Category Archives: FISH

Separating cells is hard

I write this entry to accompany my short talk at meeting ( #physiologicalirrelevantconference ) .

A few years ago we published a paper in PNAS in which we showed that full-length mRNAs transfer between mammalian cells via a unique type of structure called membrane nanotubes, or tunneling nanotubes (TNTs). This work was started at Rob Singer’s lab, continued at the Gerst lab and in collaboration with Arjun Raj.

I wrote a “behind the scenes” post, detailing how that paper came to be, and some of the problems I had along the way.

I next published a method paper, which also included some new information – in particular that the transferred mRNA is encapsulated in an unknown protein shell. I wrote a “behind the paper” post at the Springer Nature blogs. There, I told about all the problems I had just because a simple change of the cell fixation conditions of my FISH protocol.

The problem is depicted on the right side of my slide:


Very briefly – because the regular FISH protocol leads to TNTs breakage and loss, I decided to increase TNTs stability by adding glutaraldehyde to the fixation buffer. This led to a four-fold increase in TNT preservation. But the transferred mRNA disappeared! It took me a very long time to figure out what’s going on there and partially solve this – at the expense of TNTs’ stability again.  I still have hopes to find a fixative that will preserve the TNTs without affecting the FISH quality.

The left side of the slide depicts our grad student’s greatest achievement – something we’ve been trying to get at over the past six (6!) years. The idea is very simple – co-culture human and mouse cells. After some time, separate then to pure human or mouse cell populations and send for RNA-seq. This should reveal the entire transferome – which human mRNAs are found in the mouse cells and vice versa. As a control, we have a mix of human & mouse cells which were cultured separately, mixed and immediately separated in parallel to the co-culture.

The issue is that we need very high purity. This is because we estimated the amount of transferred mRNA as 1% or less of the endogenous. So if we have 1% donor cell contamination, it will obscure the transferred mRNAs.

For about 2-3 years, I tried to separate the cells with flow cytometry, using various labeling strategies and conditions. But I never managed to get a clear signal of our positive control (MS2-labeled mouse beta-actin mRNA) in co-culture over mix. Then Sandipan Dasgupta joined our lab and instead of FACS sorting, he used affinity purification with magnetic beads to sort the cells. It seemed to be going fairly well. So much so that we also designed an in vivo experiment in mice. We then sent our samples to sequencing only to find out that the sequencing facility had made some mistakes, or there was another problem and all our samples were either contaminated with mouse RNA, or just mixed somehow. That facility closed (we were last in queue ) so there was no way to solve it. But, we also learnt that we probably would not have had enough coverage anyway.

So, Sandi repeated the (in vitro) experiment in order to collect new samples for RNA seq – but we noticed, based on more quality control experiments we did, that the separation was not good enough for us. Although the mouse cells were very pure (99.9%), the human cells always had a small level of mouse cells (98.5% purity of the human cells). Since our expected signal is about 1-2% of the mRNAs being transferred, we could barely see a signal in co-culture compared to mix (1.3-fold).

So, Sandi worked really hard, playing with the conditions until he solved it, and got consistent 99.9% purity of the human cells – just a few months ago. The qRT-PCR result in the slide shows 4-5 fold more human beta-actin mRNA in mouse cells in co-culture compared to mix (we have similar results for the mouse beta-actin mRNA in human cells). The samples were shipped for deep RNA seq (150 million reads per sample) and we are waiting for the results.

We also have more experiments going on – but these stories are for another time.

Maybe we should open a falafel stand” is an actual text from Jeff when we discussed one Saturday evening on Whatsapp about all the problems we encounter in our experiments.



Quantum dots for smFISH

Single molecule FISH is currently the best method to get accurate measurements of mRNA levels at single molecule, single cell level in cell culture or tissue slices with a spatial resolution of ~200 nanometer (or less). One of the drawbacks of this method is the deterioration of the fluorescent signal (bleaching) of the organic dyes that are used to label the probes. Andrew Smith’s lab from University of Illinois now show how FISH can work with quantum dots instead of organic dyes. This provides better fluorophore stability and also the possibility to have more colors with less overlap of the emission spectra.

Continue reading

MS2 mRNA imaging in yeast – problem solved

Previously, on the story of MS2 labeling of mRNA in yeast: Roy Parker published a short letter to the editor, indicating that the MS2 system might cause accumulation of 3′ fragments. We wrote a response, showing that it is not always the case for endogenously expressed mRNAs, but it is exaggerated when over-expressed (Part 1)*. Later, Karsten Weis’s group confirmed Parker’s initial observation but their report still had some questions unanswered, and no solution to the problem; I was unhappy (Part 2).  Now, Evelina Tutucci and Maria Vera together with Jeet Biswas (all from Rob Singer’s lab) seem to have resolved the issue and solved the problem, with the development of the MBS version 6Continue reading

Intercellular mRNA transfer through membrane nanotubes – behind the scenes.

My paper was recently published. I suggest that you read it before reading this post (it is an open access paper). In this paper we show that full-length mRNA molecules can be transferred between mammalian cells through membrane nanotube-like extensions that connect the cells.

Continue reading

New CRIPSR-based RNA imaging tool

About a year and a half ago I wrote here about new uses of CRISPR/Cas9 as an imaging tool. In particular, I was excited about the possibility to use enzyme-dead Cas9 (dCas9) as an RNA binding protein for live imaging of mRNA. Unfortunately, in my hands this did not work (the dCas9 has exited the nucleus with non-targeting guide RNA at the same rate as with the specific guide RNA).

Last week, a new CRISPR tool was published in Nature, from Feng Zhang’s lab.

Continue reading

MS2 mRNA imaging in yeast: more evidence for artefacts

Previously, on the story of MS2 in yeast: Last year, Roy Parker published a short article, in which he claimed that using the MS2 system in yeast causes the accumulation of 3′ RNA fragments, probably due to inhibition of mRNA degradation by the 5′ to 3′ exoribonuclease Xrn1. He argued that these findings put in question all the work on mRNA localization in yeast using the MS2 system. About a year later, we wrote a response to that article. We argued that, yes, such fragments exist, but 1. most of it stems from over-expression of the labeled mRNA. Parker agreed with that. 2. That these fragments accumulate in P-bodies, and are distinguishable from single mRNAs and we can discard cells which show these structures. 3. We argued that this might not be the case for every mRNA and should be tested on a case by case basis.  4. We and Parker agreed that the best way to determine if such fragments exist is by performing single-molecule FISH (smFISH) with double labeling – a set of probes for the length of the mRNA and a set of probes for the MS2 stem-loops. Now, a new paper from Karsten Weis’ lab shows more evidence, by doing smFISH, for the existence of these fragments.

Continue reading

Does bound MS2 coat protein inhibit mRNA decay?

Roy Parker recently sent a  “Letter to the Editor“, published in RNA journal, in which he suggested that the MS2 system might not be best suited for live imaging of mRNA in budding yeast. According to Parker, the MS2 system inhibits the function of Xrn1, the major cytoplasmic  5′ to 3′ RNA exonuclease in budding yeast, causing us to image mostly the remaining 3’UTR fragments. Thus, he claims, it is possible that interpertation of mRNA localization data using this system in yeast can be faulty. We wrote a response to his letter which just opened the debate even further.

But lets start with his Letter:

Continue reading