2001 NSF Participants-Department of Biological Sciences - Carnegie Mellon University

2001 National Science Foundation (NSF)-supported Participants

Ashlee BakeAshlee Baker, Muskingum College
(Mentor: Dr. Alan Waggoner)

Optical biosensors: Investigations of the solvatochromic properties of fluorescent transducer dyes
The investigation of the solvatochromic properties of dyes is part of an interdisciplinary project that involves the development of a fundamental sensor unit technology. This fluorescent-based technology will provide an almost generic tool for detecting a wide range of important biological regulatory molecules in cells and in the interstitial spaces between cells. The fundamental sensor unit requires bringing together two components:

1. A structure that confers both recognition and selective binding of the target molecule at its physiological concentration
2. A "transducer" component that reports a fluorescence signal change upon the binding of a target to the selective binding component (ScFv).
a. Screening for transducer dyes: Twenty-two dye candidates, each composed of an acidic and a basic hererocycle, were selected to test. An Ultraviolet (UV) absorption spectrum was taken of each dye in water and in methanol. These solvents were chosen to detect polarity sensitivity of the dyes. The fluorescence emission was measured for those dyes with large wavelength shifts. Two dyes were found to be polarity sensitive fluorescent dyes and good candidates to continue studying. These dyes were:

Baker Image 44 Screening for commercially available thiol-reactive, environmentally sensitive dyes: Commercially produced dyes were next used to label protein 213C. It was hypothesized that the binding of the target would change the microenvironment of the protein enough to detect a fluorescent shift.

The first dye that was used was fluorescein which is a pH sensitive dye. The structure is:

Baker Image 45 A maleimide-functionalized fluorescein derivative was reacted with protein 213C. The protein was labeled with the dye then purified using size exclusion chromatography. The fluorescence was tested before and after the target antigen was introduced. A change in fluorescence was not detected before and after the binding. This may be because there wasn’t a significant pH change that occurred following the binding.

Next two polarity sensitive dyes were used to label the protein. These dyes were CPM and IADEANS.

Baker Image 46The IADEANS did not label the protein very well. The protein was labeled with the CPM, however the fluorescence measurements did not detect a large enough signal to show any emission changes.

The polarization of fluorescein was tested as well. Polarization is related to the molecular tumbling rate, which will be slowed when the receptor binds and restricts the motion of the dye. Polarization measurements did not show this behavior. Since fluorescein is highly hydrophobic it may have been pulled into the binding site, restricting its movement before the target was introduced.

c. Developing thiol-reactive dyes based on the screening data: Using the polarity studies previously conducted, a new dye was synthesized which will be used in future studies. Dye number 158, which was found to be polarity sensitive was chosen as the dye to develop in a thiol-reactive reagent. The dye had to have a maleimide moiety which would make it possible to bind to the protein. The distance of the maleimide group to the core-chromophore is a critical factor for the location of the sensor on the protein and thus on the dye’s sensing abilities. Different length linkers with a maleimide moiety can be synthesized and reacted with a carboxy-functionalized dye thus allowing the study of the optimal linker. In the first approach we synthesized a carboxy-functionalized merocyanine dye following the synthetic route given in the scheme:

Baker Image 47

Baker Image 48

The carboxy-functional dye was isolated and purified and its absorption measured. The dye was then activated and reacted with the maleimide-bearing linker. The resulting maleimide-functionalized dye was purified and its structure confirmed by 1H-NMR spectroscopy. Reacting it with papein, a protein which contains a free thiol group, tested the dye’s labeling ability. With the successful labeling of papein, protein 213C can now be labeled and the fluorescence measured.

