title: In vivo imaging of the actin polymerization state with two-photon fluorescence anisotropy.
abstract: 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.
authors.affiliation: Department of Neuroscience, Columbia University, New York, New York, USA.
authors.forename: Harshad D
authors.initials: HD
authors.lastname: Vishwasrao
authors.forename: Pierre
authors.initials: P
authors.lastname: Trifilieff
authors.forename: Eric R
authors.initials: ER
authors.lastname: Kandel
mesh_terms: D000199:Actins / Q000737:chemistry*; D000818:Animals; D002470:Cell Survival; D004735:Energy Transfer; D005454:Fluorescence Polarization / Q000379:methods*; D057809:HEK293 Cells; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D010590:Phalloidine / Q000378:metabolism; D017785:Photons; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D015203:Reproducibility of Results; D013569:Synapses / Q000378:metabolism
chemical_list: D000199:Actins; D010590:Phalloidine