BS
Brian D. Slaughter
研究方向
  • Molecular biology
Super-resolution Microscopy-based Bimolecular Fluorescence Complementation to Study Protein Complex Assembly and Co-localization
基于高分辨率显微镜的双分子荧光互补用于蛋白复合体装配和共定位
作者:Jingjing Chen, Zulin Yu, Jay R. Unruh, Brian D. Slaughter and Sue L. Jaspersen日期:02/20/2020,浏览量:4500,Q&A: 0
Numerous experimental approaches exist to study interactions between two subunits of a large macromolecular complex. However, most methods do not provide spatial and temporal information about binding, which are critical for dissecting the mechanism of assembly of nanosized complexes in vivo. While recent advances in super-resolution microscopy techniques have provided insights into biological structures beyond the diffraction limit, most require extensive expertise and/or special sample preparation, and it is a challenge to extend beyond binary, two color experiments. Using HyVolution, a super-resolution technique that combines confocal microscopy at sub-airy unit pinhole sizes with computational deconvolution, we achieved 140 nm resolution in both live and fixed samples with three colors, including two fluorescent proteins (mTurquoise2 and GFP) with significant spectral overlap that were distinguished by means of shifting the excitation wavelength away from common wavelengths. By combining HyVolution super-resolution fluorescence microscopy with bimolecular fluorescence complementation (SRM-BiFC), we describe a new assay capable of visualizing protein-protein interactions in vivo at sub-diffraction resolution. This method was used to improve our understanding of the ordered assembly of the Saccharomyces cerevisiae spindle pole body (SPB), a ~1 giga-Dalton heteromeric protein complex formed from 18 structural components present in multiple copies. We propose that SRM-BiFC is a powerful tool for examination of direct interactions between protein complex subunits at sub-diffraction resolution in live cells.