Collaborative Research: A Nanostructured Model of the Apoptotic Cell Surface

摘要合作研究：模拟细胞膜的凋亡细胞表面传感器的纳米结构模型有助于理解蛋白质与膜的相互作用。然而，凋亡细胞的细胞膜在脂质组成和结构上不同，含有氧化脂质，导致纳米级和微尺度的突出。膜的形状是蛋白质识别的潜在位点，也是机体识别受损细胞并将其清除的关键途径。通过模拟细胞凋亡膜的形状，可以制造出更准确反映细胞凋亡的传感器。纳米结构的膜将使用支撑双层技术制造。这些凋亡细胞表面的纳米结构模型将允许分离控制膜曲率和脂质组成。蛋白质与这些传感器的结合将使用表面敏感的荧光显微镜技术进行监测。利用这种方法设计的传感器将有助于理解蛋白质如何与弯曲的膜表面相互作用。本研究的第一个目标是构建支持的双层膜，允许控制脂质组成以及纳米级表面结构。将制备嵌入荧光团的二氧化硅纳米颗粒，并涂覆一系列氧化和非氧化脂质。这些纳米颗粒将附着在玻璃表面，并在其上形成脂质双分子层。这项研究的第二个目标是证明这些纳米结构膜传感器测量蛋白质与曲面结合的能力。曲率对识别凋亡细胞的蛋白结合的影响将使用全内反射荧光显微镜进行测量。c反应蛋白与膜的结合将被可视化和定量测量，以确定凋亡膜的哪些特征对蛋白质识别至关重要。这些信息将指导未来传感器的设计，以检测身体对凋亡细胞的反应。知识价值：目前尚不清楚脂质、膜曲率或两者的结合是否决定了蛋白质与凋亡细胞的结合。这项工作将回答这个问题，并提高对凋亡细胞识别的理解。反过来，这将允许设计传感器识别凋亡细胞的生理反应，这可能是诊断心血管疾病的关键。这里开发的凋亡细胞膜的工程模拟方法将成为基于光学、电子或基于质量的信号转导的传感器的基础。更广泛的影响：拟议的研究将影响教学，培训和推广。一名博士后科学家和几名学生将在纳米科学、生物学、化学和光谱学的界面上接受新兴技术的培训。此外，该项目的教育影响将超出这两个校区，并将涉及代表性不足的少数民族。来自奥罗拉社区学院、丹佛社区学院和丹佛大都会州立学院“迈向鼓励科学专业”项目的学生将参与这项拟议的研究。第二种推广方法是通过落基山中学数学和科学伙伴关系将中学科学教师包括在内。

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.