Kevin Edwards - US grants

Cell communication, proliferation, and motility are governed by a complex web of protein-protein interactions and phosphorylation events at the cell cortex. Protein tyrosine kinases and protein tyrosine phosphatases (PTPs) provide critical control points for these processes and thus play central roles in normal development as well as diseases such as cancer. The fly Drosophila melanogaster provides a powerful model system to study the role of PTPs in development and signal transduction. The investigator has sequenced clones encoding two novel Drosophila PTPs, named Pez and Meg after two of their human orthologs. Pez and Meg are members of the FERM-PTP family, modular proteins of unknown biological function that contain amino-terminal FERM (4.1, ezrin, radixin, moesin-related) domains, carboxy-terminal PTP domains, and distinct protein-protein interaction motifs in their central portions. Most other FERM domain proteins are structural components and/or regulators of the membrane cytoskeleton or focal adhesions. Thus the domain structures of Pez and Meg suggest that they participate in multiprotein complexes important in cell motility or adhesion, and therefore could play a role in tumor metastasis. To test this hypothesis, mutations will be generated in the Pez gene and their effects on proliferation, motility, adhesion, and differentiation will be examined by a variety of phenotypic assays available in Drosophila. Further, Pez substrates will be pursued by expressing a "substrate trapping" mutant form of Pez. Trapped substrates will identified by genetic and biochemical approaches. These experiments are designed to elucidate the biological function of Pez and identify the signaling pathways) in which Pez resides. This work will initiate a long term molecular genetic investigation of the role of the membrane cytoskeleton in signaling and cell movement.

DESCRIPTION (provided by applicant): Protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) are major control switches in the protein networks that govern cell signaling, movement, differentiation, and other processes. These enzymes thus play central roles in normal development as well as in cancer, diabetes, and other diseases. The fly Drosophila melanogaster provides a powerful model system to study the role of PTPs in cancer-related processes such as cell migration, growth factor signaling, hematopoiesis, and apoptosis. Genetic analysis is proposed to elucidate the functions of two novel Drosophila PTPs, named Pez and Meg after their human orthologs. Pez and Meg are FERM-PTPs, modular proteins that contain a FERM (4.1, ezrin, radixin, moesin-like) domain, a PTP domain, and additional protein-protein interaction motifs. Like other FERM domain proteins, Pez and Meg may reside at the cell cortex and participate in multiprotein complexes important for cell motility or adhesion, processes that are relevant to tumor metastasis. To test this hypothesis, three aims will be pursued. 1) Mutant animals will be generated that lack only Pez, and the resulting phenotypes will be documented to determine if Pez is essential for development. 2) Genetic modifier tests will be performed to identify the pathways regulated by Pez and Meg. Small deletions removing either the Pez or the Meg gene will be crossed into flies with activated or inhibited PTK pathways; a functional interaction is indicated if the deletion modifies the PTK phenotype. 3) To shed light on their biological roles, the developmental expression pattern and subcellular location of Pez and Meg will be documented by in situ RNA hybridization and immunolocalization studies. Since FERM-PTPs have not been analyzed by gene knockout in any organism, these experiments will provide novel insights into the role of this family in normal and abnormal cell behaviors. This work is part of a long-term molecular genetic investigation of the role of membrane-cytoskeleton proteins in signaling and cell movement.

[unreadable] DESCRIPTION (provided by applicant): Cytochrome P450 monooxygenases are heme-containing enzymes that insert oxygen into a wide range of xenobiotics, such as pollutants, pesticides, and drugs, promoting their clearance from the body. Although they provide a vital defense system against environmental toxins, P450s may also catalyze the formation of products that are more toxic or carcinogenic than their precursors. P450s perform essential roles in development and homeostasis as well, including steroid hormone synthesis. It is unclear how these defensive and developmental roles of the P450 system are coordinated and regulated; to address this problem in vivo, a genetic analysis was initiated in the model organism Drosophila melanogaster. P450s that reside on the endoplasmic reticulum (ER) generally require a redox partner, Cytochrome P450 Reductase (CPR). Most animals have dozens of P450 genes, but CPR is often encoded by a single gene, providing a unique "Achilles' heel" for genetically inactivating the ER P450 system. The principal investigator's lab generated an allelic series of mutations in the Drosophila CPR gene (Cpr), including 6 novel lethal alleles. Flies were constructed which conditionally express a Cpr cDNA; this transgene can rescue the lethal mutations, and it causes visible phenotypes when overexpressed. These genetic tools provide the means to elucidate the numerous functions of the highly pleiotropic Cpr gene. 3 aims are proposed: I) Characterize embryonic and larval phenotypes associated with Cpr, to identify its developmental functions. II) Develop an assay system to determine the degree to which metabolism of a compound depends on CPR. The sensitivity (or resistance) of the mutants to several xenobiotics and mixtures will be tested. A method for generating CPR-deficient adults will be established, to facilitate compound testing and analysis of adult Cpr phenotypes. Ill) Test for feedback induction of the P450s. Toxin and drug interactions are often caused by P450 induction. It was recently hypothesized that vertebrates have a large-scale system to overexpress P450s when CPR is compromised. P450 mRNAs will be quantified in Cpr mutants to determine if this system exists and can be investigated in Drosophila. [unreadable] [unreadable] [unreadable]

