In this particular experiment, it is not possible to perform any permeabilization protocol that weakens the sarcolemma or cytoskeleton, so I would have to rely on finding a Phalloidin that can permeate the cell without any such protocol being performed beforehand.
Thanks for any input on this!
Phalloidin enters liver and kidney cells via cell membrane transporters (I don't remember which). It does not permeate the plasma membrane. I have no idea if heart cells will express the same transporters. You might want to consider SiR-Actin as an alternative. SiR-Actin is a pretty bright fluorescent jasplakinolide derivative. Its main drawback is that its emission peak is @670 nm, which means that you won't see it with your own eyes!
Edit: I forgot to mention that some people report using liposomes to deliver fluorescent phalloidin to live cells (see Neurosci Lett (1996) 207:17-20). However, I cannot vouch for these methods because we have never used them in the lab.
Thanks very much for your reply, Guillermo! The reason I'd like to use Phalloidin is because it depolymerizes Actin filaments, but I'll look more into alternatives!
Yes, we tried with injecting small amounts of phalloidin in cell and image the F-actin. The results were interesting, but it looked like phalloidin strongly affected the actin dynamics and changed cell morphology. A better way is to try with TxR labeled G-actin injection and use the fluorescence speckle microscopy. That is, injecting tiny amounts of labeled actin and then imaging using a high sensitivity camera. GFP-actin did not give good results as it changed the actin dynamics to a substantial degree. I guess it has to do with the fact that GFP is a relatively larger molecule compared to G-actin. Taking everything we got, I thing the best is probably TxR or Rhodamine labeled actin with Fluorescence Speckle Microscopy (FSM). Hope this helps.
Regards
Thank you Nurul, for a very helpful answer! I've been looking into the possibility of microinjecting it so knowing that you succeeded is great.
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