Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025 Feb;35(2):149-152.
doi: 10.1038/s41422-024-01061-9. Epub 2024 Dec 10.

Photoactivated SOPP3 enables APEX2-mediated proximity labeling with high spatio-temporal resolution in live cells

Affiliations

Photoactivated SOPP3 enables APEX2-mediated proximity labeling with high spatio-temporal resolution in live cells

Dajun Qu et al. Cell Res. 2025 Feb.
No abstract available

PubMed Disclaimer

Conflict of interest statement

Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Photoactivated SOPP3 triggers APEX2-mediated proximity labeling with high spatio-temporal resolution.
a Schematic illustration of APEX2 + SOPP3-mediated photoactivated-proximity labeling (photo-PL). B, biotin. b Photosensitizer screening for APEX2 activation. Streptavidin blot showed photo-PL efficiency. Anti-V5 and anti-HA blots indicated expression levels of APEX2 and photosensitizers, respectively. α-Tubulin, internal loading control for western blot. Three bands in the negative control lanes correspond to endogenous biotinylated proteins (at 130 kDa, 75 kDa and 72 kDa). c Evaluation on the generation of singlet oxygen using Singlet Oxygen Sensor Green (SOSG) probe (10 μM). ****P < 0.0001; data are shown as mean ± SEM (n = 3). Statistical significance was determined using two-way ANOVA analysis. d Schematic illustration of APEX2 + SOPP3-mediated PL to map proteome at ER–Mito contact sites. BP, biotin-phenol. e Evaluation on the efficiency of photo-PL at ER–Mito contact sites. Anti-HA and anti-V5 blots indicated expression levels of SOPP3-ERM and OMM-APEX2, respectively. f Left: confocal fluorescence imaging of APEX2 + SOPP3-mediated photo-PL at ER–Mito contact sites. OMM-localized APEX2 and ERM-localized SOPP3 were visualized by anti-V5 antibody in combination with Alexa Fluor 647-conjugated secondary antibody and anti-HA antibody in combination with Alexa Fluor 488-conjugated secondary antibody, respectively. Biotinylation signals at the contact sites were visualized by Alexa Fluor 555-conjugated streptavidin. Illumination time, 5 s. Scale bar, 9 μm. Right: the “Surface” tool in Imaris software was used to create a 3D rendering from each channel of confocal images of boxed region. Contact area algorithm was further performed to determine the interface (yellow) between ER (green) and Mito (cyan). Scale bar, 0.8 μm. g Validation on MAM proteins from streptavidin-enriched PL samples. h, i Schematic diagram and construct designs (h) and evaluation on the photo-PL efficiency at cell–cell contact sites via western blotting analysis (i). Anti-V5 and anti-HA blots indicated expression levels of APEX2-TM and SOPP3-TM, respectively. j Confocal fluorescence imaging of APEX2 + SOPP3-mediated photo-PL at cell–cell contact sites, stained with Alexa Fluor 555-conjugated streptavidin. Scale bar, 2 μm. Zoomed image from boxed region showed biotin labeling precisely at cell–cell contact sites. Scale bar, 1 μm. Illumination time, 10 s. k Schematic diagram and construct design of chimeric APEX2-SOPP3-mediated photo-PL in ERM. l Evaluation on the efficiency of photo-PL mediated by chimeric APEX2-SOPP3 targeted to ERM via western blotting analysis. Anti-HA, anti-Flag and anti-V5 blots indicated expression levels of chimeric APEX2-SOPP3-ERM, SOPP3-ERM and OMM-APEX2, respectively. m Confocal fluorescence imaging of photo-PL via APEX2-SOPP3 targeted to ERM. Chimeric APEX2-SOPP3-ERM and biotinylation signals were visualized by anti-HA antibody in combination with Alexa Fluor 488-conjugated secondary antibody and Alexa Fluor 555-conjugated streptavidin, respectively. Illumination time, 5 s. Scale bar, 10 μm. n, o Schematic diagram and construct designs (n) and evaluation on the efficiency of photo-PL mediated by chimeric APEX2-SOPP3 at cell–cell contact sites via western blotting analysis (o). Anti-HA blot indicated expression level of chimeric APEX2-SOPP3-TM. p Confocal fluorescence imaging of chimeric APEX2-SOPP3-mediated photo-PL on cell surface. Chimeric APEX2-SOPP3 was visualized by anti-HA antibody in combination with Alexa Fluor 488-conjugated secondary antibody. Biotinylation signals were visualized by Alexa Fluor 555-conjugated streptavidin. Scale bar, 8 μm. Illumination time, 5 s. q Flow cytometry analysis on p (30,000 cells per condition). After photo-PL, cells were stained with Alexa Fluor 555-conjugated streptavidin, followed by FACS gating for quantification of biotin labeling on the surface of APEX2-SOPP3-TM cells (anti-HA positive, q upper) and neighboring WT cells (anti-HA negative, q lower). Blue light-activated SOPP3 was more efficient to facilitate APEX2-mediated proximity labeling compared to 1 mM H2O2 treatment. Illumination time, 10 s.

References

    1. Lam, S. S. et al. Nat. Methods12, 51–54 (2015). - PMC - PubMed
    1. Kotani, N. et al. Proc. Natl. Acad. Sci. USA105, 7405–7409 (2008). - PMC - PubMed
    1. Roux, K. J., Kim, D. I., Raida, M. & Burke, B. J. Cell Biol.196, 801–810 (2012). - PMC - PubMed
    1. Kim, D. I. et al. Mol. Biol. Cell27, 1188–1196 (2016). - PMC - PubMed
    1. Branon, T. C. et al. Nat. Biotechnol.36, 880–887 (2018). - PMC - PubMed

LinkOut - more resources