2010 Summer Scholar Participants-HHMI Undergraduate Program - Carnegie Mellon University

2010 HHMI Summer Scholar Participants

Turi Alcoser

Turi Alcoser, Carnegie Mellon University

Mentor: Dr. Kris Dahl

Nuclear Organizational Response to Compressive Forces in HeLa & Saos-2 Cells.

Tissues and cells within our body experience a wide range of forces which affect their phenotype. The nucleus of a cell shows unique viscoelastic deformation under applied stress through coordinated deformation of nuclear subunits. Understanding the deformation of the genome may correlate to gene placement in the nucleus and further gene expression. HeLa cells were transfected with Upstream Binding Factor-1 tagged with green fluorescent protein (UBF1-GFP) for use as fiducial particles which were tracked every 2 minutes for 2 hours using live cell imaging. Cells were also transfected with DsRed-progerin to artificially stiffen the nuclear lamina shell. Compressive forces from 20 and 100 grams weights were applied to the cells and the corresponding changes in subnuclear movements were measured. Under 20 grams of compression cells expressing progerin showed no significant difference with control cells cells in average particle mean square displacement (MSD; p < 0.05 by Student’s t-test). Alternatively, 100 grams of compression caused a significant difference in the MSD for the first 18 minutes and last 18 minutes of the first hour of compression between control and progerin-expressing cells. This suggests that progerin expression reduces the cells ability to reorganize its genome under high force. Also, the MSD profiles for progerin exhibit discrete organizational response rather than a continuous MSD profile in control cells, consistent with previous observations of unique failure modes (“cracks”) in patient cells expressing progerin. Under low and high compressive forces the nucleus is able to adapt by reorganizing its subunits through mechanotransduction of the nuclear membrane. Future work includes introducing our model of compression in successfully transfected Saos-2, osteosarcoma cells to measure nuclear organizational response in cells conditioned for compressive response.

Allyson Dill

Allyson Dill, Carnegie Mellon University

Mentor: Dr. Mark Macbeth

Structural Analysis of Adenosine Deaminases that Act on RNA

Adenosine deaminases that act on RNA (ADARs) catalyze the deamination of adenosine to inosine in double-stranded RNA substrates. Since inosine is interpreted as guanosine during translation, ADAR activity diversifies an organism’s proteome. In fact, ADARs are required for proper neuronal function. My research focuses on human ADAR1 (hADAR1), which consists of a C-terminal catalytic domain, three double-stranded RNA binding motifs, and two N-terminal Z-DNA binding motifs. The goal of my project is to use X-ray crystallography to solve the structure of hADAR1 in order to illuminate the enzyme’s mechanisms of substrate specificity and catalysis. I am currently working with two constructs: M296, a native truncation that lacks the Z-DNA binding motifs and functions in the nucleus, and S823, which consists only of the catalytic domain. Each construct has been cloned with an N-terminal histidine tag followed by a Tobacco Etch Virus (TEV) protease cleavage site. A galactose promoter allows for overexpression of the enzymes to be induced in S. cerevisiae strain BCY123. The purification procedure employs nickel, cation exchange, and gel filtration chromatography. Perhaps due to both misfolding and precipitation, attaining a high yield of either construct has been a struggle. I’ve experimented with various buffers for each purification step, and have learned that high salt helps to solubilize the protein. This summer I was able to get the more difficult construct, M296, into storage and achieved a yield of pure S823 sufficient for crystal trays. With that sample I have screened 2592 different conditions for crystals, and am currently monitoring the trays in hopes of identifying a promising condition to focus on in the future. I will soon clone a shorter construct, L833, to eliminate a random coil predicted by a secondary structure algorithm. This should increase the enzyme’s stability and aid in crystal formation. Solving the structure of hADAR1 will give us a wealth of information with which to understand how this RNA-editing enzyme functions.

