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Super-resolution imaging and tracking of protein-protein interactions in sub-diffraction cellular space.

Liu Z, Xing D, Su QP, Zhu Y, Zhang J, Kong X, Xue B, Wang S, Sun H, Tao Y, Sun Y - Nat Commun (2014)

Bottom Line: Imaging the location and dynamics of individual interacting protein pairs is essential but often difficult because of the fluorescent background from other paired and non-paired molecules, particularly in the sub-diffraction cellular space.The super-resolution imaging shows interesting distribution and domain sizes of interacting MreB-EF-Tu pairs as a subpopulation of total EF-Tu.The single-molecule tracking of MreB, EF-Tu and MreB-EF-Tu pairs reveals intriguing localization-dependent heterogonous dynamics and provides valuable insights to understanding the roles of MreB-EF-Tu interactions.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biomembrane and Membrane Biotechnology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871, China.

ABSTRACT
Imaging the location and dynamics of individual interacting protein pairs is essential but often difficult because of the fluorescent background from other paired and non-paired molecules, particularly in the sub-diffraction cellular space. Here we develop a new method combining bimolecular fluorescence complementation and photoactivated localization microscopy for super-resolution imaging and single-molecule tracking of specific protein-protein interactions. The method is used to study the interaction of two abundant proteins, MreB and EF-Tu, in Escherichia coli cells. The super-resolution imaging shows interesting distribution and domain sizes of interacting MreB-EF-Tu pairs as a subpopulation of total EF-Tu. The single-molecule tracking of MreB, EF-Tu and MreB-EF-Tu pairs reveals intriguing localization-dependent heterogonous dynamics and provides valuable insights to understanding the roles of MreB-EF-Tu interactions.

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BiFC-PALM super-resolution imaging of EFTu-MreB-PPIs.(a) BiFC-PALM imaging of EFTu-MreB-PPIs in two fixed E. coli cells. (b) Schematic illustration indicating that EF-Tu can interact with multiple proteins in the cell. EF-Tu molecules that interact with MreB can be visualized by both Snap-Alexa647 (upper path) and complemented mEos3.2, whereas ET-Tu molecules that interact with other proteins or free EF-Tu can be visualized by Snap-Alexa647 (lower path); (c) EFTu-MreB-PPIs distribution obtained by BiFC-PALM (green) as a subpopulation of total EF-Tu labelled with Alexa647 (red) in two fixed cells. Scale bar, 1 μm.
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f3: BiFC-PALM super-resolution imaging of EFTu-MreB-PPIs.(a) BiFC-PALM imaging of EFTu-MreB-PPIs in two fixed E. coli cells. (b) Schematic illustration indicating that EF-Tu can interact with multiple proteins in the cell. EF-Tu molecules that interact with MreB can be visualized by both Snap-Alexa647 (upper path) and complemented mEos3.2, whereas ET-Tu molecules that interact with other proteins or free EF-Tu can be visualized by Snap-Alexa647 (lower path); (c) EFTu-MreB-PPIs distribution obtained by BiFC-PALM (green) as a subpopulation of total EF-Tu labelled with Alexa647 (red) in two fixed cells. Scale bar, 1 μm.

Mentions: We first used pull-down assay to confirm that MreB and EF-Tu also have specific interaction in E. coli (Supplementary Fig. 3), consistent with an interactome study of E. coli using mass spectrometry1. The detection and quantification of EFTu-MreB-PPIs in E. coli were obtained using BiFC-PALM of mEosN-EF-Tu and MreB-mEosC in fixed cells. The expression levels of both fusion proteins were much lower than their endogenous counterparts (Supplementary Fig. 4). The specificity of the BiFC signal was confirmed by two non-interacting pairs, EFTu-MreC and EFTu-MreD1, as well as a truncated EF-Tu with MreB (Supplementary Fig. 5)30. In the super-resolution images, the EFTu-MreB-PPIs seemed to exist in two forms, clusters and small dots, which did not seem to have distinct patterns (Fig. 3a and Supplementary Figs 6–8). We analysed 143 bacterial cells, among which 100 demonstrated rod shapes with normal aspect ratios (length/width ratio between 2.5 and 6), similar with the bacteria in Fig. 3a. We performed cluster analysis on 15 such type of bacteria (Supplementary Fig. 6). The pairwise distance distributions of all dots (Supplementary Fig. 6b) and clusters (Supplementary Fig. 6c) in 15 bacterial cells were similar, all approximating a generalized beta distribution31. This suggests that only a small fraction of EFTu-MreB-PPIs formed clusters, whereas the majority did not form higher-order structures and distributed rather randomly. The number of clusters in each bacterium was nearly proportional to the cell size with a density of ~80 clusters per μm2 (Supplementary Fig. 6d,e). The 15 bacterial cells were not only similar in cluster density and distance, but also similar in cluster area (Supplementary Fig. 6f,g).


