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09 17, 2024;123 (18): 3051-3064.

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

Hirushi Gunasekara, Thilini Perera, Chih-Jia Chao, Joshua Bruno, Badeia Saed, Jesse Anderson, Zongmin Zhao, Ying S Hu
Author Information
  1. Hirushi Gunasekara: Department of Chemistry, College of Liberal Arts and Sciences, University of Illinois Chicago, Chicago, Illinois.
  2. Thilini Perera: Department of Chemistry, College of Liberal Arts and Sciences, University of Illinois Chicago, Chicago, Illinois.
  3. Chih-Jia Chao: Department of Pharmaceutical Sciences, College of Pharmacy, University of Illinois Chicago, Chicago, Illinois.
  4. Joshua Bruno: Department of Biological Sciences, College of Liberal Arts and Sciences, University of Illinois Chicago, Chicago, Illinois.
  5. Badeia Saed: Department of Chemistry, College of Liberal Arts and Sciences, University of Illinois Chicago, Chicago, Illinois.
  6. Jesse Anderson: Department of Chemical Engineering, College of Engineering, University of Illinois Chicago, Chicago, Illinois.
  7. Zongmin Zhao: Department of Pharmaceutical Sciences, College of Pharmacy, University of Illinois Chicago, Chicago, Illinois.
  8. Ying S Hu: Department of Chemistry, College of Liberal Arts and Sciences, University of Illinois Chicago, Chicago, Illinois. Electronic address: yshu@uic.edu.
PMID: 38961624 DOI: 10.1016/j.bpj.2024.07.003

Abstract

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.

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Grants

  1. R35 GM146786/NIGMS NIH HHS

MeSH Term

Phalloidine
Actins
Animals
Mice
Dendritic Cells
Humans
Nanotechnology
Cell Line, Tumor

Chemicals

Phalloidine
Actins
Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

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