Lyndsey Gray

Lyndsey Gray, Carnegie Mellon University

Mentor: Dr. Gordon Rule

Human Tear Lipocalin for Fluorogen-based Cellular Imaging

One of the more recent approaches for constructing new fluorescent imaging tools in living cells is the use of fluorogen-activating single-chain antibodies (FAPs). FAP domains have demonstrated successful specificity for fluorogens and, upon binding, produce fluorescence enhancements that create clearer cellular images and simplified real-time tracking of protein trafficking. However, their domains require intra-cellular disulphide bonds, thereby decreasing the FAP’s overall utility as a fluorogen receptor. Based on their structurally similarity to antibodies, small size, ligand specificity, and reduced dependence on disulphide bond formation, it is believed that lipocalins could serve as a viable candidate to supplement single-chain variable fragment-based FAP domains. More specifically, it is hypothesized that the human tear lipocalin (Tlc), which is characterized by its promiscuity in ligand binding, could undergo mutagenesis so that its binding pocket could accommodate the fluorogenic dyes malachite green or thiazole orange. After generation of a coding sequence with yeast optimized codons, the immediate goal is to successfully insert this coding sequence into a yeast display vector, pPNL6. This assesses the ability to express Tlc on the surface of yeast cells, a necessary step if one is to select variants that could possibly bind fluorogenic dyes. Through this process, it is expected to either discover a specific Tlc mutant that can be used to improve the fluorogen imaging technique or, at the least, to garner further information towards Tlc’s ligand specificity, a relatively new and unknown topic in protein biochemistry.

Katlin Griswold

Katlin Griswold, Carnegie Mellon University

Mentor: Dr. Aaron Mitchell

The Effects of pH on the Localization of Rim101 during Activation by Proteolytic Cleavage

In fungi, Rim101 is a transcriptional repressor that regulates cellular response to environmental pH. This project examines the subcellular localization of Rim101 during its interaction with the protease Rim13 and regulator Rim20, two key proteins essential for activation of Rim101 by proteolysis. Previous research indicates that Rim20 and various additional components required to form this trimeric complex localize on endosomes, the potential site for Rim101 activation. We hypothesized that Rim101 indeed localizes on the endosomal surface, perhaps transiently, as a part of this complex and the cleavage event of Rim101 triggers the protein’s dissociation. Thus, presumably, non-cleavable versions of the protein would remain on the endosome for longer periods of time, enabling in vivo visualization. We created fusion constructs containing GFP with either a cleavable or non-cleavable form of Rim101 by gap repair and employed restriction enzyme digest, sequencing, and western blot for verification. The plasmids were then transformed into wild type and rim13Δ yeast strains and analyzed using live fluorescence microscopy. The data obtained indicated that Rim101 cannot be observed on the wild type endosomes because it is cleaved too quickly and/or due to transient binding. In the rim13Δ yeast, both versions of Rim101 can be partially localized under neutral and alkaline conditions. Further experiments to colocalize Rim101 with genuine endosomal proteins are underway.

Stephanie Guerra

Stephanie Guerra, Carnegie Mellon University

Mentor: Dr. Charles Ettensohn

Gene Expression Patterns in the Non-Skeletogenic Mesoderm of Lytechinus variegatus

The non-skeletogenic mesoderm (NSM) of Lytechinus variegatus gives rise to four cell subpopulations including the pigment, blastocoelar, coelomic pouch, and circumesophageal muscle cells. At the hatched blastula stage, the NSM cells are surrounded by the endoderm and are present in a ring at the vegetal plate surrounding the presumptive primary mesenchyme cells (PMCs). Much has been revealed regarding the segregation of the endomesoderm prior to PMC ingression. However, not much is known concerning the specification of cell types within this mesoderm population. Observation of genes specifically expressed in the pigment cells (gcm, pks) and blastocoelar cells (scl, GATAC) was conducted using two-color fluorescent whole-mount in situ hybridization (F-WMISH) with the ultimate goal of elucidating temporal gene expression patterns. The preliminary results confirm previous findings that prior to mesoderm specification, gcm is expressed in a ring at the vegetal plate, followed by its restriction to an aboral crescent. Scl is first expressed in the mesenchyme blastula in an oral crescent. This project has found that pigment cells and blastocoelar cells form a complete and equally divided ring at the mesenchyme blastula vegetal plate.

