Sensors 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.
The research performed during the course of this collaborative project has both advanced our ability to identify how proteins recognize membrane curvature and improved our understanding of specific proteins that are critical to the recognition of apoptotic cells. To achieve this, several new biosensing tools were designed using nanomaterials. The development of new tools for characterizing protein recognition of curvature will have an impact in fields beyond apoptotic cell clearance. Membrane curvature plays a role in neurotransmission, endocytosis, exocytosis, viral lipid coat formation, and will be critical to developing methods for drug delivery. Two types of biosensors were designed for studying proteins at interfaces that contain membranes with nanoscale features. First, for the study of individual proteins interactions with membrane curvature, a two dimensional substrate was synthesized that contains topographical features with sub-100 nanometer dimensions. This assay is amenable to fluorescence microscopy methods and single molecule imaging. By adapting nanoparticle coating methods to lipid membrane surfaces we have established robust techniques for creating substrates on which protein binding and protein conformational changes can be studied. Second, by using lipid-coated nanoparticles in solution we have developed techniques for studying en masse the interactions of proteins with highly curved membranes. This approach is complementary to our two dimensional nanotemplated substrate and enables an analysis of ensembles of proteins. By using dynamic light scattering, plasmonics, and fluorescence spectroscopy it is possible to observe protein binding events at nanoparticle surfaces in this solution based assay. With the addition of probes selective for conformational states of proteins it is possible to simultaneously observe protein binding and protein structure. In addition to the technical advances that this research provided, our work provides insight into how apoptotic cell surfaces are recognized and distinguished from healthy cells in the body. This is a critical step to understanding how the body is able to identify cells for removal by the immune system. We have determined that C-reactive protein, which plays a critical role in the innate immune response, identifies cellular features based on changes in curvature. This expands the current view of how proteins can distinguish apoptotic cells from healthy cells and demonstrates that membrane shape can be a recognition feature of the immune system. Our research confirms that C-reactive protein can identify cells that contain no oxidized lipids but that are highly curved as an indirect result of membrane oxidation and apoptotic processes. Furthermore, we have shown that the binding of C-reactive protein results in conformational changes to the protein that trigger the activation of the immune clearance of these membranes. Beyond the scientific progress made during this project, a number of broader impacts have resulted from this work. A large number of undergraduate and graduate students have been trained in research techniques and several contributed as co-authors on publications. Additionally, a number of high school teachers have participated in this research during a summer research program. As a result of this, the impact of this project extended beyond the laboratories in which the research was performed and reached the classrooms of these teachers.
