I transfected a protein in to cells. This protein promotes cell migration. When I co-transfected the cells with paxillinI, I can observe a decrease of paxillin in the transfected cells compared to control samples. I want to test the adhesion turn over but I don't know how to do it. Is there anyone with experience that can teach me how to do this analysis?
HI, I've never done that, but I suppose you can do a timelapse immunofluorescence with an antibody directed toward actin, and keep track of the focal adhesion point.
To assess the rate of migration, I'll suggest also a wound healing/transwelling assay.
Hi I 've never done it myself but you can take a look at this paper http://www.ncbi.nlm.nih.gov/pubmed/14576354 they are experts in adhesions turnover.
the one weird thing you mention though is that you see decreased levels of paxillin: normally what changes during adhesion maturation is the level of phosphorylation (mainly at Tyr31 and Tyr118) not the total levels of paxillin.
Hope this helps!
Hi, there are a numbers of ways to analyze adhesions turnover. As suggested by Marco, one of the most informative approaches is the set up of a time-lapse analysis to follow focal adhesion persistance over time. In my opinion, the more reproducible way is to use a cell line with stable reporter (such as GFP- or RFP-paxillin). I used the lentiviral particles sold by Millipore (17-10154 | LentiBrite™ Paxillin-GFP Lentiviral Biosensor, http://www.emdmillipore.com/GB/en/product/LentiBrite%E2%84%A2-Paxillin-GFP-Lentiviral-Biosensor,MM_NF-17-10154) to establish a stable HeLa cell line with this reporter and then use it for my experiments. For the time lapse analysis, I used TIRF microscopy to specifically observe basal fluorescence staining. Finally, you would have to track FAs and analyze the data: you could do it by ImageJ or Matlab softwares.
Good luck with your experiments!
I agree with other colleages: life cell imaging (TIRF, confocal, WF with transient or stable fluorescently labeled markers: paxillin - early adhesion, zyxin - late adhesion, p130Cas, etc.) As an analysis you may want to try - ImageJ Kymograph .
Thank you very much for wonderful suggestion friends. I read several papers related to focal adhesion, I found one lab that worked on focal adhesion so I wrote an email for them to ask the way to analyze focal adhesion turn over. They also mentioned TIRF or confocal microscope. I did confocal microscope for this experiment. When I analyzed the data, it requires a lot of patient and labor to work on that. I will try the website that Julien Elric suggested. Thank you very much for helping me solve this problem.
Impedance based measurements can also provide some readout for adhesion in this case.
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