Category Archives: Cell-Cell communication

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.

 

 

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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Counting exosome secretion

Last month I wrote a post about exosome internalization by recipient cells.  One of the topics I discussed was the lack of good quantitative data in the exosomal field, and what the current data tells us about the efficiency and capacity of exosome-mediate cell-to-cell communiation.

Today I came across an interesting paper in which the researchers try to get quantitative data of exosome secretion by the donor cells.

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The wild ride of the exosomes

Exosomes are extracellular vesicles that are thought to mediate cell-to-cell communication in eukaryotes. Briefly, exosomes are 50-100 nanometer (nm) sized vesicles produced by the endosomal system. They are exported out of the cell and can be found in every bodily fluid: plasma, saliva, milk, urine and more. These vesicles then enter recipient cells, and the cargo they carry (proteins, RNA molecules and lipids) modulate the physiology and/or gene expression of the recipient cell. Exosomes catch a lot of attention lately because of their clinical significance. First, exosomes might be used as biomarkers for some diseases (most importantly tumors). Second, they are being considered for therapeutics as a delivery system.

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Tracking membranes by imaging – mCLING and surface glycans

Living cells exhibit many types of membranes which participate in most biological precesses, one way or another. Imaging membranes is usually acheived by two types of reagents: chemical dyes or fluorescent proteins that are targeted to the membrane itself or inside an organelle.

The chemical dyes are usually targeted to an organelle based on a specific chemical property of that organelle.

For example:

Rhodamine 123, tetramethylrosamine, and Mitotracker  are dyes that preferentially target mitochondria, due to its membrane potential. Mitotracker has thiol groups that allow it to bind to matrix proteins, thus making it more resistant to disruption of the membrane potential (e.g. by fixation).

Lysotracker are lypophilic, mildly basic dyes, which accumulate in the acidic lysosomes.

ER-tracker is a BODIPY (boron-dipyrromethene; a group of relatively pH insensitive dyes that are almost all water insoluble) based dyes which are linked to glibenclamide – a sulfonylurease – which binds to ATP sensitive Potassium channels exclusively resident in the ER membrane.

Long chain carbocyanines like DiL, DiO and DiD are lipophylic fluorescent molecules, which are weakly fluorescent in water, but highly fluorescent when incorporetaed into membranes, particularly the plasma membrane.

FM lipophylic styryl dyes bind the plasma membranes in a reversible manner and are also incorporated into internal vesicles.

On the other hand, fluorescent proteins (FP) are targeted to membranes or organelles by fusing them to either whole proteins that localize to a specific organelle, or to short peptides that carry a localization signal. Thus, a nuclear localization signal (NLS) targets the to the nucleus, mitochondrial targeting signal (MTS) to the mitochondria and a palmitoylation signal to the plasma membrane and endocytic vesicle.

There are advantages and disadvantages to each system, relating to ease of use, specificity, photostability etc… I do not want to go into that.

Here, I would like to mention two new methods to image the plasma membrane.

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Update

Hello dear readers,

As you’ve probably noticed, I’ve been away for a couple of months. Just overload of work and other stuff that came in the way of updating. I hope to return to my regular reports on interesting papers (which have accumulated on my desk).

The meeting last month on trafficking was very interesting, though not as much microscopy talks as I thought. Nothing really ground breaking in terms of imaging methods.

Orna Amster Choder showed beautiful images of mRNA localization in bacteria, and Eitan Bibi talked about the targeting of mRNA and ribosomes to the E. coli plasma membrane.

Yaron Shav-Tal used imaging to look at transcription dynamics of Cyclin D1, as well as upstream events (i.e. the signal transduction process leading to transcription activation).

Jeff Gerst & Yoav Arava talked about localization of mRNAs and local translation of mRNAs to mitochondria* in yeast.

* Actually, the mito people at the meeting said that nowadays it should be called “mitochondrion” (a single mitochondria) since this is just one organelle spread throughout the cell.

Hagai Abeliovich talked about mitophagy, which is apparently dependent on mitochondrial dynamics and relates to hypo and hyper-polarization of mitochondria “daughters” during fission events.

Importantly, I learnt from him that GFP is stable in the yeast vacuole (the equivalent of lysosome) which is very interesting and maybe useful info in yeast imaging.

Felippe court and Jose Sotelo talked about movement of RNA from Glia cells to Axons. Very exciting stuff, but still missing a stronger evidence for the movement, and particularly biological effect (translation…).

I have learnt that the mito makes contacts with the vacuole and the ER membranes, where they may exchange lipids and proteins.

And there were many more interesting talks. It was a good meeting.

The next meeting I’m going to attend is FISEB 2014 where I will also present my work in an oral presentation (its called “oral poster”).

Cells reach out their “hands” to create new limbs

Communication between cells takes many forms. There could be communication by sending out microvesicles with important messages inside, by sending out free molecules (like hormones) or by special structures (e.g. synapses).

Sonic hedgehog (SHH) is a signaling protein that is important for the development of vertebrate limbs. It was thought to be release from a small group of cells at the posterior end of the limb bud, and is recognized by receptors on cells a long distance away.

Not this Sonic Hedgehog… (image taken from http://sonic.wikia.com)

A new paper publish in Nature from Maria Barna’s lab shows that SHH actually remains bound on the external side of the cells that produce it. The cells simply send very long thin protrusions (here named filopodia) that reach all the way to similar filopodia of the receiving cells.

I think that not only the story is very novel and interesting, but the images are very pretty.

Several “technical” issues:

In order to study the SHH signaling in live chick embryos, they designed a custom made live in ovo microscopy system: a temperature controlled plate; on it an egg container chamber, and an objective that is dipping into the yoke.

They show that standard fixation methods (e.g formaldehyde) destroy these filopodia. Also, a volume marker (in this case sfGFP) that just fills the cytoplasm does not give a strong enough signal to detect these filopodia (possibly since they are very thin, and packed with actin filaments  and other proteins, so there’s very little free volume left).

So, they used palmitoylated fluorescent proteins, pmeGFP (green) and pmKate2 (red) that target them to the plasma membrane. This enabled them to visualize these very thin and long filopodia. Here’s a video movie from their paper.

They use a variety of cytoskeletal proteins fused to EGFP or to mKate2 to learn about the structure of these filopodia. Their conclusion is that these structures contain only a specialized form of actin filaments.

They show beautifully that SHH (fused to EGFP) travels to the tip of these filopodia:

They used a split GFP technology to show that SHH is actually found on the outside of the cell membrane.  In split GFP, two fusion proteins are produced, each one is fused to “half” of a GFP protein (its not exactly half but let not go into that now). If the two fused proteins are in close proximity, the two halves associate to produce an GFP that fluoresce irreversibly. The two separate halves do no fluoresce. So one half was fused to SHH and the other was anchored to the extracellular leaflet of the plasma membrane, and when both were expressed, they got green fluorescence.

In total – a very nice and pretty paper.
ResearchBlogging.orgSanders TA, Llagostera E, & Barna M (2013). Specialized filopodia direct long-range transport of SHH during vertebrate tissue patterning. Nature, 497 (7451), 628-32 PMID: 23624372