Teal Russell, North Carolina State University

Mentor: Philip LeDuc

Endothelial Cell Micropatterning for Engineering Vascularized Tissue Constructs

Bioengineered tissues are often limited to thin-layer and avascular tissues (i.e. skin and cartilage) because it is extremely challenging to create a functional vascular network that supplies nutrients and oxygen to the cells that comprise complex organs. As a step towards engineering synthetic vascular networks, we have developed a novel method to fabricate microfluidic channels with circular cross-sectional geometries that are mimetic of microvascular networks. To build upon this progress, the goal of this project was to utilize these microfluidic channels to micropattern endothelial cells within a fibroblast-seeded scaffold, forming a vascularized co-culture system. Our first aim was to mold a polymer solution within the microfluidic channel to create a molded gel construct, which would serve as the structural template for forming a microvascular network. For initial proof of concept, a 20% gelatin solution was molded within a PDMS microchannel, and subsequently cross-linked upon demolding. Human umbilical vein endothelial cells (HUVECs) were seeded on the gelatin surface at a high density to promote attachment and cultured to form the microvascular network. Bright field and fluorescent confocal microscopy were used to assess molding precision and cell attachment and morphology. The second aim was to enclose the formed microvascular network within a cell-laden hydrogel, to create a vascularized tissue construct. The molded gelatin was fully enclosed within a collagen gel seeded with GFP+ rat dermal fibroblasts, successfully transferring the vascular-mimetic microchannel template to a biocompatible hydrogel system. These results help to establish a means for developing functional synthetic vascular networks towards the ultimate goal of engineering complex organ tissues.

Rosa Saad

Hala Rosa Saad, Brooklyn College

Mentor: Veronica Hinman

Characterizing the Role of Wnt Signaling at the Blastula Stage in Patiria miniata

The Hinman lab is trying to understand how axial patterning has evolved in sea urchins and starfish. The role of Wnt signaling in axial development is well understood in sea urchins but not in sea stars. At least 4 wnt genes are expressed at the vegetal pole endomesoderm in starfish, similar to where they are expressed in the sea urchin. In order to understand the role of Wnt signaling in starfish, we are altering canonical Wnt signaling in embryos. Overexpression of Δ-cadherin was used to prevent the nuclearization of b-catenin (the output of canonical signaling), and LiCl will be used to increase canonical signaling in embryos. Treated embryos will be examined using an in situ hybridization method, which assesses the spatial localization of mRNAs in embryos. We hypothesized that canonical Wnt signaling would be required for endomesoderm formation and would function to limit the extent of ectoderm. The results we obtained corroborated with our hypothesis. The Δ-cadherin treatments decreased the expression of genes expressed in the endomesoderm, while the expression of ectodermal genes increased. The LiCl treated embryos had an expansion of endomesodermal territories but a decrease in ectodermal, which is the exact opposite of what occurred in the Δ-cadherin treated embryos. This data shows conservation in the general role of the Wnt signaling pathway in endomesoderm formation in sea urchins and starfish. Subtle differences in the effects of Wnt signaling on the different germ layers in these two organisms suggest that this pathway is used differently in sea urchins and starfish. For future studies, we would want to individually turn off each Wnt gene in order to study the role of each gene separately.

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