PARF analysis or Permeabilization Activated Reduction in Fluorescence analysis allows researchers to describe the rate of dissociation of a fluorescently tagged protein of interest from intracellular structures. I will primarily be spending most of my time conducting PARF analysis to determine the effect of depleted SPECC1L on focal adhesions.
In our experiments, we transfect, or introduce, fluorescently tagged vinculin, which is one protein known to be found in focal adhesions. We visualize the focal adhesions using TIRF microscopy, and take control videos for each condition. Primarily, however, we are interested in the rate of dissociation of the fluorescently tagged vinculin from focal adhesions. This rate of dissociation corresponds to the size and relative strength of the focal adhesions. To cause this dissociation, we add digitonin to the cell media as we are filming to induce permeabilization of the cell membrane. To see more, look at the bottom of the post for a supplemental video!
This permeabilization results in an unbalanced gradient of fluorescent vinculin, with a high concentration within the cell, and a low concentration in the surrounding media. This gradient favors the dissociation of the vinculin into the external media. We measure the loss of fluorescence for around two minutes and then can fit this to an exponential decay. Using both a positive and negative control that causes larger and smaller focal adhesions, we can determine the effect of depleted SPECC1L on a cell’s focal adhesions.
Supplementary Material:
Supplemental Figure 1. This technique can be applied to various other proteins of interest. In the above video, we permeabilize these S2R+ cells expressing both fluorescent Naus GFP and fluorescent mCherry cortactin with digitonin after 20 seconds. We can then visualize the loss of fluorescent cortactin as it dissociates from the cell into the surrounding media.
Note: To view the video, you have to download it! If you have a Mac, click on the link while pressing Ctrl, and download the file.
Citations:
- Singh PP, Hawthorne JL, Quintero OA. Permeabilization Activated Reduction in Fluorescence (PARF): a novel method to measure kinetics of protein interactions with intracellular structures. Cytoskeleton (Hoboken). 2016 June ; 73(6): 271–285. doi:10.1002/cm.21306.