University of Notre Dame
Department of Biology
NSF Summer Undergraduate Research Program

Summer 2002 Symposium Abstracts

Characterization of G-Protein Couple Receptors in the Malaria Mosquito Anopheles gambiae

Perciliz L. Tan, Catherine Hill, Frank H. Collins

In numerous African countries, millions of people die of the malaria parasite, Plasmodium spp., transmitted by the vector mosquito, Anopheles gambiae. Programs to eradicate malaria were initiated in the 1950's and have been successful in some parts of the world. Unfortunately, these programs have proven to be ineffective in other third-world locations, particularly Sub-Saharan Africa. The genome of the A. gambiae has recently been sequenced in an effort to understand the biology of the mosquito, its relationship to the parasite, and to identify new mechanisms for control of both these organisms. G-protein coupled receptors (GPCRs) are known to be the molecular target of many drugs. GPCRs, the largest family of proteins, play an essential role in biology, physiology, and pathophysiology, and are expressed throughout various tissues. Generally characterized by their unique seven transmembrane-domain structure, once activated by ligands, GPCRs initiate numerous cascades of signaling events that involve various pathways. We have specifically identified, annotated, and catalogued the repertoire of A. gambiae GPCRs using bioinformatic approaches. To gain a further comprehension of the GPCRs in A. gambiae, a bioinformatic algorithm was employed to identify "novel" GPCRs that were not found by amino acid similarity searches. Further, A. gambiae orthologs of Drosophila melanogaster GPCRs were used in a series of RT-PCR experiments. The ultimate goal of this research is to identify and characterize A. gambiae GPCRs as chemotherapeutic targets that may lead to the development of novel control mechanisms for both A. gambiae and the Plasmodium parasite.

Analysis of Cytoplasmic Dynein Subunit Expression in Mouse Macrophages: Cloning and Characterization of Intermediate Chain and Light Chain Subunits

Matthew Bayer

Cytoplasmic dynein is a multisubunit microtubule motor protein responsible for centripetal organelle transport during interphase and chromosome segregation during mitosis. Previous cloning and biochemical analysis revealed that the heavy chain (HC) subunits mediate the motor activity of the complex, whereas intermediate chain (IC), light intermediate chain (LIC) and light chain (LC) subunits are thought to mediate cargo binding. The Vaughan laboratory is interested in how the dynein motor binds cargo, and how cargo binding is regulated. Because of recent findings, we have focused on the ICs and LCs as cargo-binding subunits. As one model system to test models of cargo binding, the Vaughan lab uses latex beads-loades phagosomes purified from mouse macrophages. To define the IC and LC isoform expression complexity in mouse macrophages, we performed RT-PCR analysis using diagnostic PCR primers that straddle the coding sequences of the LCs and regions of alternative splicing in the ICs. Total RNA was harvested from the J774 line of mouse macrophage cells, and used to prepare cDNA. RT-PCR was performed using IC1, IC2, tctex, LC7, and LC8-specific primers derived from previous cloning. The resulting cDNAs were subcloned into a plasmid vector, amplified in E. coli host cells, sequenced, and aligned by BLAST and BESTFIT analysis. For the ICs, only the IC2c isoform was expressed, suggesting limited complexity. For the LCs, PCR products suggested expression of a single member of each class. Together these results suggest limited dynein subunit isoform complexity in mouse macrophages, and provide a foundation for dynein reconstitution assays using the cloned sequences.

“Guilt by Association: Establishing a Hierarchy Among Microtubule “Tip-Tracking” Proteins During Interphase”