Jennifer BaranowskiJennifer Baranowski, Grove City College
(Mentor: Dr. Peter Berget)

cDNA Analysis of CD-tagged NIH 3T3 Cell Lines
Central Dogma-, or CD-, tagging is a technique designed to study proteomics by inserting a CD-cassette containing an exon of interest into a cell's genome. When the CD-cassette is properly oriented in an intron of an expressed gene, it results in the addition of a unique guest exon to the gene primary transcript and the mature mRNA, as well as the addition of a unique guest peptide to the encoded protein. The triple tagging procedure opens many lines of research, as the tag provides a handle by which to collectively study the central dogma at all three levels. This summer project utilized a specific CD-cassette, which is carried by the stealth 1.0 retroviral vector and includes exons for both a short peptide epitope and enhanced green fluorescent protein (EGFP). The EGFP exon forms a fluorescent protein tag, which allows in vitro visualization of the tagged protein's cellular localization by fluorescent microscopy. The goal of this project was to perform molecular analyses on the mRNA of various tagged NIH 3T3 cells to identify the gene into which the stealth 1.0 vector has integrated in each cell line. First, the total RNA was extracted from the tagged cells, and then cDNA copies of the mRNA were synthesized. The cDNA was used as template in nested RT-PCR to amplify the EGFP exon and the downstream cellular gene that was tagged. PCR products were analyzed by agarose gel electrophoresis. Single band PCR products were sequenced directly; multiple band PCR products were cloned via a vector and transformed into E. coli cells to isolate the desired PCR product for sequencing. For each DNA sequence, a database search was performed to identify the tagged gene and encoded protein. In cases where the protein has been previously characterized, its functions can be compared with its localization, determined by fluorescent microscopy. For each uncharacterized tagged protein, the known cellular localization of the protein can lead to hypotheses of its function. In future research, experiments can be performed on each tagged cell line as well as on purified, tagged protein to further study gene expression and function.

Tasha BreauxTasha Breaux, Northwestern State University
(Mentor: Dr. Amy Csink)

A Comparison of Satellite Sequences Found in Drosophila melanogaster and its Sibling Species and an Analysis of these Species' Ability to Interspecifically Mate
One goal of the Csink lab is to determine the reason for the association of the brownDominant gene with the heterocromatin. Because the brownDominant gene is essentially the wild-type brown gene with a 1.6 Mb insert of the heterocramatic sequence AAGAG, this association could be the result of either of two things. The association could be due either to a simple association of heterocromatin or to the association of the satellite sequence AAGAG, which appears in the pericentric heterochromatin of the second chromosome of D. melanogaster. To test this, it is first necessary to obtain Drosophila which contain the brownDominant gene but do not contain the satellite sequence AAGAG.

The goals of this project have been two-fold. First D. melanogaster was crossed with two sibling species (D. simulans and D. mauritiana) and the ability of this cross to produce progeny was examined. While crosses of this type have been previously performed, they have not been done with the brownDominant gene present. These hybrid flies were then backcrossed with the sibling species to create a fly of a sibling species to D. melanogaster that contains the brownDominant gene but not the satellite sequence AAGAG, since this particular sequence is not seen in the pericentric heterochromatin of the second chromosome in the sibling species. To verify this, the second aspect of this project used fluorescent in situ hybridization to determine the presence or absence of various satellite sequences in the specific fly lines that were involved in the interspecific mating.

Despite the presence of the brownDominant gene, F1 progeny were obtained from the interspecific mating of D. melanogaster with each of the sibling species. At this time, the backcross of these interspecific flies with the sibling lines is underway. Also, a great deal of variation among the satellite sequences was detected between the different species of flies examined as well as between different lines of flies within the same species.

Tara BrownleeTara Brownlee, Lebanon Valley College
(Advisor: Dr. Frederick Lanni)

Imaging and Mathematical Analysis of Collagen Gel Deformations
All multi-cellular organisms are composed of both cells and an extracellular matrix (ECM). The ECM is formed and remodeled by cells, especially during embryonic development. Collagen is the major constituent of the ECM and connective tissue. It makes up 25% of the total protein mass in mammals. Type I collagen, the most common type and the one used in this project, makes up 90% of the body’s collagen, and is the collagen of skin and bones. Type I collagen molecules assemble to form a right-handed triple helix made up of left-handed glycine and proline-rich helices.