Illinois State University has been awarded a grant to acquire an environmental scanning electron microscope for research and training in the Biology, Chemistry, Geography-Geology, and Physics programs. The microscope provides capabilities for observation of fine details of diverse specimens and for analysis of their elemental composition. In environmental mode, living organisms can be examined with no preparation. This makes it possible to observe delicate specimens that would be altered by conventional preparation methods. Faculty and students use the scope in research projects that include documenting physiological stress in plants; identifying single-celled algae that are indicators of past environmental conditions; characterizing mutant fruit flies; using elemental analysis to assess the chemistry of coordination complexes of the element vanadium; characterizing nanospheres; documenting surface structures in plants with defective cell expansion; examining structure of bacteria-host interactions and bacterial biofilms, evaluating geochemical interactions in fossils, making nanoscale measurements of thermoelectric lattices, comparing flower structure and development, evaluating synthesis of lithium cobalt oxide; and characterizing the material composition of new electrodes being developed for an improved scanning probe microscope. The results of these diverse studies will be published in peer-reviewed journals and integrated into courses and faculty websites as appropriate. Software for remote control operation of the scope will allow students in classrooms to observe specimens remotely. The scope will be featured at various community outreach events, and it will be available to other academic researchers in central Illinois.

An award is made to Illinois State University (ISU) to acquire a multichannel laser scanning confocal microscope. The instrument will provide advanced imaging capabilities to ISU students and faculty in Physics, Chemistry, and Biological Sciences, as well as institutions across the Midwest region. As the centerpiece of the ISU Biological Sciences Microscopy Core Facility, the microscope will greatly enhance ISU's research infrastructure, enabling high-resolution three dimensional reconstructions of cells and tissues, time-lapse imaging of dynamic cellular processes in live samples, and advanced fluorescence techniques. ISU's diverse population of undergraduates will be able to use the facility as part of mentored independent research projects; over 50 undergraduate researchers per year are projected to learn widely useful STEM skills through this training. K-12 students will be exposed to concepts in cells, tissues, light microscopy, biotechnology, and neuroscience, through several ongoing outreach programs such as the Illinois Summer Research Academy. Students from groups underrepresented in science will be recruited for training via ISU's Louis Stokes Alliance for Minority Participation. Future and current K-12 science teachers will train on the instrument to gain practical research experience, for example in the NSF-funded project "Noyce Scholarships for STEM Teachers of Under-Represented Groups". 20-30 graduate students/year will use the facility to pursue new avenues of research, ultimately contributing new skills to the Illinois STEM workforce. Positive societal impacts of the project also include agricultural and environmental advances. It will promote the USDA-funded development of pennycress as a new winter cover crop, reducing soil erosion and nitrogen runoff from barren fields while profitably producing oilseed for generating biofuels.

The microscope will support the projects of seven main research labs, advancing knowledge in genetics, cell biology, development, neuroscience, and plant science. Projects include: regulation of protein quality control in response to aging and oxidative stress; interactions among proteins that control directional growth of plant cells; mechanisms of gene silencing and meiotic drive in fungi; effects of newly synthesized chemicals on parasitic Leishmania; how nematode worms detect and respond to magnetic fields; in vivo roles of the cell's cortical cytoskeleton in organismal development and tissue maintenance; and regulation of neural activity by sensory inputs and neuromodulators. New capabilities provided by the award will allow users to expand the scope of their research; the system will feature a broad range of excitation wavelengths; rapid scanning; high-sensitivity, low noise photon detectors; sub-200 nm spatial resolution; and fluorescence lifetime data. The users engage in nation-wide and international research collaborations, which will be expanded and strengthened by the added capabilities.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.