Heather L. Wieman, Patricia S. Vaughan, and Kevin T. Vaughan

Recent work from several groups suggests that microtubule binding proteins at the kinetochore assemble into a hierarchy which is essential for normal chromosome segregation. The same proteins assemble at microtubule plus-ends during interphase and exhibit microtubule “tip-tracking” behavior, however the hierarchy of these proteins is not known. To gain insight into this hierarchy, we used three novel approaches and analyzed microtubule binding of several proteins. First, applying recent knowledge of regulated binding of p150^GLUED to microtubules, we used p150^GLUED mutants to explore the role of regulation in the hierarchy of these “tip-tracking” proteins. Using immunofluorescence microscopy, we found that transfection of the “dephospho” mutant resulted in diminished EB1 and CLIP-170 microtubule binding, whereas transfection of the “phospho” mutant had no impact. To determine if dynactin was required for the binding of other proteins, we utilized the small interfering RNA (siRNA) technique and designed several constructs to ablate p150^GLUED gene expression. Fluorescence microscopy of cells cotransfected with GFP and siRNA constructs revealed diminished p150^GLUED and EB1 at microtubule tips, but normal levels of CLIP-170. To test the function of APC at microtubule tips, we analyzed GFP-EB1 and GFP-p150^GLUED by live cell imaging in human colon cancer cells harboring APC mutations. GFP-EB1 labeling was shorter than normal in both cell lines, however SW480 cells displayed longer “comet tails” than the metastatic SW620 cell line. Both cell lines exhibited microtubule decoration by GFP-p150^GLUED, indicating abnormal phosphorylation kinetics in these cells. Our data suggests that an assembly hierarchy does exist among proteins that bind to the plus-end of microtubules, and that APC influences regulation of microtubule binding.

Identification of 25-hydroxyvitamin D₃ 1-a-Hydroxylase in Breast Cancer

Kelly Smith

JoEllen Welsh, advisor

The active form of vitamin D₃, 1a,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] is known to cause differentiation, growth arrest, and apoptosis in mammary epithelial cells. This has created interest in the therapeutic use of 1,25(OH)₂D₃ for breast cancer. However, the side effects of treatment with 1,25(OH)₂D₃, including high serum calcium levels, limit the feasibility of therapy with this form of vitamin D₃. Therefore the prohormone, 25-dihydroxyvitamin D₃ (25-OH-D₃) has arisen as another possibility for cancer treatment. The mitochondrial cytochrome P450 enzyme 1-a-hydroxylase (1aOHase) converts 25-OH-D₃ to the active 1,25(OH)₂D₃ and is normally expressed in the kidney. Recently several extrarenal tissues have been found to express this enzyme activity, allowing cells to generate 1,25(OH)₂D₃ which can function as an autocrine or paracrine regulator of cell growth. This study analyzed the presence of 1-a-OHase and the in vitro effects of 25-OH-D₃ and 1,25(OH)₂D₃ in cells derived from human and mouse mammary gland and breast cancer. Both human lines, MCF7 and hTERT-HME, showed high levels of 1-a-OHase expression; however, only the normal cell line significantly responded to treatment with 25-OH-D₃. The murine mammary cancer cell line, WT145, weakly expressed 1-aOHase and responded only to high doses of 25-OH-D₃. The primary normal murine mammary epithelial (MEC) line also responded only to high concentrations of either form of D₃. Based on the finding that non-malignant human breast cells express active 1-a-OHase, 25-OH-D₃ represents a possible preventative treatment for women with a high risk of cancer. Additionally, loss of 1-a-OHase in mammary cells may be associated with development of malignancy. Therefore, further investigation into the role and expression of 1-a-OHase is necessary and warranted for the treatment and prevention of breast cancer.

EFFECTS OF CASODEX ON PC346C ORTHOTOPIC XENOGRAFT MODEL IN ATHYMIC MICE

G. Patricia Ayala, Allen B. Peetz, Edmund Lee, Martin Tenniswood

Prostate cancer is the most common form of primary cancers in men in the United States. Anti-androgens such as Casodex are designed to treat advanced stage prostate cancer by interfering with androgen receptor-mediated cell survival and by initiating cell death. A xenograph model was obtained by orthotopically injecting PC346C^RFP human prostate cancer cells into male NCR nu/nu immuno-compromised mice. These PC346C^RFP cells form large, well vascularized orthotopic tumors, which reach between 1.5 and 2.0 grams after 5 weeks if left untreated. These orthotopic tumors are sensitive to Casodex, administered by implantation of 50mg, 90-days slow release pellets, starting two weeks after injection of the cells. This also causes shrinkage of the tumors and seminal vesicles. In addition, treatment with Casodex resulted in the loss of cellurarity in tumors and induced a dose-dependent decrease of cell proliferation rate as determined by BrdU incorporation. Furthermore, treatment with Casodex caused a dose-dependent increase of active cell death as determined by TUNEL. This demonstrates that PC346C^RFP cells are sensitive to Casodex treatment and can be used to study the mechanism of Casodex-induced cell death in vivo.