Interestingly, genes initially expressed in the PMCs, ets1 and erg, are later expressed in cells of the NSM. Ets1 is responsible for the epithelial-mesenchymal transition (EMT) of the PMC cells. Since both pigment cells and blastocoelar cells exhibit a similar though later EMT, it would be assumed that they also express ets1 at some point. From previous analyses it appears that pigment cells do not express ets1. This project conducted F-WMISH of erg, a member of the ETS family of proteins to see if pigment cells instead used this related gene for EMT. The images collected showed that erg expression, while present in blastocoelar cells, was absent in pigment cells. This suggests a different mode of EMT for pigment cells.

David Huang

David Huang, Carnegie Mellon University

Mentor: Dr. Aaron Mitchell

Identification of New Candida albicans Adherence Genes

The diploid fungus Candida albicans is a commensal fungus that is benign for most people most of the time. However, C. albicans becomes pathogenic when immune function is impaired or if an environmental niche becomes available. Pathogenicity of C. albicans is attributed to its ability to form surface-bound microbial communities called biofilms. Biofilm formation on medical devices causes severe impacts for human health by providing both an entry to the body and a sanctuary for invasive pathogens. In addition, biofilms have increased resistance to many antifungal agents compared to free floating planktonic cells. C. albicans biofilm formation begins with the adhering of yeast form cells to a substrate, such as a medical device or a catheter. This adherence provides the foundation for the development of a mature biofilm. The goal of my project was to identify genes that function in C. albicans’ adherence to a substrate. To accomplish this goal, I focused on two groups of genes: transcription factors and cell wall genes. Transcription factors play a significant role in the regulation of adherence. The role of cell wall genes in adherence can be linked with its cell wall structure. Using an established adherence assay, three transcription factor mutants exhibited significant reduction in adherence in yeast form cells. The three mutant strains were then assayed for biofilm formation and other stress assays to test for defects in cell wall formation and integrity. Hopefully, the knowledge gained from the identification of new C. albicans adherence genes can identify targets for drug development that will aid in the treatment of patients who suffer from systemic C. albicans infection.

Daiji Kano

Daiji Kano, Carnegie Mellon University

Mentor: Dr. Mark Macbeth

A Structural Approach to Determining the Mechanism of Substrate Recognition by Adenosine Deaminases that Act on Ribonucleic Acids

The goal of my project is to determine the structure of Human Hepatitis Delta Virus RNA editing substrate (HDV), one of the substrates for Adenosine Deaminase that acts on RNA (ADAR), by x-ray crystallography, in hopes to better understand the mechanism by which ADAR deaminates its substrates. ADAR only deaminates specific adenosines and we suspect that this high specificity is dependent on RNA structure (rather than sequence) such as a unique hairpin pattern. In order to crystallize the HDV RNA, I fused the U1 RNA loop, which binds to the U1A protein, to the HDV RNA in order to facilitate the crystallization of the entire complex. The production of HDV-U1 was done by in vitro transcription and nickel column purification of the construct (glmS construct) composed in the following manner—from 5’ to 3’—HDV-U1, glmS ribozyme, and MS2. The RNA purification process is summarized as follows: the MS2 region binds to the hexahistidine-tagged MBP-MS2 coat fusion protein (HMM), which binds to the nickel column for immobilization of the entire construct. The glmS ribozyme is activated upon the addition of glucosamine-6-phosphate (GlcN6P) and it cleaves off the 5’ HDV-U1 RNA, which is then eluted and ethanol-precipitated for storage. So far, I have optimized the purification protocol of my RNA and purified enough RNA and U1A protein for crystallization attempts; I was able to greatly improve the cleavage efficiency of the glmS ribozyme, but not the premature transcription termination. Currently, I am determining the optimal crystallization conditions for HDV-U1. Future goals include the selection and x-ray crystallography of high-quality crystals of HDV-U1 in hopes to solve the 3D structure of the RNA, which in turn may help elucidate the mechanism of ADAR activity.

