丹麦哥本哈根大学的研究人员采用荧光共振能量转移法(FRET),成功地实现了神经细胞囊泡融合过程的实时成像。这一技术的运用不仅会增进人们对神经系统疾病和病毒感染的了解,还可能有助于开发出治疗神经疾病和精神疾病(如精神分裂症、抑郁症、帕金森氏症、早老性痴呆病)的新疗法。该研究成果刊登在最近出版的《美国国家科学院院刊》上。
囊泡是装载神经递质的微小容器,这种只有纳米级大小的小囊是神经细胞彼此沟通的桥梁。囊泡与神经细胞的膜融合,会向周围释放神经递质,从而被下一个神经细胞检测到,神经信号以此方式进行传递。神经细胞的这种沟通过程一旦中断,会造成多种疾病和精神紊乱,如抑郁症。然而,目前科学家们对于这种囊泡融合是如何进行的依然缺乏详细了解。
哥本哈根大学神经学与药理学系和纳米科学中心的研究人员使用了一种称为荧光共振能量转移的方法。这种方法众所周知,但研究人员的使用方式却与众不同。他们在实验室中制作出含有荧光供体分子的囊泡和固定在一个表面的含荧光受体分子的细胞膜。只有当两个不同的荧光分子彼此接近对方时,才会发出荧光,研究人员据此检测囊泡融合情况。研究人员称,这种方法可实时判定囊泡的形状,其清晰度达纳米级。
哥本哈根大学副教授迪米特里奥斯·斯塔莫指出,囊泡与膜的联系接触是很多重要生理过程的基本步骤。过去一直缺乏有效方法来测量纳米级尺度上囊泡融合的实时情况,现在也仅是有可能获取这个进程的高清静止图片,或者是解析度很低的实时影像。而使用这个新方法,研究人员就能以极高的分辨率实时观测融合过程中囊泡形态的变化,量化囊泡间的接触区域,判定囊泡的大小和形状。这有助于研究人员了解囊泡融合过程中的分子特性,为神经系统和感染性疾病的研究提供了一个广阔的前景。(生物谷Bioon.com)
生物谷推荐原始出处:
PNAS July 28, 2009 vol. 106 no. 30 12341-12346
Quantification of nano-scale intermembrane contact areas by using fluorescence resonance energy transfer
Poul Martin Bendix, Mette S. Pedersen and Dimitrios Stamou,1
Bio-Nanotechnology Laboratory, Department of Neuroscience and Pharmacology and Nano-Science Center, University of Copenhagen, 2100 Copenhagen, Denmark
Nanometer-scale intermembrane contact areas (CAs) formed between single small unilamellar lipid vesicles (SUVs) and planar supported lipid bilayers are quantified by measuring fluorescence resonance energy transfer (FRET) between a homogenous layer of donor fluorophores labeling the supported bilayer and acceptor fluorophores labeling the SUVs. The smallest CAs detected in our setup between biotinylated SUVs and dense monolayers of streptavidin were ≈20 nm in radius. Deformation of SUVs is revealed by comparing the quenching of the donors to calculations of FRET between a perfectly spherical shell and a flat surface containing complementary fluorophores. These results confirmed the theoretical prediction that the degree of deformation scales with the SUV diameter. The size of the CA can be controlled experimentally by conjugating polyethylene glycol polymers to the SUV or the surface and thereby modulating the interfacial energy of adhesion. In this manner, we could achieve secure immobilization of SUVs under conditions of minimal deformation. Finally, we demonstrate that kinetic measurements of CA, at constant adhesion, can be used to record in real-time quantitative changes in the bilayer tension of a nano-scale lipid membrane system.