The Role of PI-3 Kinase in Mitogen - Activated Protein Kinase Activation Following Mycobacterial Infection of Primary Macrophages

Loren L. Martinez, Shannon K. Roach, and Jeffrey Schorey

Mycobacteria are intramacrophage pathogens and are the etiological agents of numerous disease, including tuberculosis, MAC, leprosy, and paratuberculosis. Following infection, a series of signaling pathways are elicited within the macrophage, including the mitogen-activated protein kinase (MAPK) signaling cascade. Previous studies in our lab have shown a differential regulation of the MAPK cascades following infection with pathogenic vs. nonpathogenic mycobacteria. The signaling pathways upstream of the MAPK potentially responsible for this differential regulation following mycobacterial infection are unknown. However, PKC, PKA and PI-3 kinase are known activators of the MAPKs in different systems. Therefore, this study focuses on the upstream involvement of the PI-3 kinase following pathogenic and nonpathogenic mycobacterial infection using the PI-3 kinase inhibitor LY 294002. Using primary murine bone marrow macrophages, inhibition of PI-3 kinase caused a significant increase in p38 and SAPK/JNK phosphorylation after infection with pathogenic Mycobacterium avium 724, which was also seen in resting cells. However, MKK 3/6 was not activated by PI-3 kinase, and treatment with inhibitor downregulated MEK1/2 and ERK1/2 phosphorylation The activation of the transcription factors CREB and NF-kB also appears to be dependent on PI-3 kinase. These results suggest that PI-3 kinase plays an important role in the regulation of the MAPK cascade. However, the exact involvement of PI-3 kinase after mycobacterial infection requires further investigation.

A Computational Analysis of Recombination in Plasmodium falciparum

Matthew D. Wilkerson, Michael T. Ferdig

One of the planet’s deadliest diseases, malaria is a leading cause of sickness and death in developing countries. The parasite Plasmodium falciparum is the causative agent of the most common and most lethal form of malaria. A genetic linkage map was previously constructed and several drug loci for drug resistance genes have been placed on it. This study seeks to discover and gain new knowledge of resistance gene-linkage relationships. Additionally, sequencing of the P. falciparum genome is near completion. This study also seeks to gain knowledge of genetic to physical map relationship such as marker order, marker hotspotting, and recombination frequencies. To achieve these objectives, computer programs were written and implemented on previously recorded data. Genotype data of progeny from a drug sensitive and drug resistant cross was used to discover balancing resistance genotypes and lethal genotypes. Physical map ePCR data of the genetic map markers was used to relate the physical and genetic maps graphically and numerically. In this two part study, specific knowledge of P. falciparum recombination was gained and trends and behaviors were recorded. Notably, a novel locus on chromosome 3 was determined to be significant with several drug resistance genotypes and also exist as a recombinational hotspot.

Functional Analysis of zASIP in Retinal Development

Scott Nelson

The zebrafish retina is a great model to study the molecular mechanisms that control retinal patterning. A previous study indicated that nagie oko, a gene involved in maintaining cellular polarity in the retinal epithelium, is also critical for controlling cellular patterning in the retina. This suggested that other polarity genes are also required for generating the proper cellular organization of the retina. To test this hypothesis, we isolated the zebrafish gene zASIP, which is a homolog of the ASIP/Bazooka/Par-3 gene that is conserved from worms to humans. The zASIP protein possesses the same protein binding domains as the other homologues, which suggests that they are involved in assembling similar protein complexes. We studied the functions of zASIP protein during retinal development using morpholino knock-down technology. Morpholinos are modified DNA oligonucleotides that anneal to mRNAs and reduce their translation. Injection of zASIP morpholinos into 1-2 cell zebrafish embryos revealed that the loss of zASIP function disrupted cellular patterning, but not cell specification in the zebrafish retina. Further experiments utilizing morpholino knock-down of other polarity genes will give more insight into understanding the genetic pathway involved in cellular patterning.

