Laminar organization of molecular complexes in a clathrin coat revealed by nanoscale protein colocalization

IF 4.4 2区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Structure Pub Date : 2025-04-23 DOI:10.1016/j.str.2025.03.012

Tai Kiuchi, Ryouhei Kobayashi, Shuichiro Ogawa, Louis L.H. Elverston, Dimitrios Vavylonis, Naoki Watanabe

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引用次数: 0

Abstract

Super-resolution microscopy achieves a few nanometers resolution, but colocalization analysis in a molecular complex is limited by its labeling density. Here we present a method for quantitative mapping of molecular complexes using multiplexed super-resolution imaging, integrating exchangeable single-molecule localization (IRIS). We developed antiserum-derived Fab IRIS probes for high-density labeling of endogenous proteins and protein cluster coloring (PC-coloring), which employs pixel-based principal component analysis and clustering. PC-coloring maps regions of distinct ratios of multiple proteins, and in each region, correlation between two proteins is calculated for evaluating the complex formation. PC-coloring revealed multi-layered complex formation in a clathrin-coated structure (CCS) prior to endocytosis. Upon epidermal growth factor (EGF) stimulation, EGF receptor (EGFR)-dominant, EGFR-Grb2-complex, and Grb2-dominant regions lined up from outside the CCS rim. Along the interior of Grb2-dominant regions, CCS components (Eps15, FCHo1/2 and intersectin-1) formed a complex with Grb2 away from EGFR. The Grb2-dominant region and Grb2-CCS component complex formation probably determine EGFR recruitment sites in the CCS rim.

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纳米级蛋白共定位揭示了网格蛋白外壳中分子复合物的层流组织

超分辨显微镜可达到几纳米的分辨率，但分子复合物的共聚焦分析却受到其标记密度的限制。在此，我们提出了一种利用多重超分辨率成像定量绘制分子复合物图谱的方法，即整合可交换单分子定位（IRIS）。我们开发了抗血清衍生的 Fab IRIS 探针，用于内源蛋白质的高密度标记和蛋白质聚类着色（PC-coloring），后者采用了基于像素的主成分分析和聚类。PC着色绘制出多种蛋白质比例不同的区域，并在每个区域中计算两种蛋白质之间的相关性，以评估复合物的形成。PC-着色显示了内吞之前在凝集素包被结构（CCS）中形成的多层复合物。在表皮生长因子（EGF）刺激下，表皮生长因子受体（EGFR）优势区、EGFR-Grb2-复合物区和 Grb2 优势区从 CCS 边缘外侧排开。沿着 Grb2 优势区的内部，CCS 成分（Eps15、FCHo1/2 和 intersectin-1）与 Grb2 形成复合物，远离表皮生长因子受体。Grb2优势区和Grb2-CCS成分复合物的形成可能决定了表皮生长因子受体在CCS边缘的招募位点。

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来源期刊

Structure 生物-生化与分子生物学

CiteScore

8.90

自引率

1.80%

发文量

155

审稿时长

3-8 weeks

期刊介绍： Structure aims to publish papers of exceptional interest in the field of structural biology. The journal strives to be essential reading for structural biologists, as well as biologists and biochemists that are interested in macromolecular structure and function. Structure strongly encourages the submission of manuscripts that present structural and molecular insights into biological function and mechanism. Other reports that address fundamental questions in structural biology, such as structure-based examinations of protein evolution, folding, and/or design, will also be considered. We will consider the application of any method, experimental or computational, at high or low resolution, to conduct structural investigations, as long as the method is appropriate for the biological, functional, and mechanistic question(s) being addressed. Likewise, reports describing single-molecule analysis of biological mechanisms are welcome. In general, the editors encourage submission of experimental structural studies that are enriched by an analysis of structure-activity relationships and will not consider studies that solely report structural information unless the structure or analysis is of exceptional and broad interest. Studies reporting only homology models, de novo models, or molecular dynamics simulations are also discouraged unless the models are informed by or validated by novel experimental data; rationalization of a large body of existing experimental evidence and making testable predictions based on a model or simulation is often not considered sufficient.