Limits...
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.

Show MeSH

Related in: MedlinePlus

BiFC-PALM single-molecule tracking of individual EFTu-MreB-PPIs.(a) Two-dimensional trajectories of individual EFTu-MreB-PPIs in a live E. coli cell. The colour for each trajectory was randomly picked to distinguish nearby traces. (b) Mean speed histogram reveals that EFTu-MreB-PPIs had two populations with different mobility. Data were from three cells. (c) Spatial view of EFTu-MreB-PPIs with different mobility in a live E. coli cell. The red trajectories represent EFTu-MreB-PPIs that were moving faster than 6 μm s−1 and the slower ones are presented in green colour.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4109008&req=5

f4: BiFC-PALM single-molecule tracking of individual EFTu-MreB-PPIs.(a) Two-dimensional trajectories of individual EFTu-MreB-PPIs in a live E. coli cell. The colour for each trajectory was randomly picked to distinguish nearby traces. (b) Mean speed histogram reveals that EFTu-MreB-PPIs had two populations with different mobility. Data were from three cells. (c) Spatial view of EFTu-MreB-PPIs with different mobility in a live E. coli cell. The red trajectories represent EFTu-MreB-PPIs that were moving faster than 6 μm s−1 and the slower ones are presented in green colour.

Mentions: BiFC has actually been used for single-molecule tracking of G-protein-coupled receptor dimers in mammalian cells, but that was not in a crowded, diffraction-limited cellular space32. We took the advantage of photo-controlled convertibility of split mEos3.2 to repeatedly convert and track minimal number of EFTu-MreB-PPIs in live E. coli cells using BiFC-PALM (Fig. 4a, Supplementary Fig. 11 and Supplementary Movie 2). Analysis of the single-molecule trajectories can provide information not only about the mobility of each molecule but also their spatial distribution (Supplementary Figs 12–14). For instance, the mean frame-to-frame speed histogram of all trajectories show that EFTu-MreB-PPIs had two populations with different mobility (Fig. 4b). Interestingly, spatial view of the two populations reveals clear dependence of molecule mobility on their locations, with slow-moving ones localizing near the cell periphery and faster ones within the cell (Fig. 4c). The two motility populations are also found to be related to the polymerization state of MreB because addition of MreB perturbing compound A22 was found to change the fractions of slow and fast mobility populations dramatically (Supplementary Fig 15). In contrast, separate super-resolution single-molecule tracking of MreB and EF-Tu dynamics reveals that both of them had three different mobility populations, which were also localization dependent (Fig. 5). It needs to note that the distribution of EFTu-MreB-PPIs (Fig. 4b) could also be fitted with a triple Gaussian function that is statistically equivalent to the double Gaussian fit at 5% level of significance, see Supplementary Fig. 16 for more discussion and the corresponding fractions of three mobility populations are given in Supplementary Table 2.


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 single-molecule tracking of individual EFTu-MreB-PPIs.(a) Two-dimensional trajectories of individual EFTu-MreB-PPIs in a live E. coli cell. The colour for each trajectory was randomly picked to distinguish nearby traces. (b) Mean speed histogram reveals that EFTu-MreB-PPIs had two populations with different mobility. Data were from three cells. (c) Spatial view of EFTu-MreB-PPIs with different mobility in a live E. coli cell. The red trajectories represent EFTu-MreB-PPIs that were moving faster than 6 μm s−1 and the slower ones are presented in green colour.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: BiFC-PALM single-molecule tracking of individual EFTu-MreB-PPIs.(a) Two-dimensional trajectories of individual EFTu-MreB-PPIs in a live E. coli cell. The colour for each trajectory was randomly picked to distinguish nearby traces. (b) Mean speed histogram reveals that EFTu-MreB-PPIs had two populations with different mobility. Data were from three cells. (c) Spatial view of EFTu-MreB-PPIs with different mobility in a live E. coli cell. The red trajectories represent EFTu-MreB-PPIs that were moving faster than 6 μm s−1 and the slower ones are presented in green colour.
Mentions: BiFC has actually been used for single-molecule tracking of G-protein-coupled receptor dimers in mammalian cells, but that was not in a crowded, diffraction-limited cellular space32. We took the advantage of photo-controlled convertibility of split mEos3.2 to repeatedly convert and track minimal number of EFTu-MreB-PPIs in live E. coli cells using BiFC-PALM (Fig. 4a, Supplementary Fig. 11 and Supplementary Movie 2). Analysis of the single-molecule trajectories can provide information not only about the mobility of each molecule but also their spatial distribution (Supplementary Figs 12–14). For instance, the mean frame-to-frame speed histogram of all trajectories show that EFTu-MreB-PPIs had two populations with different mobility (Fig. 4b). Interestingly, spatial view of the two populations reveals clear dependence of molecule mobility on their locations, with slow-moving ones localizing near the cell periphery and faster ones within the cell (Fig. 4c). The two motility populations are also found to be related to the polymerization state of MreB because addition of MreB perturbing compound A22 was found to change the fractions of slow and fast mobility populations dramatically (Supplementary Fig 15). In contrast, separate super-resolution single-molecule tracking of MreB and EF-Tu dynamics reveals that both of them had three different mobility populations, which were also localization dependent (Fig. 5). It needs to note that the distribution of EFTu-MreB-PPIs (Fig. 4b) could also be fitted with a triple Gaussian function that is statistically equivalent to the double Gaussian fit at 5% level of significance, see Supplementary Fig. 16 for more discussion and the corresponding fractions of three mobility populations are given in Supplementary Table 2.

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