The Role of APP in Protein Trafficking in Drosophila Photoreceptor Neurons

Dennis Terrell Sansom

The Amyloid Precursor Protein, (APP), is ubiquitously expressed in human neuronal tissue and plays a vital role in the progression of the Alzheimer's disease. It is assumed that trafficking of one of its peptide derivatives, A§42, leads to plaque formation in the brain leading to neuronal degeneration. The Amyloid Precursor Protein Like ,(APPL), found in Drosophila melangaster, is homologous to APP. We utilized Drosophila photoreceptor cells to determine the role of APP and APPL in intracellular trafficking by observing rhodopsin localization. We over expressed APP and APPL exclusively in photoreceptor cells utilizing the UAS-GAL4 system. We also observed Golgi and ER distribution using the NAGT, Rab6, and PDI markers when APP and APPL were over expressed. Results show that rhodopsin trafficking is not affected due to an over expression of APP and APPL, however, a redistribution of ER and Golgi occurs. These results indicate that APP and APPL may play an intrinsic role in intracellular trafficking.

Characterization of Three Zebrafish Phosphatidylinositol Transfer Proteins

Matthew O'Rourke, Thomas Vihtelic, David Hyde

Phosphatidylinositol transfer proteins (PITPs) are small, soluble proteins found in all eukaryotic cell types. Based on their ability to transfer phosphatidylinositol (PI) and phosphatidylcholine (PC) between membrane bilayers in vitro, these proteins may play essential roles in lipid-mediated signaling pathways and secretory vesicle trafficking. However, the exact in vivo function of these proteins remains elusive. Mammals express two PITPs; PITPa and PITPb. While both are able to transfer PI and PC in vitro, PITPb may possess the unique ability to transfer sphingomyelin. Based on cDNA library screening and EST database analysis, we obtained three zebrafish PITP cDNAs. Sequence analysis of these three cDNAs revealed that zebrafish PITPs are highly homologous to the previously identified PITP molecules in other species. Based on pairwise amino acid identities and multiple protein sequence alignments, the zebrafish molecules were designated zfPITPa, zfPITPb#1, and zfPITPb#2. To examine the subcellular distribution of these zfPITP isoforms, I created expression constructs. In these constructs, sequences corresponding to the different zfPITP open reading frames were placed under the control of the CMV promoter and fused at their C terminus to the myc epitope. These constructs were individually transfected into COS7 cells. Immunohistochemical detection of the myc epitope revealed that each zfPITP isoform localized to a distinct region in transfected COS7 cells. While zfPITPa was found within the nucleus, both zfPITPb isoforms were excluded form the nucleus. All three zebrafish PITPs were also expressed in the cytosol. While zfPITPb#1 was expressed throughout the cytoplasm, zfPITPb#2 expression was limited to the perinuclear region, perhaps the Golgi apparatus. These results are consistent with mammalian PITP localization patterns and suggest that zebrafish will be an ideal model system for further study of in vivo PITP function.

Analysis of Apoptotic Mechanisms in norpA Induced Retinal Degeneration in Drosophila

Ka’ohimanu Dang, Michelle A. Whaley, Joseph E. O’Tousa

Apoptosis, while necessary for normal development, is also involved in retinal degeneration, a neurodegenerative disease in which death of photoreceptor cells lead to blindness. In order to define critical pathways of apoptosis, a better understanding of signal processes in retinal degeneration is necessary. In Drosophila, a large number of genes have been identified and characterized as being involved in retinal cell death. One of these genes, norpA, causes light-dependent retinal degeneration. Initial data indicates the p35 gene from the baculovirus Autographa californica does not completely suppress apoptosis in the norpA mutant. This suggests a caspase-independent pathway may be involved in norpA cell death. The focus of this project was on studying the retinal cell death pathway in norpA degeneration. Previous TEM data gave evidence of cytoplasmic condensation and engulfment of cells indicating the hallmarks of apoptosis. In order to further verify apoptosis, TUNEL was performed on white-eyed mutants and compared with wild type (control) flies. Results indicate norpA mutants show TUNEL positive nuclei. To better understand the pathways and mechanisms involved in norpA-mediated degeneration, two known genes in the apoptotic cascade were introduced into a norpA background. Dronc, a non-mainstream caspase, was introduced as a dominant negative mutant and DIAP1, a known inhibitor of apoptosis, was overexpressed in norpA. While data for Dronc are not currently available, initial data indicates DIAP1 has a minimal effect in delaying norpA retinal degeneration. In order to identify new genes involved in norpA retinal cell death, microarray technology will be incorporated using Affymetrix gene chips. norpA mutants and norpA;p35 aged three days under dark and light conditions will be compared to determine differences in gene expression.