In Dr. Lanni’s lab the interest is in the mechanics of cells within a collagen gel. This involves how cells move and reshape the ECM. Collagen is used as a model ECM and, in order to understand how cells shape the ECM, there first needs to be an understanding of collagen in terms of its mechanical properties.

This summer project involved making collagen gels of known concentration, imaging the gel network under a microscope, and then producing deformations in the gel. A glass microneedle in a motorized micromanipulator was used to apply a highly localized load (force) tangent to the surface of the gel. After imaging the deformations with a video camera attached to the microscope and imaging software called STC-View, the next step was to input the image files into the Deformation Quantification Algorithm (DQA), a computer program developed by Steven Vanni in Dr. Lanni’s lab. The DQA takes the sequential image pairs and produces a field of vectors that show how the gel moved when the load was applied.

The main goal this summer was to produce deformations that would (1) give the best output from the DQA and (2) correspond to known idealized solutions of the elasticity equations. Different types of movements with the microneedle were used to get deformations that the DQA was capable of tracking well. The reason for getting good DQA output was to enable my collaborator on the project, Jennifer Airone, to match my data to her simulated materials with greater accuracy.

From the different types of movements made with the needle and through much trial and error, it was discovered that to produce the best deformations, and subsequently the best DQA output, the needle needed to be (1) moved to the edge of the field of view and (2) moved perpendicular to the edge of the field of view as opposed to parallel. Also, the best output was achieved when a mask was used to block out the site of the needle for the DQA. Lastly, the most important thing discovered was that small, controlled movements, such as those made by using the step feature on the micromanipulator, produced the best output from the DQA.

After discovering how to produce the DQA output, the next step will be to match the actual data to computed mathematical simulations of the movement of the collagen gel. Once the mechanical properties of collagen are known, they will be used to predict the pattern of movement for cells in the collagen gel. This will help researchers to better understand the movement of fibroblasts and other non-muscle locomoting cells not only in collagen gel but also in real tissues and organs.

Pauline ChughPauline Chugh, Millikin University
(Mentor: Dr. Jonathan Minden)

Investigation of developmental cell death in wildtype and mutant Drosophila embryos

In this project, I studied the process of apoptosis during Drosophila melanogaster embryogenesis. Apoptosis is a highly regulated process that is required for proper development. During development, apoptosis ensures that tissues develop with the correct number of cells. The regulation of apoptosis may be linked to cell proliferation. For example, if there is ectopic proliferation, cell death may increase to compensate whereas a reduction in proliferation would result in a corresponding reduction in cell death. Dead cells are removed by phagocytic cells (macrophage) by a process called phagocytosis, or engulfing of the cells.

I used two different Drosophila mutants to investigate regulation of cell death and phagocytosis. The first of these mutants is the string (stg) mutant. The string protein is responsible for regulating cell division after the 14th division. The first 14 divisions are regulated by maternal proteins. However, cell division in embryos after the 14th division is regulated by the zygotic string protein. String mutants lack the zygotic string protein and therefore there are no more cell divisions following division 14. I used Acridine orange, a fluorescent marker that labels dead cells, to sudy cell death in stg embryos to see if cell death compensates for less cell division in these embryos. The fluorescent marker was injected into both wild-type and mutant embros and they were then analyzed using fluorescence 4D-microscopy.

The second mutant, myoblast city (mbc), has cytoskeletal defects that result in a lack of myoblast fusion. The mbc gene encodes a protein that is nearly homologous to the human protein DOCK180, which is also involved in myoblast fusion. When the mbc gene is missing, the fusion of myoblasts into multinucleate muscles is virtually nonexistent. I hypothesized that the cytoskeletal abnormalities would cause a defect in the removal of dead cells by inhibiting the ability of phagocytic cells to wrap around and engulf dead cells. I studied two different mbc mutations, mbcC1 and mbcC2, and examined the rate of engulfment and macrophage function. Vgal, a fluorescent marker that labels phagocytic cells, was injected into both wild-type and mutant embryos to observe the process of phagocytosis. The injected embryos were then analyzed using fluorescence time-lapse microscopy. The time-lapse movies indicate that macrophage in mbc mutant embryos take nearly twice the amount of time to engulf dead cells and show a reduction in movement compared to wild-type embryos. These results suggest that mbc mutants exhibit defects in phagocytosis as well as myoblast fusion.

