Zou Shan, Ramirez Daniel M Carter, Vanderlip Sapphire, Ogilvie William, Jakubek Zygmunt J, Johnston Linda J
Steacie Institute for Molecular Sciences, National Research Council of Canada, Ottawa, Ont., Canada.
J Struct Biol. 2009 Oct;168(1):78-89. doi: 10.1016/j.jsb.2009.03.014. Epub 2009 Apr 5.
The effect of enzymatic generation of ceramide on phase separated bilayers with a mixture of co-existing fluid and liquid-ordered phases has been examined using a combination of atomic force microscopy (AFM) and fluorescence imaging. Supported lipid bilayers prepared from a DOPC/sphingomyelin/cholesterol mixture were imaged prior to, during and after incubation with sphingomyelinase by total internal reflection fluorescence (TIRF) microscopy. Enzyme treatment resulted in the growth of large dye-excluded regions. The growth kinetics for these patches are consistent with activity of a variable number of enzyme molecules in different regions of the bilayer. Correlated AFM and fluorescence imaging shows that some of the large dye-excluded patches form around the original liquid-ordered domains, which become heterogeneous in height with many raised ceramide-rich regions around their periphery. However, some of the dye-excluded patches correspond to areas of the bilayer where the initial domains have largely or partially disappeared. The dye-excluded patches observed by fluorescence are shown to be areas of increased adhesion in lateral deflection AFM images and are postulated to form by incorporation of both cholesterol and ceramide in the original fluid phase and to vary in composition throughout the bilayer. This is evident from the observation that the dye-excluded areas are all detected as areas of increased friction, but do not always show a distinct height difference in topographic images. These results highlight the utility of a multi-modal imaging approach for understanding the complex membrane restructuring that occurs upon enzymatic generation of ceramide.
利用原子力显微镜（AFM）和荧光成像相结合的方法，研究了神经酰胺酶促生成对具有共存流体相和液相有序相混合物的相分离双层膜的影响。由二油酰磷脂酰胆碱（DOPC）/鞘磷脂/胆固醇混合物制备的支持脂质双层膜，在与鞘磷脂酶孵育之前、期间和之后，通过全内反射荧光（TIRF）显微镜成像。酶处理导致大的染料排斥区域生长。这些斑块的生长动力学与双层膜不同区域中可变数量酶分子的活性一致。相关的AFM和荧光成像表明，一些大的染料排斥斑块在原始液相有序域周围形成，这些域在高度上变得不均匀，其周边有许多富含神经酰胺的凸起区域。然而，一些染料排斥斑块对应于双层膜中初始域已大部分或部分消失的区域。荧光观察到的染料排斥斑块在横向偏转AFM图像中显示为粘附增加的区域，并推测是通过胆固醇和神经酰胺在原始流体相中的掺入形成的，且在整个双层膜中组成不同。这从以下观察中很明显：染料排斥区域都被检测为摩擦增加的区域，但在地形图中并不总是显示出明显的高度差异。这些结果突出了多模态成像方法在理解神经酰胺酶促生成时发生的复杂膜重构方面的实用性。