Effect of Environmental versus Genetic Factors at Quantitative Trait Loci (QTL) on the Body Size of Laboratory Reared Aedes Aegypti Mosquitoes

Teshad Delandro

The body size of the Ae. Ageypti mosquito has been a major center of study because of its potential to impact vector competence. We hatched field-collected eggs and reared the larvae under low-diet and high-diet conditions. The wing sizes of emerged adults were compared and a size distribution was determined for both diet groups. From previous study, it is known that the QTL for body size is present between A13L975and LF335 region on the Ae. Ageypti chromosome. Using PCR analysis, three SNP markers (LF 264,Chym, PGK) were examined and specific restriction enzymes were used to cut amplified DNA fragments at specific nucleotide regions. With the digested product, the scored mosquitoes can be compared along with the environmental and sibling data to determine which has a more influential effect on mosquito body size.

Elucidation of the intracellular signaling pathway leading to Src-mediated activation of ARF6 during epithelial cell scattering.

Shatomi J.S. Kerbawy, Felipe Palacios and Crislyn D'Souza-Schorey

The dissociation of the adherens junctions is intimately linked to epithelial to mesenchymal transitions and increased cell motility. Adherens junction disassembly is accompanied by the activation of the oncogenic tyrosine kinase, v-Src. Recent work from our lab has shown that Src activation during cell scattering results in activation of the Ras-related GTPase, ARF6. In support of this contention we have shown that (1) dominant negative ARF6 blocks Src-induced cell scattering and (2) Src activation enhances GTP loading of endogenous ARF6. ARF6-GTP facilitates cell scattering by inducing the endocytosis of adherens junction components and by promoting ruffling of the lateral membrane. Here we have focused on the signaling pathway leading to ARF6 activation by examining v-Src-mediated phosphorylation of the exchange factor for ARF6 (EFA6). EFA6 has the structural requirements for Src-mediated phosphorylation. These investigations were conducted using an MDCK cell line stably expressing a temperature sensitive mutant of v-Src, MDCK-pp60^v-src. Low levels of protein expression have hindered our attempts to successfully immunoprecipitate EFA6 from lysates of. MDCK-pp60^v-src cells and assay for tyrosine phosphorylation of EFA6. Our current efforts are directed at investigating the role of EFA6 and EFA6 mutants defective in ARF6 nucleotide exchange during cell scattering.

COMPARATIVE GENOMICS OF AFRICAN MALARIA VECTORS, ANOPHELES FUNESTUS AND AN. GAMBIAE: EST SEQUENCE CONSERVATION

Andrew Serazin

Anopheles funestus Giles and An. gambiae Giles are the two most important malaria vectors in Africa. As a consequence of the activity of An. funestus during the dry season, when An. gambiae is normally inactive, the malaria transmission season is extended. Yet, An. funestus has received much less attention than An. gambiae, currently the model for all anophelines. Although both species are highly competent vectors of malaria, any malaria control strategy must account for the biological differences between An. gambiae and An. funestus. What are the similarities and differences in both gene sequence and gene order that may help to explain the larger biological differences? To help answer this question and to accelerate genetic research on Anopheles funestus, we have constructed and analyzed a cDNA library made from pupal, larval, and adult developmental stages. Approximately 3500 cDNA clones were sequenced from the 5’ end, processed automatically into clusters and consensus sequences, and BLASTed against Genbank databases. Manual annotation was performed on >500 ESTs with significant BLAST results, revealing a diverse set of predicted proteins, among which were stage-specific, digestive, and immune-related proteins. A subset of these ESTs were physically mapped to An. funestus polytene chromosomes, which allowed for an analysis of large-scale chromosome rearrangements relative to An. gambiae. We discovered the highest rate of chromosome rearrangement ever calculated for any eukaryote. At the sequence level, we examined the degree of conservation between An. funestus and An. gambiae orthologs with respect to predicted protein function, physical location, and natural selection, as evidenced by relative rates of synonymous/nonsynonymous substitutions. These results suggest substantial molecular differences between An. funestus and An. gambiae orthologs. From this information, we hope to identify common targets for interrupting malaria transmission, and to gain an understanding of the genetic basis of ecological and behavioral differences between these species.