Membrane Biophysics and Atomic Force Microscopy

本项目通过几个合作项目，利用原子力显微镜（AFM）和其他技术研究脂质膜的物理化学性质和生物学作用。(1)在与NIAID合作者(dr。J.A. Dvorak和F. Tokumasu)对脂质膜的非均质性进行了研究，我们扩展了对2-二myristoyl- n-glycero-phosphocholine （DMPC）的主要相变的定量研究，揭示了一个直径约4.2 nm的固有结构域，为双棕榈酰磷脂酰胆碱（DPPC）、二酰磷脂酰胆碱（DLPC）和胆固醇（chol）的三脂混合体系。这种三脂系统更接近于模拟生物膜，并已由康奈尔大学的G. W. Feigenson教授（也是本次研究的合作者）通过光学显微镜进行了研究。对于这个三脂系统，我们量化了大单层囊泡（LUV）表面吸附和融合的中等膜-云母吸引支持。然后，我们的AFM成像显示了广泛的纳米级膜结构域（c.f.膜筏），范围从26到38纳米不等，这取决于胆固醇水平，DLPC和DPPC的比例，以及其他相空间参数。我们的总体目标是应用新颖的AFM成像和数学分析来全面了解受其组成和天然相互作用影响的细胞膜。(2)同时与LPD， NIAID合作者(博士。J.A. Dvorak和T. Arie)，我们开发了新的视频显微镜和图像分析来表征恶性疟原虫疟疾感染过程中的红细胞闪烁和边缘抖动现象。我们发现寄生虫感染显著改变了细胞膜动力学，并根据寄生虫的发育阶段对这些变化进行量化，这可能与人类微循环中的疟疾疾病机制有关。(3)进一步扩大上述两项合作，并与其他NIAAA研究合作者(dr。B. J.利特曼和s . l。Niu)，我们正在使用AFM和差示扫描量热法（DSC）来表征新的三组分DDPC/DPPC/胆固醇系统的微域和纳米结构，模拟视觉通路中的杆外节膜。不寻常的膜性质被确定并与双不饱和酰基链在DDPC脂质的存在相关。(4)最后，与NIAID的另一位合作科学家（J. Silver博士）合作，正在开发纳米尺度的膜能量和结构描述，以了解融合孔动力学对病毒入侵和细胞运输过程的重要作用。膜侧组织在这些过程中的潜在作用尚不清楚。

The physical-chemical properties and biological roles of lipid membranes are being investigated by atomic force microscopy (AFM) and other technologies in this project via several collaborations. (1) In a continuing work with NIAID collaborators (Drs. J.A. Dvorak and F. Tokumasu) on heterogeneities of lipid membranes, we have extended our quantitative studies of the main phase transition in 2-dimyristoyl-sn-glycero-phosphocholine (DMPC), revealing an intrinsic domain size of about 4.2 nm in diameter, to tri-lipid mixture system of dipalmitoyl phosphatidylcholine (DPPC), dilauroyl phosphatidylcholine (DLPC), and cholesterol (chol). This tri-lipid system mimics more closely biological membranes and has been previously investigated via light microscopy by Prof. G. W. Feigenson (Cornell University) who is also a collaborator for the current study. For this tri-lipid system, we quantified a moderate membrane-mica attractive support from large unilamellar vesicle (LUV) surface adsorption and fusion. Then, our AFM imaging revealed extensive nanoscopic membrane domains (c.f. membrane rafts) that range from 26 to 38 nanometers depending on cholesterol level, DLPC and DPPC ratio, and other phase space parameters. Our overall aim is to apply novel AFM imaging and mathematical analyses toward a comprehensive understanding of the cellular membrane under influence of its composition and native interactions. (2) Also with LPD, NIAID collaborators (Drs. J.A. Dvorak and T. Arie), we developed new video microscopy and image analyses to characterize the red blood cell flicker and edge dithering phenomena during the Plasmodium falciparum malaria infection process. We have found that the parasitic infection markedly modifies cell membrane dynamics and are quantifying these changes according to parasitic developmental stages, in potential relevance to malaria disease mechanism in human microcirculations. (3) In a further expansion of above two collaborations and with in addition NIAAA research collaborators (Drs. B. J. Litman and S.-L. Niu), we are using AFM and Differential Scanning Calorimetry (DSC) to characterize micro-domain and nanoscopic structures in a new three-component DDPC/DPPC/cholesterol system mimicking rod outer segment membranes in the vision pathway. Unusual membrane properties are being identified and correlated to presence of the double unsaturated acyl chains in the DDPC lipid. (4) Finally, with another NIAID collaborating scientist (Dr. J. Silver), nanoscopic energetic and structural descriptions of membranes are being developed to understand fusion pore dynamics important to virus invasion and cellular trafficking processes. The potential role of membrane lateral organizations in these processes is yet to be understood.