Curvature and Composition Regulation in Tubular Lipid Membranes: A Biophysical Investigation

近年来，在揭示与脂质和脂质/蛋白膜形状有关的现象方面取得了显著进展。生物科学已经认识到膜曲率的重要性：而不是一个被动的矩阵，由外部因素塑造，它积极参与的过程，如膜分类和运输已经实现。理论膜物理学和细胞膜生物学的发现已经产生了填补定量理论（缺乏实验实现）和定性描述（缺乏生物物理测量）之间空白的冲动。目前的僵局在以最近发现的曲率产生和传感模块BAR域为中心的研究中尤为明显。BAR域具有潜在的普遍重要性。这种香蕉形状的蛋白质结构域被定性地观察到导致膜呈现具有不同曲率的圆柱形管的形状，但其机制尚不完全清楚，并且缺乏对所涉及的力学的阐明。这些和其他类型的隔离模块将作为系统研究膜形成因素的起点。膜曲率研究的一个空白一直集中在多个微米大小（巨型）囊泡的微观可分辨曲率半径上。技术上更具挑战性（因此未被充分开发），但与生物膜曲率相关的是膜管的机械方面的研究，其中纳米尺度曲率的高精度测量可以方便地与脂质/蛋白质相互作用调节的机械力/力矩平衡的测量相结合。从巨大的囊泡中抽出的膜管代表了一个机械可控的膜系统，具有陡峭的、精确可调的曲率梯度。这些管道将允许解决第二个基本问题，即理论和生物模型没有通过定量数据充分联系起来。有待探讨的问题是，膜组分在膜曲率梯度中分选到何种程度，以及基于何种分子细节，以阐明这种生物物理分选如何参与细胞膜内分选和运输。从曲率依赖的荧光脂质横向分选开始，延伸到外周和跨膜蛋白质分选，PI将量化曲率分选的各个方面，并表征和开发可能用于细胞环境的曲率传感器。与PI的研究目标相辅相成的是参与和教育，特别是本科生参与研究项目，为高中生（包括少数群体）和教师提供研究机会，这一活动和谐地融入了NSF资助的宾夕法尼亚大学MRSEC中心长期建立的推广计划，以及ACS资助的项目SEED。进一步的教育目标包括将科学伦理教学整合到本科生和研究生的课堂中。这方面将包括讨论媒体报道的科学不端行为的例子，以及常见的灰色地带案例，如图像处理技术的机会和边界。此外，PI将整合使用科学软件Matlab的方法来说明统计概念，以及教学和研究中图像分析的基本方面。

Remarkable progress has recently been made in uncovering phenomena that are related to the shape of lipid and lipid/protein membranes. Biological science has recognized the importance of membrane curvature: rather than being a passive matrix that is shaped by external factors, its active participation in processes as important as membrane sorting and trafficking has been realized. Findings from theoretical membrane physics and cell membrane biology have generated an urge to fill the gap between quantitative theories (with insufficient experimental realization) and qualitative descriptions (with missing biophysical measurements). The current impasse is particularly evident in research centered on a recently discovered, potentially ubiquitously important, curvature generating and sensing module, the BAR domain. This banana shaped protein domain is qualitatively observed to cause membranes to assume the shape of cylindrical tubes with varying curvature, but with incompletely understood mechanism and absence of elucidation of the mechanics involved. These and other types of isolated modules will serve as a starting point for systematic investigation of membrane shaping factors. A hiatus of membrane curvature research has been the focus on microscopically resolvable curvature radii of multiple micron sized (giant) vesicles. Technically significantly more challenging (and hence under-explored), but relevant for biological membrane curvatures, is the investigation of mechanical aspects of membrane tubes, where high precision measurement of nanometer scale curvature can be conveniently combined with the measurement of mechanical force/moment equilibria modulated by lipid/protein interactions. Membrane tubes pulled from giant vesicles represent a mechanically controllable membrane system with a steep, precisely adjustable curvature gradient. These tubes will allow addressing a second fundamental problem where theoretical and biological models have not been sufficiently connected through quantitative data. The question to be explored regards to what extent, and based on which molecular details, membrane components are sorted in membrane curvature gradients, to clarify how this biophysical sorting could be involved in intracellular membrane sorting and trafficking. Beginning with curvature dependent lateral sorting of fluorescent lipids, and extending to peripheral and transmembrane protein sorting, the PI will quantify aspects of curvature sorting and characterize and develop curvature sensors that may be used in the cellular context. Synergistically coupled to the PI's research objectives is the involvement and education particularly of undergraduate level students in research projects, enriched by research opportunities for high school students, including minority groups, and teachers, an activity which is harmoniously integrated into long established outreach programs of the NSF funded UPenn MRSEC center, as well as an ACS funded project SEED. Further educational objectives include the integration of the teaching of science ethics into classes directed towards undergraduate and graduate students. This aspect will include discussion of scientific misconduct examples that received media coverage, as well as common gray area cases like opportunities and boundaries of image processing techniques. Furthermore, the PI will integrate methods to use the scientific software Matlab for illustrating both statistical concepts, as well as fundamental aspects of image analysis in teaching and research.