One- and two-photon photoactivation of a paGFP-fusion protein in live Drosophila embryos

Janine Nicole Post, Keith A. Lidke, Bernd Rieger, Donna J. Arndt-Jovin

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68 Citations (Scopus)

Abstract

We constructed a photoactivatable Drosophila histone 2 A variant green fluorescent fusion protein (H2AvD-paGFP) for tracking chromatin loci in living Drosophila embryos. Activation of paGFP was achieved by irradiation from a single-photon diode laser at 408 nm, but activated nuclei failed to divide. Photoconversion could also be achieved by two-photon fs pulses in the range of 780–840 nm. Viability in whole-mount embryos could only be maintained at 820 nm, at which we could activate, simultaneously track and quantitate the mobility of multiple fluorescent loci. This report constitutes the first demonstration of two-photon activation of paGFP and the use of a paGFP-fusion protein in investigations of whole organisms. Keywords: Green fluorescent protein; Histone; Chromatin dynamics; Nuclear architecture; Two-photon microscopy; Photoactivation Abbreviations: eGFP, enhanced green fluorescent protein; paGFP, photoactivatable GFP; wtGFP, wild-type GFP; H2AvD, histone 2 A variant Drosophila; FRAP, fluorescence recovery after photobleaching; DIC, differential interference contrast; MSD, mean square displacement; SNR, signal-to-noise ratio; FWHM, full-width at half-maximum
Original languageUndefined
Pages (from-to)325-330
JournalFEBS letters
Volume579
Issue number2
DOIs
Publication statusPublished - 2005

Keywords

  • IR-77332

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