Quantitative Superresolution Imaging of F-Actin in the Cell Body and Cytoskeletal Protrusions Using Phalloidin-Based Single-Molecule Labeling and Localization Microscopy.

Autor:	Gunasekara H; Department of Chemistry, College of Liberal Arts and Sciences, University of Illinois Chicago, Chicago, IL, 60607, USA., Perera T; Department of Chemistry, College of Liberal Arts and Sciences, University of Illinois Chicago, Chicago, IL, 60607, USA., Chao CJ; Department of Pharmaceutical Sciences, College of Pharmacy, University of Illinois Chicago, Chicago, IL, 60612, USA., Bruno J; Department of Biological Sciences, College of Liberal Arts and Sciences, University of Illinois Chicago, Chicago, IL, 60607, USA., Saed B; Department of Chemistry, College of Liberal Arts and Sciences, University of Illinois Chicago, Chicago, IL, 60607, USA., Anderson J; Department of Chemical Engineering, College of Engineering, University of Illinois Chicago, Chicago, IL, 60607, USA., Zhao Z; Department of Pharmaceutical Sciences, College of Pharmacy, University of Illinois Chicago, Chicago, IL, 60612, USA., Hu YS; Department of Chemistry, College of Liberal Arts and Sciences, University of Illinois Chicago, Chicago, IL, 60607, USA.
Jazyk:	angličtina
Zdroj:	BioRxiv : the preprint server for biology [bioRxiv] 2024 Mar 06. Date of Electronic Publication: 2024 Mar 06.
DOI:	10.1101/2024.03.04.583337
Abstrakt:	We present single-molecule labeling and localization microscopy (SMLLM) using dye-conjugated phalloidin to achieve enhanced superresolution imaging of filamentous actin (F-actin). We demonstrate that the intrinsic phalloidin dissociation enables SMLLM 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-based SMLLM are better preservation of cellular structures sensitive to mechanical and shear forces during standard sample preparation and more consistent F-actin quantification at the nanoscale. In a proof-of-concept study, we employed SMLLM to super-resolve F-actin structures in U2OS and dendritic cells (DCs) and demonstrate more consistent F-actin quantification in the cell body and structurally delicate cytoskeletal proportions, which we termed membrane fibers, of DCs compared to direct stochastic optical reconstruction microscopy ( d STORM). Using DC2.4 mouse dendritic cells as the model system, we show F-actin redistribution from podosomes to actin filaments and altered prevalence of F-actin-associated membrane fibers on the culture glass surface after lipopolysaccharide exposure. While our work demonstrates SMLLM for F-actin, the concept opens new possibilities for protein-specific single-molecule labeling and localization in the same step using commercially available reagents. Competing Interests: Competing Interests The authors declare no competing financial interests.
Databáze:	MEDLINE
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