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Cited by 12Advanced Fluorescence Microscopy TechniquesCell Image Analysis TechniquesForce Microscopy Techniques and Applications

The DOI matches its Crossref record; 35 of the 35 references it cites were found in a registry or an index.

Live‐cell super‐resolution imaging of actin using LifeAct ‐14 with a PAINT ‐based approach

Open access
Bhaskar, Haresh, Kleinjan, Dirk‐Jan, Oi, Curran, Gidden, Zoe, Rosser, Susan J., Horrocks, Mathew H., Regan, Lynne·2023·Protein Science
DOI 10.1002/pro.4558PMC PMC9878614

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Abstract

We present direct‐LIVE‐PAINT, an easy‐to‐implement approach for the nanoscopic imaging of protein structures in live cells using labeled binding peptides. We demonstrate the feasibility of direct‐LIVE‐PAINT with an actin‐binding peptide fused to EGFP, the location of which can be accurately determined as it transiently binds to actin filaments. We show that direct‐LIVE‐PAINT can be used to image actin structures below the diffraction‐limit of light and have used it to observe the dynamic nature of actin in live cells. We envisage a similar approach could be applied to imaging other proteins within live mammalian cells.

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Figures

FIGURE 1 (a) Cryo‐EM structure of LifeAct (17 AA) bound to F‐actin. Five monomers of actin are shown in space‐filling representation in shades of gray. Each actin monomer is shown bound to the LifeAct peptide, which is shown in magenta in the ribbon representation. The three residues at the C‐terminus, colored yellow, indicate the amino acids that are missing from LifeAct‐14. Structure 7AD9 was retrieved from the PDB (Belyy et al., 2020). (b) Cartoons showing a predicted structure of the LifeAct‐14‐EGFP construct (LifeAct‐14, magenta; EGFP, green) (Jumper et al., 2021) and illustrating the reversible nature of the binding between LifeAct‐14‐EGFP and actin filaments. EGFP sequence retrieved from FPbase (ID: R9NL8) (Lambert, 2019). Structures visualized using PyMOL (the PyMOL molecular graphics system, version 2.0 Schrödinger, LLC.). (c) A HEK293 cell, transiently transfected with a plasmid expressing LifeAct‐14‐EGFP. The distinctive actin filament structure is clearly visible in the region of interest (ROI, yellow box). The diagonal line separates diffraction‐limited and super‐resolution (SR) imaging. SR image colored “red hot.” Diffraction‐limited image was obtained by Z‐projecting the time‐course images (100 s, 50 ms exposure). 5 𝜇m scale bar
FIGURE 1 (a) Cryo‐EM structure of LifeAct (17 AA) bound to F‐actin. Five monomers of actin are shown in space‐filling representation in shades of gray. Each actin monomer is shown bound to the LifeAct peptide, which is shown in magenta in the ribbon representation. The three residues at the C‐terminus, colored yellow, indicate the amino acids that are missing from LifeAct‐14. Structure 7AD9 was retrieved from the PDB (Belyy et al., 2020). (b) Cartoons showing a predicted structure of the LifeAct‐14‐EGFP construct (LifeAct‐14, magenta; EGFP, green) (Jumper et al., 2021) and illustrating the reversible nature of the binding between LifeAct‐14‐EGFP and actin filaments. EGFP sequence retrieved from FPbase (ID: R9NL8) (Lambert, 2019). Structures visualized using PyMOL (the PyMOL molecular graphics system, version 2.0 Schrödinger, LLC.). (c) A HEK293 cell, transiently transfected with a plasmid expressing LifeAct‐14‐EGFP. The distinctive actin filament structure is clearly visible in the region of interest (ROI, yellow box). The diagonal line separates diffraction‐limited and super‐resolution (SR) imaging. SR image colored “red hot.” Diffraction‐limited image was obtained by Z‐projecting the time‐course images (100 s, 50 ms exposure). 5 𝜇m scale barCC BY · PubMed Central
FIGURE 2 Intensity fluctuations and localizations of LifeAct‐14‐EGFP targeting F‐actin in HEK293 cells in response to a range of DNA transfection amounts. Diffraction‐limited images shown on the left are median intensity Z‐projections where the first column shows an entire field of view (FOV) taken over 2000 frames (100 s, 50 ms exposure, 10 𝜇m scale bar) and the adjacent column shows an ROI from the region in the dashed box shown in yellow over a 300‐frame subset (15 s). The time‐lapse montage column shows intensity fluctuations within the ROI across the 300‐frame subset (12‐frame step [0.6 s], time ‐ left to right and top to bottom). The SR images constructed from the detected localizations in the ROI (0.2 𝜇m scale bar) and then across the entire FOV (10 𝜇m scale bar, 2000 frames) are shown in the far right. Precision threshold <30 nm
FIGURE 2 Intensity fluctuations and localizations of LifeAct‐14‐EGFP targeting F‐actin in HEK293 cells in response to a range of DNA transfection amounts. Diffraction‐limited images shown on the left are median intensity Z‐projections where the first column shows an entire field of view (FOV) taken over 2000 frames (100 s, 50 ms exposure, 10 𝜇m scale bar) and the adjacent column shows an ROI from the region in the dashed box shown in yellow over a 300‐frame subset (15 s). The time‐lapse montage column shows intensity fluctuations within the ROI across the 300‐frame subset (12‐frame step [0.6 s], time ‐ left to right and top to bottom). The SR images constructed from the detected localizations in the ROI (0.2 𝜇m scale bar) and then across the entire FOV (10 𝜇m scale bar, 2000 frames) are shown in the far right. Precision threshold <30 nmCC BY · PubMed Central
FIGURE 3 F‐actin structures are dynamic within timescales of imaging and can be detected through direct‐LIVE‐PAINT. (a) SR images of F‐actin imaged with LifeAct‐14‐EGFP at a range of integration times in a HEK293 cell. 10 𝜇m scale bar. (b) Resolution and localization number in response to increasing integration times. Frame subsets were sampled randomly at each integration time for resolution and localization number calculation. Standard deviation error is shown as shaded region (n = 3). Precision threshold <20 nm. (c) SR images are shown as a time montage in an ROI (dashed green box from [a]), tracking an actin filament (dashed white box) through the same timeframe. 2 𝜇m scale bar. (d) Map of localizations color‐coded based on time of acquisition across the same timeframe. 2.5 s split, time scales from red to yellow, to green, to blue. Red box indicates the same filament of interest that changes in shape during this timeframe
FIGURE 3 F‐actin structures are dynamic within timescales of imaging and can be detected through direct‐LIVE‐PAINT. (a) SR images of F‐actin imaged with LifeAct‐14‐EGFP at a range of integration times in a HEK293 cell. 10 𝜇m scale bar. (b) Resolution and localization number in response to increasing integration times. Frame subsets were sampled randomly at each integration time for resolution and localization number calculation. Standard deviation error is shown as shaded region (n = 3). Precision threshold <20 nm. (c) SR images are shown as a time montage in an ROI (dashed green box from [a]), tracking an actin filament (dashed white box) through the same timeframe. 2 𝜇m scale bar. (d) Map of localizations color‐coded based on time of acquisition across the same timeframe. 2.5 s split, time scales from red to yellow, to green, to blue. Red box indicates the same filament of interest that changes in shape during this timeframeCC BY · PubMed Central

