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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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Comparison between two-colour STORM/PALM and BiFC-PALM.(a) Optical diffraction and fluorescent background from non-interacting proteins make it difficult to image specific protein–protein interactions. The red and green spots are the point spread functions of individual protein A and protein B molecules, respectively. The spatial resolution is about 200 nm. (b) Two-colour STORM/PALM co-localization imaging shows uncertainty on overlapping non-interacting molecules. The red and green spots are the single-molecule localizations of individual protein A and protein B molecules, respectively. The spatial resolution is about 20 nm. The high density of both proteins results in large uncertainty for identification of interacting protein pairs by co-localization. (c) BiFC-PALM can locate specific interacting pairs with high spatial resolution, given almost zero background from non-interacting molecules. The yellow spots are the single-molecule localizations of interacting pairs of protein A and protein B molecules. The spatial resolution is about 20 nm.
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f1: Comparison between two-colour STORM/PALM and BiFC-PALM.(a) Optical diffraction and fluorescent background from non-interacting proteins make it difficult to image specific protein–protein interactions. The red and green spots are the point spread functions of individual protein A and protein B molecules, respectively. The spatial resolution is about 200 nm. (b) Two-colour STORM/PALM co-localization imaging shows uncertainty on overlapping non-interacting molecules. The red and green spots are the single-molecule localizations of individual protein A and protein B molecules, respectively. The spatial resolution is about 20 nm. The high density of both proteins results in large uncertainty for identification of interacting protein pairs by co-localization. (c) BiFC-PALM can locate specific interacting pairs with high spatial resolution, given almost zero background from non-interacting molecules. The yellow spots are the single-molecule localizations of interacting pairs of protein A and protein B molecules. The spatial resolution is about 20 nm.

Mentions: A typical example is the prokaryotic cell, which, although lacking internal membrane systems, is recently discovered to have subcellular domains and higher-order organization2. All of the current imaging approaches have limitations for studying PPIs in such small and crowded systems. For instance, electron microscopy is unsuitable for dynamic imaging of a particular PPI subpopulation because of its poor specificity and low temporal resolution, albeit its exceeding spatial resolution. Fluorescent imaging techniques have high specificity, but optical diffraction disqualifies conventional fluorescence microscopy for imaging the subcellular distribution and dynamics of high-density molecules. Recently developed super-resolution optical imaging techniques, such as stochastic optical reconstruction microscopy (STORM)3 and (fluorescent) photoactivated localization microscopy (FPALM/PALM)45, have redefined the resolution barrier and allowed cellular ultrastructures being resolved at ~7 nm resolution6. Regarding PPI imaging, two-colour co-localization can be in principle used to identify particular PPIs, but often suffers high background from non-interacting proteins (Fig. 1a) and uncertainty on seemingly overlapped pairs even in super-resolution images due to finite spatial resolution (Fig. 1b). Förster resonance energy transfer (FRET) is a powerful fluorescent approach for studying PPIs, particularly their dynamic processes7. However, because of spectral bleed-through and limited energy transfer efficiency, imaging individual FRET pairs in live cells is difficult. It is thus challenging to implement FRET on single-molecule localization-based super-resolution imaging methods such as STORM and FPALM/PALM.


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)

Comparison between two-colour STORM/PALM and BiFC-PALM.(a) Optical diffraction and fluorescent background from non-interacting proteins make it difficult to image specific protein–protein interactions. The red and green spots are the point spread functions of individual protein A and protein B molecules, respectively. The spatial resolution is about 200 nm. (b) Two-colour STORM/PALM co-localization imaging shows uncertainty on overlapping non-interacting molecules. The red and green spots are the single-molecule localizations of individual protein A and protein B molecules, respectively. The spatial resolution is about 20 nm. The high density of both proteins results in large uncertainty for identification of interacting protein pairs by co-localization. (c) BiFC-PALM can locate specific interacting pairs with high spatial resolution, given almost zero background from non-interacting molecules. The yellow spots are the single-molecule localizations of interacting pairs of protein A and protein B molecules. The spatial resolution is about 20 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Comparison between two-colour STORM/PALM and BiFC-PALM.(a) Optical diffraction and fluorescent background from non-interacting proteins make it difficult to image specific protein–protein interactions. The red and green spots are the point spread functions of individual protein A and protein B molecules, respectively. The spatial resolution is about 200 nm. (b) Two-colour STORM/PALM co-localization imaging shows uncertainty on overlapping non-interacting molecules. The red and green spots are the single-molecule localizations of individual protein A and protein B molecules, respectively. The spatial resolution is about 20 nm. The high density of both proteins results in large uncertainty for identification of interacting protein pairs by co-localization. (c) BiFC-PALM can locate specific interacting pairs with high spatial resolution, given almost zero background from non-interacting molecules. The yellow spots are the single-molecule localizations of interacting pairs of protein A and protein B molecules. The spatial resolution is about 20 nm.
Mentions: A typical example is the prokaryotic cell, which, although lacking internal membrane systems, is recently discovered to have subcellular domains and higher-order organization2. All of the current imaging approaches have limitations for studying PPIs in such small and crowded systems. For instance, electron microscopy is unsuitable for dynamic imaging of a particular PPI subpopulation because of its poor specificity and low temporal resolution, albeit its exceeding spatial resolution. Fluorescent imaging techniques have high specificity, but optical diffraction disqualifies conventional fluorescence microscopy for imaging the subcellular distribution and dynamics of high-density molecules. Recently developed super-resolution optical imaging techniques, such as stochastic optical reconstruction microscopy (STORM)3 and (fluorescent) photoactivated localization microscopy (FPALM/PALM)45, have redefined the resolution barrier and allowed cellular ultrastructures being resolved at ~7 nm resolution6. Regarding PPI imaging, two-colour co-localization can be in principle used to identify particular PPIs, but often suffers high background from non-interacting proteins (Fig. 1a) and uncertainty on seemingly overlapped pairs even in super-resolution images due to finite spatial resolution (Fig. 1b). Förster resonance energy transfer (FRET) is a powerful fluorescent approach for studying PPIs, particularly their dynamic processes7. However, because of spectral bleed-through and limited energy transfer efficiency, imaging individual FRET pairs in live cells is difficult. It is thus challenging to implement FRET on single-molecule localization-based super-resolution imaging methods such as STORM and FPALM/PALM.

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