Claire Koechlein

Claire Koechlein, Carnegie Mellon University

Mentor: Dr. Veronica Hinman

Investigating Cell Migration in the Development of Sea Star Larval Nervous System

The sea star ciliary bands are comprised of two rows of ciliated ectodermal cells that surround the mouth and are associated with the larval nervous system. Neurons are scattered throughout the ectoderm in the early development and later seemingly aggregate to the two ciliary bands. Cell migration to the ciliary bands has not been directly shown. However, in vertebrates, which share a distant common ancestry with the sea star, cell migration over long distances is known to occur. For example, the vertebrate neural crest (NC) is a migratory population of cells derived from ectoderm. The goals of this research are to: 1) determine if cell migration occurs within the ectoderm of the sea star and 2) examine the expression of sea star orthologs of the NC using whole mount in situ hybridization (WMISH). Using lineage tracing, cell migration was observed in the mesoderm and possibly in the apical-most ectoderm. WMISH revealed that most of the NC orthologs are expressed in the sea star mesoderm. Two NC orthologs ap2 and id showed strong expression in the ectoderm. Id was detected in the apical ectoderm and a ring of ap2 expression at the border between the oral and aboral ectoderm was observed. These results suggest that the potential for migration within the ectoderm may not be equal, as cells might migrate from the apical-most ectoderm but not from other regions. Furthermore, as orthologs of NC regulators generally do not appear to be expressed in the ectoderm, it is likely that primitive NC cells arose after the divergence of vertebrates and sea star.

Jesse Lawrences

Jesse Lawrence, Carnegie Mellon University

Mentor: Dr. Javier Lopez

Effect of Recursive Splice Sites on Processivity of RNA Polymerase II

Recursive splice sites (RSSs) consist of overlapped 3’ and 5’ splice sites that function sequentially and cotranscriptionally for stepwise removal of long introns (>10kb). Deletion of a RSS in the Ubx gene of Drosophila produces a loss-of-function phenotype that is specifically enhanced by heterozygosity for a mutant allele of RNA Polymerase II (RPII-215C4) that has reduced ability to overcome blocks to elongation. These results suggest that RSSs may stimulate transcriptional processivity. This hypothesis is also consistent with the observation that RSSs are generally located approximately 1kb upstream of peaks in Pol-II occupancy that may reflect blocks to elongation. Since homozygous RPII-215C4 does not enhance the RP3 deletion phenotype, the enhancement in heterozygotes may result from interference between normal and mutant polymerases transcribing the same template. I am using a cell culture system to investigate the relation between RSSs, transcriptional elongation and the RPII-215C4 effect. The system involves two Drosophila cell lines: Schneider-2 (S2), which has only wild-type RPII-215, and a derivataive (S2C4) that is stably transfected with RPII-215C4. The C4 polymerase is resistant to inhibition by α-amanitin; wild-type polymerase is sensitive. Thus, I can compare transcription of endogenous or transfected genes by wild-type polymerase (S2 cells), by C4 polymerase (S2C4 with α-amanitin), or by a mixture of wild-type and C4 polymerases (S2C4 without α-amanitin). Preliminary results with a transfected Ubx minigene indicate that accumulation of their mRNAs is reduced upon transcription by mixed C4 and wild-type polymerase compared to either polymerase alone, supporting the hypothesis of interference. To investigate the role of RSSs, I will perform similar experiments with a Ubx minigene from which the RSS has been deleted and I will compare transcripts from endogenous genes that have long introns with or without RSSs.