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Metadata

Authors
Bhaskar, H., Kleinjan, D., Oi, C., Gidden, Z., Rosser, S. J., Horrocks, M. H., & Regan, L.
Affiliations
  • University of Edinburgh (GB)
  • University of Washington (US)
Corresponding author
  • Horrocks, Mathew H.
  • Regan, Lynne
ORCID
Venue
Protein Science
Publisher
Wiley
Published
2023-01-26
Volume
32
Issue
2
Pages
e4558-e4558
ISSN
  • 0961-8368 (ISSN-L)
  • 1469-896X
DOI
10.1002/pro.4558
PMC
PMC9878614
OpenAlex
W4313340328
Cited by
12
References
35
Citations by year
2026: 2 · 2025: 5 · 2024: 4 · 2023: 1
FWCI
1.10
Type
journal article
Language
en
Open access
Licence
Locations
  • Protein Science (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Edinburgh Research Explorer (University of Edinburgh) (submittedVersion, cc-by)
Funders
  • Wellcome Trust (226437/Z/22/Z)
  • University of California Berkeley
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
  • Physical Sciences › Physics and Astronomy › Atomic and Molecular Physics, and Optics › Force Microscopy Techniques and Applications
Keywords
F-actin, super-resolution imaging, live-cell imaging, EGFP fusion protein, actin dynamics, diffraction limit
MeSH terms
Actins, Animals, Mammals, Actin Cytoskeleton, Protein Binding

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