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利用工程化标签进行四色单分子成像解析质膜中信号复合物的分子结构。

Four-color single-molecule imaging with engineered tags resolves the molecular architecture of signaling complexes in the plasma membrane.

机构信息

Department of Biology and Center for Cellular Nanoanalytics (CellNanOs), Osnabrück University, Osnabrück, Germany.

Division of Cell Signalling and Immunology, University of Dundee, School of Life Sciences, Dundee, UK.

出版信息

Cell Rep Methods. 2022 Feb 4;2(2):100165. doi: 10.1016/j.crmeth.2022.100165. eCollection 2022 Feb 28.

DOI:10.1016/j.crmeth.2022.100165
PMID:35474965
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9017138/
Abstract

Localization and tracking of individual receptors by single-molecule imaging opens unique possibilities to unravel the assembly and dynamics of signaling complexes in the plasma membrane. We present a comprehensive workflow for imaging and analyzing receptor diffusion and interaction in live cells at single molecule level with up to four colors. Two engineered, monomeric GFP variants, which are orthogonally recognized by anti-GFP nanobodies, are employed for efficient and selective labeling of target proteins in the plasma membrane with photostable fluorescence dyes. This labeling technique enables us to quantitatively resolve the stoichiometry and dynamics of the interferon-γ (IFNγ) receptor signaling complex in the plasma membrane of living cells by multicolor single-molecule imaging. Based on versatile spatial and spatiotemporal correlation analyses, we identify ligand-induced receptor homo- and heterodimerization. Multicolor single-molecule co-tracking and quantitative single-molecule Förster resonance energy transfer moreover reveals transient assembly of IFNγ receptor heterotetramers and confirms its structural architecture.

摘要

通过单分子成像对单个受体进行定位和跟踪，为揭示质膜中信号复合物的组装和动态提供了独特的可能性。我们提出了一种全面的工作流程，用于在活细胞中单分子水平上进行多达四种颜色的受体扩散和相互作用的成像和分析。两种工程化的、单体 GFP 变体，可被抗 GFP 纳米体正交识别，用于用光稳定荧光染料在质膜中对靶蛋白进行高效和选择性标记。这种标记技术使我们能够通过多色单分子成像定量解析活细胞质膜中干扰素-γ (IFNγ) 受体信号复合物的化学计量和动态。基于多功能的空间和时空相关分析，我们确定了配体诱导的受体同型和异型二聚体。多色单分子共跟踪和定量单分子Förster 共振能量转移进一步揭示了 IFNγ 受体异四聚体的瞬时组装，并证实了其结构架构。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d88/9017138/deddd6b32808/gr5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d88/9017138/e26fb2dd8650/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d88/9017138/cbe035d10985/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d88/9017138/84786d4461d9/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d88/9017138/2f0d498b0601/gr3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d88/9017138/09efc59ca1dd/gr4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d88/9017138/deddd6b32808/gr5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d88/9017138/e26fb2dd8650/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d88/9017138/cbe035d10985/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d88/9017138/84786d4461d9/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d88/9017138/2f0d498b0601/gr3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d88/9017138/09efc59ca1dd/gr4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d88/9017138/deddd6b32808/gr5.jpg

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