LifeAct is a 17aa peptide sequence that could be conjugated with a fluorophore that could bind actin. Though Lifeact is being widely used for live cell imaging, it is reported that there are several concentration dependent effect on cellular. So it is worthy to look in to the drawbacks of the worlds most trusted live cell actin markers.
Whatever probe you add to your life cells, you can be sure there will be an effect. The best way to minimise this is to confirm your results in different assays.
In my experience, and for live cell imaging of actin filaments, Lifeact is the best. The expression is very straight forward and the background signal, i.e., the fluorescence signal arising from fluorophores not bound to actin filaments, is lower than when using eGFP.actin (which if you over-express will also modify the dynamics of your cytoskeleton.
Dear Athul,
Sebastian and Susana's answers above addressed your question very nicely.
I would like to add that in spite of the drawbacks of lifeact overexpression, it is relatively easy to detect those cells developing artifacts cause by the overexpression and therefore discriminate them from your studies.
When lifeacts is overexpressed you'll start seeing lot's of actin filament bundling, excessive filopodia production, apparently, at expenses of lamellipodia and increased bending of actin filaments (kinking) at the base of the filopodia. Also, filopodia* tends to retract by bending rather than collapsing by depolymerization.
*Wheter apparent stabilization of filopodia arises from direct stabilization or from an increased polymeryzation rate is still under discussion.
To be in the safe side, I would suggest to choose cell were signal not too strong (just enough for analyzing dynamics) so you are as far as possible from high concentrations that could interfere with the dynamics you are trying to investigate.
You can eventually deconvolve your images to increase sharpness.
To facilitate the reproducibility of your experiments, I would always work with a unique lifeact stock that should be titrated in your system to select the best concentration to use. This is, prepare a high quality, well measured stock of lifeact plasmid (enough for a long series of studies), and to titrate the best amount of DNA to use in your cell system maintaining fix your number of cells and rising the amount of plasmid from 0.25 ug until 2-3ug to then check for those concentration giving good signal with low rate of cells showing overexpression artifacts.
Hope this helps,
Best
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Peptides