Phalloidin-PAINT: Enhanced quantitative nanoscale imaging of F-actin.

Autor:	Gunasekara H; Department of Chemistry, College of Liberal Arts and Sciences, University of Illinois Chicago, Chicago, Illinois., Perera T; Department of Chemistry, College of Liberal Arts and Sciences, University of Illinois Chicago, Chicago, Illinois., Chao CJ; Department of Pharmaceutical Sciences, College of Pharmacy, University of Illinois Chicago, Chicago, Illinois., Bruno J; Department of Biological Sciences, College of Liberal Arts and Sciences, University of Illinois Chicago, Chicago, Illinois., Saed B; Department of Chemistry, College of Liberal Arts and Sciences, University of Illinois Chicago, Chicago, Illinois., Anderson J; Department of Chemical Engineering, College of Engineering, University of Illinois Chicago, Chicago, Illinois., Zhao Z; Department of Pharmaceutical Sciences, College of Pharmacy, University of Illinois Chicago, Chicago, Illinois., Hu YS; Department of Chemistry, College of Liberal Arts and Sciences, University of Illinois Chicago, Chicago, Illinois. Electronic address: yshu@uic.edu.
Jazyk:	angličtina
Zdroj:	Biophysical journal [Biophys J] 2024 Sep 17; Vol. 123 (18), pp. 3051-3064. Date of Electronic Publication: 2024 Jul 03.
DOI:	10.1016/j.bpj.2024.07.003
Abstrakt:	We present phalloidin-based points accumulation for imaging in nanoscale topography (phalloidin-PAINT), enabling quantitative superresolution imaging of filamentous actin (F-actin) in the cell body and delicate membrane protrusions. We demonstrate that the intrinsic phalloidin dissociation enables PAINT superresolution microscopy in an imaging buffer containing low concentrations of dye-conjugated phalloidin. We further show enhanced single-molecule labeling by chemically promoting phalloidin dissociation. Two benefits of phalloidin-PAINT are its ability to consistently quantify F-actin at the nanoscale throughout the entire cell and its enhanced preservation of fragile cellular structures. In a proof-of-concept study, we employed phalloidin-PAINT to superresolve F-actin structures in U2OS and dendritic cells (DCs). We demonstrate more consistent F-actin quantification in the cell body and structurally delicate membrane protrusions of DCs compared with direct stochastic optical reconstruction microscopy (dSTORM). Using DC2.4 mouse DCs as the model system, we show F-actin redistribution from podosomes to actin filaments and altered prevalence of F-actin-associated membrane protrusions on the culture glass surface after lipopolysaccharide exposure. The concept of our work opens new possibilities for quantitative protein-specific PAINT using commercially available reagents. Competing Interests: Declaration of interests The authors declare no competing interests. (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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