Yashunsky Victor, Shimron Simcha, Lirtsman Vladislav, Weiss Aryeh M, Melamed-Book Naomi, Golosovsky Michael, Davidov Dan, Aroeti Benjamin
Racah Institute of Physics, The Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904, Israel.
Biophys J. 2009 Aug 19;97(4):1003-12. doi: 10.1016/j.bpj.2009.05.052.
We report on the application of surface plasmon resonance (SPR), based on Fourier transform infrared spectroscopy in the mid-infrared wavelength range, for real-time and label-free sensing of transferrin-induced endocytic processes in human melanoma cells. The evanescent field of the mid-infrared surface plasmon penetrates deep into the cell, allowing highly sensitive SPR measurements of dynamic processes occurring at significant cellular depths. We monitored in real-time, infrared reflectivity spectra in the SPR regime from living cells exposed to human transferrin (Tfn). We show that although fluorescence microscopy measures primarily Tfn accumulation in recycling endosomes located deep in the cell's cytoplasm, the SPR technique measures mainly Tfn-mediated formation of early endocytic organelles located in close proximity to the plasma membrane. Our SPR and fluorescence data are very well described by a kinetic model of Tfn endocytosis, suggested previously in similar cell systems. Hence, our SPR data provide further support to the rather controversial ability of Tfn to stimulate its own endocytosis. Our analysis also yields what we believe is novel information on the role of membrane cholesterol in modulating the kinetics of endocytic vesicle biogenesis and consumption.
我们报道了基于傅里叶变换红外光谱在中红外波长范围内的表面等离子体共振（SPR）技术，用于实时、无标记地检测人黑色素瘤细胞中转铁蛋白诱导的内吞过程。中红外表面等离子体的倏逝场深入细胞内部，使得能够对在细胞显著深度处发生的动态过程进行高灵敏度的SPR测量。我们实时监测了暴露于人转铁蛋白（Tfn）的活细胞在SPR模式下的红外反射光谱。我们发现，虽然荧光显微镜主要测量位于细胞质深处的再循环内体中转铁蛋白的积累，但SPR技术主要测量位于质膜附近的早期内吞细胞器的转铁蛋白介导形成。我们的SPR和荧光数据通过先前在类似细胞系统中提出的转铁蛋白内吞动力学模型得到了很好的描述。因此，我们的SPR数据为转铁蛋白刺激其自身内吞这一颇具争议的能力提供了进一步支持。我们的分析还得出了我们认为是关于膜胆固醇在调节内吞小泡生物发生和消耗动力学方面作用的新信息。
