Two-color Photoactivatable Probe for Selective Tracking of Proteins and Cells

Arkadiusz Welman,Alan Serrels,Valerie G. Brunton,Mark Ditzel,Margaret C. Frame

doi:10.1074/jbc.m110.102392

Arkadiusz Welman, Alan Serrels + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m110.102392

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Abstract

We report the development and application of photoactivatable Green Cherry (G(PA)C), the first genetically encoded "continuously red-photoactivatable green" two-color probe for live cell imaging. G(PA)C is unique in that it enables real-time tracking of selected subpopulations of proteins and organelles in the cell or of cells within tissues and whole organisms, with constant reference to the entire population of the probe. Using G(PA)C-zyxin as proof of utility, we obtained new insights into the dynamic movement of the cytoskeletal protein zyxin. We show that zyxin is continuously and rapidly recruited from the cytosol into established focal adhesions. It can also move rapidly within a given focal adhesion and "hop" between adjacent focal adhesions, emphasizing the dynamic nature of proteins within these structures. The in vivo utility of G(PA)C is exemplified by tracking hemocyte movements using a versatile transgenic Drosophila model engineered to express G(PA)C in tissues and cells of interest under the control of the GAL4-inducible promoter.

Highlights

We provide proof of principle that this is a probe with great potential, utility, and versatility by demonstrating proof of concept tracking and dynamic movement of the focal adhesion protein zyxin and by tracking hemocyte movements between distinct regions of intact larvae of Drosophila melanogaster
We generated several mCherry-photoactivatable green fluorescent protein (PAGFP) fusion constructs employing different linker sequences. This strategy was associated with some risk because green fluorescent protein and mCherry have been previously described to constitute a good fluorescence resonance energy transfer (FRET) donor acceptor pair, especially in tandem construct experiments [21]
The vast majority of currently available FHPs are visible in one fluorescence channel only [4]

Summary

Introduction

The currently available photoactivatable fluorescent proteins include photoactivatable green fluorescent protein (PAGFP) [7], photoactivatable monomeric RFP1 [8], KFP1 [9], Dronpa [10], and photoactivatable mCherry [11] Their lack of visibility in the non-activated state and bright fluorescence after activation provide a good contrast welcomed in fluorescent microscopy, a limitation that remains is to select specific intracellular subpopulations to be activated for dynamic analysis. The development of additional FHPs with new and improved properties is eagerly awaited and would contribute to further advances in the field For these reasons, we have devised a novel photoactivatable fluorescent probe, photoactivatable Green Cherry (GPAC), which enables efficient highlighting and tracking of selected pools of proteins, organelles, and cells with parallel continuous visualization of the entire population of the probe. We provide proof of principle that this is a probe with great potential, utility, and versatility by demonstrating proof of concept tracking and dynamic movement of the focal adhesion protein zyxin and by tracking hemocyte movements between distinct regions of intact larvae of Drosophila melanogaster

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