In vivo imaging of the actin polymerization state with two-photon fluorescence anisotropy.

Vishwasrao HD; Trifilieff P; Kandel ER

doi:10.1016/j.bpj.2012.01.031

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In vivo imaging of the actin polymerization state with two-photon fluorescence anisotropy.

Vishwasrao HD, Trifilieff P, Kandel ER.

Biophys J. 2012 Mar 7; 102(5):1204-14

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Abstract

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18 May 2012

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Suzanne Scarlata
F1000 Pharmacology & Drug Discovery
Stony Brook University Medical Center,
Stony Brook, NY, USA.

Technical Advance

DOI: 10.3410/f.715447852.791052804

In this study, the authors developed a method to study the polymerization of actin in living cells by fluorescence homotransfer between actin-green fluorescent protein (GFP). The authors derive a relation between actin-GFP fluorescence anisotropy and the actin polymer fraction, which enables them to follow actin polymerization in living cells, brain tissue and insect larvae. The use of fluorescence anisotropy to monitor homo-oligomerization and self-assembly offers several advantages over the more common hetero- fluorescence resonance energy transfer (FRET) measurements.

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Scarlata S: F1000Prime Recommendation of [Vishwasrao HD et al., Biophys J 2012, 102(5):1204-14]. In F1000Prime, 18 May 2012; DOI: 10.3410/f.715447852.791052804. F1000Prime.com/715447852#eval791052804

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Using two-photon fluorescence anisotropy imaging of actin-GFP, we have developed a method for imaging the actin polymerization state that is applicable to a broad range of experimental systems extending from fixed cells to live animals. The incorporation of expressed actin-GFP monomers into endogenous actin polymers enables energy migration FRET (emFRET, or homoFRET) between neighboring actin-GFPs. This energy migration reduces the normally high polarization of the GFP fluorescence. We derive a simple relationship between the actin-GFP fluorescence polarization anisotropy and the actin polymer fraction, thereby enabling a robust means of imaging the actin polymerization state with high spatiotemporal resolution and providing what to the best of our knowledge are the first direct images of the actin polymerization state in live, adult brain tissue and live, intact Drosophila larvae.

Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

DOI: 10.1016/j.bpj.2012.01.031

PMID: 22404943

Abstract courtesy of PubMed: A service of the National Library of Medicine and the National Institutes of Health.