Roslyn CrowderRoslyn Crowder, Florida A&M University
(Advisor: Dr. William Brown)

Single Chain Variable Fragments (ScFv) and the use of Fluorescent Dyes
Toluene diisocyanate (TDI) is a molecule that contains reactive isocyanate groups (N=C=O). Diisocyanates contain two of these highly reactive functional groups per molecule. They are commonly used in both industrial and commercial applications to manufacture products such as paint and polyurethane foam. The cause of occupational asthma in workers, who come in contact with these chemicals, has been contributed to these compounds.

Single chain variable fragments (ScFv) have been engineered in Dr. William Brown’s laboratory to recognize the bonds between TDI and amino acids, particularly lysine. It was then useful to screen the previously prepared ScFv library to find a specific ScFv that recognized the bond between TDI and the amino acid serine. This is important because with a hydroxyl group with pKa 14, serine is predicted to be the amino acid that is predominantly modified by TDI in vivo. The screening procedure termed phage panning utilizes the ability to specifically recognize an antigen. The antigen in this case encompasses a P-Tolylmonoisocyanate (TMI) conjugated to N-acetyl Serine that is covalently linked to Bovine Serum Albumin (BSA).

After finding such an ScFv, we used it as an in vivo biosensor to locate TDI conjugates. This required the use of fluorescent dyes to follow the ScFv. Therefore, another portion of our research was dedicated to linking Thiol-reactive fluorescent dyes at various cysteine locations engineered around the active site of the ScFvs. We also used enzyme-linked immunosorbent assays (ELISAs) to determine which binding sites can be labeled without affecting target binding. We also tested different fluorescent signals that respond to the binding events, which include pH changes, solvent polarity, and molecular tumbling rate.

TMI only contains a single isocyanate group while TDI has two. Isoelectric focusing (IEF) gels and absorbance readings at 240 nm were to make certain that both the serine and lysine conjugates were similar in conjugation. Using ELISAs with both TMI-Serine and TMI-Lysine conjugates, the ScFv #12 was tested on its binding specificity.

Jermaine JonesJermaine Jones, University of Virginia
(Mentor: Dr. Jonathan Jarvik)

Improving the Efficiency of Retroviral Infection of NIH3T3 Cells using Partially Synchronized Cultures
With the current method of CD-tagging by infecting asynchronous cultures of NIH3T3 cells with the Stealth 1.0 retrovirus, about 0.1% of the infected cells become positive for EGFP (Green Florescent Protein). My goal was to attempt to raise this efficiency by synchronizing the cells and infecting them at an opportune time in the cell cycle. The time just prior to mitosis was chosen on the basis of anecdotal reports indicating (1) that integration of the proviral DNA requires nuclear membrane breakdown, and (2) that integration-competent proviral DNA survives in the cell for no more than four hours. If true, these factors would be expected to limit the times of productive infection to the period immediately before and during mitosis.

Cultures of NIH3T3 cells were subjected to a synchronization protocol consisting of serum starvation for forty-eight hours to produce cell-cycle arrest, followed by serum restoration (stimulation) to release the cells from arrest. Twenty-one hours later, when the synchronized cells were about to enter mitosis, they were infected with the Stealth virus. The number of EGFP-positive CD-tagged clones was determined by fluorescence microscopy and compared with that of asynchronous control cells infected in parallel. Preliminary data suggest that there was a greater CD-tagging efficiency in the synchronized cells as compared to the unsynchronized controls.

To examine the effectiveness of the synchronization protocol, asynchronously growing cells were fluorescein-labeled using CMFSE and then serum-starved for two days. Flow cytometric analysis revealed that approximately half of the cells arrested in a G0-like state - but that the other half continued to divide — i.e., they were not growth-arrested and therefore not subject to synchronization when serum was restored. These results suggest that it may be possible to further improve CD-tagging efficiency by employing a more effective synchronization protocol.