Super-resolution imaging and tracking of protein-protein interactions in sub-diffraction cellular space.

Liu Z, Xing D, Su QP, Zhu Y, Zhang J, Kong X, Xue B, Wang S, Sun H, Tao Y, Sun Y - Nat Commun (2014)

BiFC-PALM super-resolution imaging of EFTu-MreB-PPIs.(a) BiFC-PALM imaging of EFTu-MreB-PPIs in two fixed E. coli cells. (b) Schematic illustration indicating that EF-Tu can interact with multiple proteins in the cell. EF-Tu molecules that interact with MreB can be visualized by both Snap-Alexa647 (upper path) and complemented mEos3.2, whereas ET-Tu molecules that interact with other proteins or free EF-Tu can be visualized by Snap-Alexa647 (lower path); (c) EFTu-MreB-PPIs distribution obtained by BiFC-PALM (green) as a subpopulation of total EF-Tu labelled with Alexa647 (red) in two fixed cells. Scale bar, 1 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109008&req=5

f3: BiFC-PALM super-resolution imaging of EFTu-MreB-PPIs.(a) BiFC-PALM imaging of EFTu-MreB-PPIs in two fixed E. coli cells. (b) Schematic illustration indicating that EF-Tu can interact with multiple proteins in the cell. EF-Tu molecules that interact with MreB can be visualized by both Snap-Alexa647 (upper path) and complemented mEos3.2, whereas ET-Tu molecules that interact with other proteins or free EF-Tu can be visualized by Snap-Alexa647 (lower path); (c) EFTu-MreB-PPIs distribution obtained by BiFC-PALM (green) as a subpopulation of total EF-Tu labelled with Alexa647 (red) in two fixed cells. Scale bar, 1 μm.
Mentions: We first used pull-down assay to confirm that MreB and EF-Tu also have specific interaction in E. coli (Supplementary Fig. 3), consistent with an interactome study of E. coli using mass spectrometry1. The detection and quantification of EFTu-MreB-PPIs in E. coli were obtained using BiFC-PALM of mEosN-EF-Tu and MreB-mEosC in fixed cells. The expression levels of both fusion proteins were much lower than their endogenous counterparts (Supplementary Fig. 4). The specificity of the BiFC signal was confirmed by two non-interacting pairs, EFTu-MreC and EFTu-MreD1, as well as a truncated EF-Tu with MreB (Supplementary Fig. 5)30. In the super-resolution images, the EFTu-MreB-PPIs seemed to exist in two forms, clusters and small dots, which did not seem to have distinct patterns (Fig. 3a and Supplementary Figs 6–8). We analysed 143 bacterial cells, among which 100 demonstrated rod shapes with normal aspect ratios (length/width ratio between 2.5 and 6), similar with the bacteria in Fig. 3a. We performed cluster analysis on 15 such type of bacteria (Supplementary Fig. 6). The pairwise distance distributions of all dots (Supplementary Fig. 6b) and clusters (Supplementary Fig. 6c) in 15 bacterial cells were similar, all approximating a generalized beta distribution31. This suggests that only a small fraction of EFTu-MreB-PPIs formed clusters, whereas the majority did not form higher-order structures and distributed rather randomly. The number of clusters in each bacterium was nearly proportional to the cell size with a density of ~80 clusters per μm2 (Supplementary Fig. 6d,e). The 15 bacterial cells were not only similar in cluster density and distance, but also similar in cluster area (Supplementary Fig. 6f,g).

Bottom Line: Imaging the location and dynamics of individual interacting protein pairs is essential but often difficult because of the fluorescent background from other paired and non-paired molecules, particularly in the sub-diffraction cellular space.The super-resolution imaging shows interesting distribution and domain sizes of interacting MreB-EF-Tu pairs as a subpopulation of total EF-Tu.The single-molecule tracking of MreB, EF-Tu and MreB-EF-Tu pairs reveals intriguing localization-dependent heterogonous dynamics and provides valuable insights to understanding the roles of MreB-EF-Tu interactions.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biomembrane and Membrane Biotechnology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871, China.

ABSTRACT
Imaging the location and dynamics of individual interacting protein pairs is essential but often difficult because of the fluorescent background from other paired and non-paired molecules, particularly in the sub-diffraction cellular space. Here we develop a new method combining bimolecular fluorescence complementation and photoactivated localization microscopy for super-resolution imaging and single-molecule tracking of specific protein-protein interactions. The method is used to study the interaction of two abundant proteins, MreB and EF-Tu, in Escherichia coli cells. The super-resolution imaging shows interesting distribution and domain sizes of interacting MreB-EF-Tu pairs as a subpopulation of total EF-Tu. The single-molecule tracking of MreB, EF-Tu and MreB-EF-Tu pairs reveals intriguing localization-dependent heterogonous dynamics and provides valuable insights to understanding the roles of MreB-EF-Tu interactions.

Show MeSH
Related in: MedlinePlus