Department of Biochemistry, 1 King's College Circle, University of Toronto, Toronto, Ontario, Canada; Institute of Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada; Terrence Donnelly Centre for Cellular & Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.
Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada.
J Biol Chem. 2022 Oct;298(10):102448. doi: 10.1016/j.jbc.2022.102448. Epub 2022 Sep 5.
Mapping the self-organization and spatial distribution of membrane proteins is key to understanding their function. Developing methods that can provide insight into correlations between membrane protein colocalization and interactions is challenging. We report here on a correlated stochastic optical reconstruction microscopy/homoFRET imaging approach for resolving the nanoscale distribution and oligomeric state of membrane proteins. Using live cell homoFRET imaging of carcinoembryonic antigen-related cellular adhesion molecule 1, a cell-surface receptor known to exist in a complex equilibrium between monomer and dimer/oligomer states, we revealed highly heterogeneous diffraction-limited structures on the surface of HeLa cells. Furthermore, correlated super-resolved stochastic optical reconstruction microscopy imaging showed that these structures comprised a complex mixture and spatial distribution of self-associated carcinoembryonic antigen-related cellular adhesion molecule 1 molecules. In conclusion, this correlated approach provides a compelling strategy for addressing challenging questions about the interplay between membrane protein concentration, distribution, interaction, clustering, and function.
绘制膜蛋白的自组织和空间分布图谱对于理解其功能至关重要。开发能够深入了解膜蛋白共定位和相互作用之间相关性的方法具有挑战性。我们在此报告了一种相关的随机光学重建显微镜/同型荧光共振能量转移（homoFRET）成像方法，用于解析膜蛋白的纳米尺度分布和寡聚状态。我们使用活细胞 homoFRET 成像技术对癌胚抗原相关细胞黏附分子 1（一种已知存在于单体和二聚体/寡聚体状态之间复杂平衡的细胞表面受体）进行研究，揭示了 HeLa 细胞表面存在高度异质的衍射受限结构。此外，相关的超分辨随机光学重建显微镜成像表明，这些结构包含了自我关联的癌胚抗原相关细胞黏附分子 1 分子的复杂混合物和空间分布。总之，这种相关方法为解决关于膜蛋白浓度、分布、相互作用、聚类和功能之间相互作用的具有挑战性的问题提供了一种引人注目的策略。
