Collaborative Research: A Nanostructured Model of the Apoptotic Cell Surface

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AbstractCollaborative Research: A Nanostructured Model of the Apoptotic Cell SurfaceSensors that mimic cellular membranes have contributed to understanding of protein-membrane interactions. However, the membranes of an apoptotic cells differ in lipid composition and structure, containing oxidized lipids that cause nanoscale and microscale protrusions. Membrane shape is a potential site for protein recognition and a critical way in which the body identifies damaged cells for removal. By mimicking the shapes of apoptotic membranes, sensors will be created that more accurately reflect apoptotic cells. Nanostructured membranes will be created using supported bilayer techniques. These nanostructured models of the apoptotic cell surface will allow for separate control of both membrane curvature and lipid composition. Binding of protein to these sensors will be monitored using a surface sensitive, fluorescence microscopy technique. Sensors designed using this approach will be useful for understanding how proteins interact with curved membrane surfaces. The first goal of this research is to construct supported bilayer membranes that allow for control of lipid composition as well as nanoscale surface structure. Silica nanoparticles embedded with fluorophores will be prepared and coated with a series of oxidized and non-oxidized lipids. These nanoparticles will be attached to glass surfaces and a lipid bilayer will be formed over them. The second goal of this research is to demonstrate the ability of these nanostructured membrane sensors to measure protein binding to curved surfaces. The effects of curvature on the binding of proteins involved with recognizing apoptotic cells for removal will be measured using total internal reflection fluorescence microscopy. Binding of C-reactive protein to the membranes will be visualized and quantitatively measured to determine what features of an apoptotic membrane are critical for protein recognition. This information will guide the future design of sensors that detect how the body responds to apoptotic cells.Intellectual Merits: It is unknown whether lipids, membrane curvature, or the combination determines protein binding to apoptotic cells. This work will answer that question and improve understanding of apoptotic cell recognition. In turn, this will allow for the design of sensors that recognize physiological responses to apoptotic cells that could be critical in diagnosing cardiovascular disease. The approaches developed here for engineering mimics of apoptotic cell membranes will become the foundation for sensors based on optical, electronic, or mass based signal transduction.Broader impacts: The proposed research will impact teaching, training, and outreach. A postdoctoral scientist and several students will be trained in emerging techniques at the interface of nanoscience, biology, chemistry, and spectroscopy. Additionally, the educational impact of this project will extend beyond the two campuses and will reach out to involve underrepresented minorities. Students from the Strides Toward Encouraging Professions in Science program at the Community College of Aurora, Community College of Denver and Metropolitan State College of Denver will be involved in the proposed research. A second method of outreach will be to include middle school science teachers through the Rocky Mountain-Middle School Math and Science Partnership.