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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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Construction and screening of complemented mEos3.2.(a) Schematic illustration of mEos3.2 complementation and its photoconversion. (b) Seven cleavage sites at different flexible loops of mEos3.2 (34F, 96E, 138K, 148V, 150D, 160A, 164E) yielded highly variable BiFC signal. Site 164E generated the highest fraction of bacteria cells with bright BiFC signal. (c) 405 nm irradiation converted 164E complemented mEos3.2 to the red form, which was excited by a 561-nm laser. Scale bar 1 μm.
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f2: Construction and screening of complemented mEos3.2.(a) Schematic illustration of mEos3.2 complementation and its photoconversion. (b) Seven cleavage sites at different flexible loops of mEos3.2 (34F, 96E, 138K, 148V, 150D, 160A, 164E) yielded highly variable BiFC signal. Site 164E generated the highest fraction of bacteria cells with bright BiFC signal. (c) 405 nm irradiation converted 164E complemented mEos3.2 to the red form, which was excited by a 561-nm laser. Scale bar 1 μm.

Mentions: BiFC-PALM requires a photoactivatable fluorescent protein, which can retain the ability of photoactivation and fluorescing when its two split fragments complement and refold (Fig. 2a). A photoswitchable fluorescent protein, Dronpa, has actually been tested for BiFC12, but its mediocre intensity contrast between the bright and dark states disqualifies Dronpa from being a super-resolution and single-molecule probe in vivo. Instead, we chose mEos3.2, a recently developed photoconvertible fluorescent protein13, as the BiFC-PALM probe. mEos3.2 is truly monomeric, which is crucial for BiFC. In addition, mEos3.2 demonstrates excellent performance in PALM imaging in terms of its brightness, maturation time and labelling density13.


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)

Construction and screening of complemented mEos3.2.(a) Schematic illustration of mEos3.2 complementation and its photoconversion. (b) Seven cleavage sites at different flexible loops of mEos3.2 (34F, 96E, 138K, 148V, 150D, 160A, 164E) yielded highly variable BiFC signal. Site 164E generated the highest fraction of bacteria cells with bright BiFC signal. (c) 405 nm irradiation converted 164E complemented mEos3.2 to the red form, which was excited by a 561-nm laser. Scale bar 1 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Construction and screening of complemented mEos3.2.(a) Schematic illustration of mEos3.2 complementation and its photoconversion. (b) Seven cleavage sites at different flexible loops of mEos3.2 (34F, 96E, 138K, 148V, 150D, 160A, 164E) yielded highly variable BiFC signal. Site 164E generated the highest fraction of bacteria cells with bright BiFC signal. (c) 405 nm irradiation converted 164E complemented mEos3.2 to the red form, which was excited by a 561-nm laser. Scale bar 1 μm.
Mentions: BiFC-PALM requires a photoactivatable fluorescent protein, which can retain the ability of photoactivation and fluorescing when its two split fragments complement and refold (Fig. 2a). A photoswitchable fluorescent protein, Dronpa, has actually been tested for BiFC12, but its mediocre intensity contrast between the bright and dark states disqualifies Dronpa from being a super-resolution and single-molecule probe in vivo. Instead, we chose mEos3.2, a recently developed photoconvertible fluorescent protein13, as the BiFC-PALM probe. mEos3.2 is truly monomeric, which is crucial for BiFC. In addition, mEos3.2 demonstrates excellent performance in PALM imaging in terms of its brightness, maturation time and labelling density13.

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