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

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.

The peripheral nervous system supports blood cell homing and survival in theDrosophilalarva

“…Based on our real-time and time-lapse studies, and consistent with the previously reported adhesion-based recruitment of circulating hemocytes to wound sites , and hemocyte dynamics in the terminal cluster (Welman et al, 2010), some of this exchange may be attributed to the detachment, circulation and subsequent re-attachment of hemocytes to resident sites. However, lateral movement of hemocytes during re-formation…”

“…Previous studies investigated the dynamics of circulating-, dorsalvessel-associated-, and terminal cluster hemocytes of the larva Welman et al, 2010). Focusing on epidermal-muscular pockets, we found that physical manipulation can perturb the pattern of resident hemocytes, which spontaneously re-formed within 30-60 minutes, independently of new hemocyte formation ( Fig.…”

Actin network disassembly powersListeria monocytogenesdissemination

“…Collectively, these results demonstrate that protrusion elongation relies on a pool of ARP2/3 that is constantly recycled from the distal network and incorporated into the newly formed network at the bacterial pole. We further examined the dynamics of the actin network by using a photo-activatable version of GFP fused to mCherry-actin (Welman et al, 2010). In the cytosol, photo-activation of the distal region of an elongating actin tails produced a GFP signal that slowly decreased in intensity as the tail disassembled (Fig.…”

“…Primary infected cells were transfected with a membrane-CFP plasmid (supplementary material Table S2) and a PAGFP-mCherry-tagged bactin construct (Welman et al, 2010). A Micropoint 404 nm laser (Andor Technology) was used to photo-activate a region of interest (ROI) situated ,2 mm from a bacterial pole in protrusions (above 7 mm in length).…”

Advances in imaging cell–matrix adhesions

“…An alternative is the use of newer PA vectors that also contain a normal fluorescent tag, thus allowing the location of a specific pool of the PA-fluorophore-tagged protein of interest to be defined. One example of this is a recent study in which zyxin was tagged with PA-GFP at one end and mCherry at the other (Welman et al, 2010). This allowed the authors to follow zyxin by imaging the mCherry tag before selecting a particular region to be activated, for example, a focal adhesion.…”