Catherine LewisCatherine Lewis, University of Virginia
(Mentor: Dr. Peter Berget)

Construction of Modified Stealth Vectors for CD-Tagging
Central Dogma-tagging, or CD-tagging, is a new approach for gene discovery and analysis that puts specific molecular tags on a gene, its RNA transcript, and its protein product. The current method of CD-tagging uses a retroviral vector, Stealth 1.0, to deliver a specialized CD cassette, which includes a universal epitope sequence and a sequence for an enhanced green fluorescence protein. The current version of the vector can only successfully GFP tag proteins if the vector is inserted into type 0 introns. This vector is only capable of GFP labeling a limited number of genes and its products. However, two new versions of the Stealth vector, Stealth 1.1 and Stealth 1.2, have been constructed. These two new vectors are capable of GFP tagging proteins in which the vector has been inserted into type 1 and type 2 introns, respectively. The target cells for this study are mouse NIH-3T3 cells. In order for CD-tagging to be successful, the insertion of the cassette must be within an intron of a gene and oriented in the same direction as transcription. At the molecular level, it is important to determine the location of the tag in the sequence of the gene that was tagged and eventually determine the function and true identity of the gene. So far, this has been successful at the mRNA level. Mouse NIH-3T3 cells that have been CD-tagged are currently available. Cells that have been CD-tagged are currently being identified using fluorescence light microscopy. This method of identifying tagged cells is very time-consuming and subject to different interpretations. As a result, it was of interest to construct a special, modified Stealth vector that encodes for puromycin drug resistance. The construction of this vector will allow for the selection of cells in culture that have been tagged. A method called polyA trapping was built into the construction of this puromycin resistant vector. The puromycin resistance gene was inserted into Stealth 1.0 at two different restriction enzyme sites in order to test which of the two would be most efficient at polyA trapping the puromycin resistance gene.

Maricel MartinezMaricel Martinez, Universidad Metropolino
(Mentor: Dr. David Hackney)

Introduction of a second microtubule binding site into conventional kinesin
Kinesins are microtubule (MT) dependent motor proteins that posses MT-stimulated ATPase activity and that are known to be involved in intracellular transport of synaptic vesicles along axons, organelle transport, mitosis and meiosis. Subfamily member BimC is an essential bipolar mitotic motor that has an extra 72 amino acid extension that is positively charged and has shown to be homologous to known microtubule binding domains. During previous investigations this extra domain affinity properties was addressed by comparing the effect on ATPase rates between bimC proteins with and without the 72 amino acid region. Results indicated strong binding to MT with this extra region. During this project the role of this extra domain was studied by different plasmid constructs containing the charged extra region but lacked the microtubule homologous region. ATPase assays were performed on protein extracted from plasmid construct starting at amino acid position 66 containing the highly charged region and demonstrated significant ATP regeneration , which is an indirect confirmation of the protein binding to microtubules at different concentrations. In comparison with the previous investigation results this highly charged region produces tighter affinity to the microtubules. This information will help to further understand and characterize the function of these dominions on these proteins therefore address different physiological conditions caused by ineffective transport of intracellular contents.

Elizabeth OttesenElizabeth Ottesen, Grinnell College
(Mentor: Dr. Gordon Rule)

Characterization of Active Site Dynamics in the Human Glutathione Transferase M2-2
The glutathione transferases are a family of enzymes that catalyze theaddition of glutathione to a wide variety of hydrophobic compounds. This process is vital for the degradation of endogenous cellular toxins, carcinogens, and environmental pollutants. However, these same processes have been implicated in the deactivation of anti-cancer drugs and the development of treatment resistant cancers. The purpose of this study was to investigate the catalytic mechanism of the human glutathione transferase M2-2. Isotopically labeled enzyme was generated and used for NMR-based characterization of active site dynamics. In addition, mutant forms of GST M2-2 were produced to further investigate the roles of four methionine residues located within the active site.