Poking holes into membranes to label proteins for live imaging

There are two major way to label inner proteins, structures or organnelles for live cell imaging. The most common method is fusing the studied protein to a fluorescent protein. A second approach is the addition of labeling agents from outside the cells. However, many labels cannot penetrate through the cell membrane. This is true to some, but not all dyes, but more importantly, to larger agents, such as antibodies or DNA/RNA oligos. To allow these agents to enter cells, researchers can use microinjection, electroporation, bead-loading, or transfection (e.g. of short oligos).

In a paper just published in eLife, a new technique is described to form temporary holed in the cell membrane. These holes allow delivery of any labeling agent into cells.

Sterptolysin O (SLO) is a streptococcal toxin that makes holes in membranes. It has been used in the past as a way to deliver goods into cells. But there was no systematic analysis of which molecules can be delivered or the health and recovery of the cells after SLO treatment. So the idea in this paper was to test the usefulness of SLO to deliver small and larger labeling agents to label specific proteins for live cell imaging and for super resolution microscopy, direct stochastic optical reconstruction microscopy (dSTORM).

the method is very simple: you treat the cells for 7-10 minutes with SLO. The incubation time depends on cell type & confluence. They also note that different batches have different activities and must be tested empirically. after that, 5min with the fluorescent label, some washes and then recovery for 15-20 min in a recovery medium (which is normal medium supplemented with excess ATP, GTP and glucose).

Using this method, they were able to show delivery of fluorescent probes of sizes ranging from very small

Some control experiments showed that the cells remain viable and able to divide. Another experiment showed that the mitochodria (labeled with mtGFP-Halotag) looks the same before and after SLO. Another control experiment was to show that a signal transduction pathway is working. They showed that stimulation with TNFα induces nuclear translocation of the transcription factor p65.

Overall, it looks like a useful method for intracellular labeling. However, there are a few points that I find lacking in their paper (or I missed it?)

  1. Although cells seem to recover nicely, there must be an effect on many parameters. The imaged organelles seem to be intact and unharmed – but these are only 3 organelles that were checked: mitochondria, peroxisomes and nucleus. What about ER? endosomes? the plasma membrane itself? Also, their cell division assay is not very convincing: they counted cells in only 2 regions after 16hr. Also, were is the control of untreated cells?
  2. The damage to the membrane probably induces stress response pathways. The recovery phase probably induces other pathways as well. This is something to consider when using this system. Probably needs transcriptomics/proteomics to assess what goes on in those damaged/recovered cells. These responses can affect your experimental outcome…
  3. One question that I really did not see being addressed, is how long do the labeling lasts? Obviously, it depends on the type of labeling and the labeled protein. Most of their imaging, if I understood correctly, took place within less than 1hr after the hole-poking. It would have been interesting to follow the cells for longer times to see what’s going on.
  4. Obviously, the function of the labeled protein itself can be affected. An antibody bound to an enzyme might inhibit its activity, alter it’s localization, affect its stability.
  5. Last, they only compared their labeling with a genetically encoded fluorescent-fusion protein. At least for Halotag experiments, they could, should, have compared to SLO-untreated cells with a Halotag cell-permeant dyes (e.g. JF dyes from Luke Lavis lab). Such an experiment would allow a true comparison of the status of the cell and the effect of SLO on the activity/localization of the Halotag-labeled protein.

All these needs to be taken into consideration. Still, because it seems to be a rather simple method, I think people should try it. I would like to. I have some ideas already…

ResearchBlogging.orgTeng KW, Ishitsuka Y, Ren P, Youn Y, Deng X, Ge P, Belmont AS, & Selvin PR (2016). Labeling proteins inside living cells using external fluorophores for microscopy. eLife, 5 PMID: 27935478

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out /  Change )

Google photo

You are commenting using your Google account. Log Out /  Change )

Twitter picture

You are commenting using your Twitter account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s