Category Archives: Transport & Trafficking

Separating cells is hard

I write this entry to accompany my short talk at Woodstock.bio 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 Woodstock.bio slide:

Gal_Haimovich

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.

 

 

Sugar-coated RNA

A new pre-print from Caroline Bertozzi’s lab shows that some RNA molecules are glycosylated. At least some of these glyco-RNA molecules might reside inside the ER lumen.

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RNA meeting 2019 – Part 1

The RNA society meeting is big. With over 150 talks and almost 700 posters, there’s a lot of new stuff to learn. This was my first RNA society meeting and I decided from the beginning to take it easy. That is why I did not live tweet like I usually do.

Instead, I thought of writing a summery blog post.

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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.

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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. Continue reading

Imaging translation of single mRNAs in live cells

Translating the information encoded in mRNAs into proteins is one of the most basic processes in biology. The mechanism requires a machinery (i.e. ribosomes) and components (mRNA template, charged tRNAs, regulatory factors, energy) that are shared by all organisms on Earth. We’ve learned a great deal about translation over the last century. We know how it works, how it is being regulated at many levels and under varuious conditions. We know the structures of the components. We have drugs that can inhibit translation. With the emergance of next-gen sequencing, we can now perform ribosome profiling and determine exatly which mRNAs are being translated, how many ribosomes occupay each mRNA species and where these ribosomes “sit” on the mRNA, on average. New biochemical approaches like SILAC and PUNCH-P can quantifiy newly synthesized proteins & peptides. Yet, all of that information comes from population studies, typically whole cell populations. Rarely, whole transcriptome/ribosome analysis of a single cell is performed. Still, there is no dynamic information of translation, since cells are fixed and/or lysed. And there is no spatial information regarding where in the cell translation occurs (poor spatial information can be determined if cell fractionation is performed, which is never a perfect separation of organelles/regions and we are still not at the stage of single organelle sequencing).

Imaging translation in single cells is intended to provide both spatial and dynamic information on translation at the single cell and, hopefully, single mRNA molecule resolution. Recently, four papers were published (on the same day!) providing information on translation of single mRNAs. Here is a summary of these papers.

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ASCB15 – part 2

I ended Part 1 after the morning session on pushing the boundaries of imaging.

After the amazing talks on imaging, I browsed the halls, visited some exhibitors, sampled a couple of exhibitor tech-talks. I later went to a mycrosymposium (#2: signaling in health & disease). This was mainly to see how this ePoster thing works, but also I promised Qunxiang Ong – with whom I discussed optogenetics the day before – to be at his presentation. He used a light-induced dimerization of signaling proteins to study the effect on neurite growth. The nice thing in his system was that the cells were plated in wells which were partly dark – so light-induction cannot take place in these regions. This allowed for analysis of neurite growth in lit vs “light-protected” regions of the same cell.

After this session, I attended my first “discussion table”. Continue reading