Category Archives: Cell-Cell communication

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

Malaria parasites send each other genes

Communication between cells takes many forms. There could be communication by direct contact, by sending out free molecules (like hormones) or by special structures (e.g. synapses).

But how can parasites, that dwell inside their host cell, communicate with one another?

A very elegant mechanism used by Malaria parasites was found, and is described in a recent Cell paper (actually, it was published in the same issue as my paper).

Malaria parasites (Plasmodium falciparum) are transferred from mosquitoes to humans, where they infect red blood cells (RBCs). Once inside the RBCs, the parasite need to sexually differentiate into sexual forms that are competent for transmission by the next mosquito.

When trying to understand which signals are transferred between the parasites that dwell in different RBCs, they mixed two cultures, each expresses a different drug resistant gene and a different fluorescent protein. Surprisingly, the mixed cultures survived when both drugs were added, and parasite cells exhibited both colors.

Further analysis showed that the parasites send out tiny vesicles (their size is ~70 nm). These vesicles are similar to endosome-derived “exosomes” ,and therefor are referred in the paper as “exosome-like” vesicles. In most papers that study exosomes, they are visualized by electron microscopy. However, in this paper, Atomic Force Microscopy (AFM) was employed.  AFM resolution is ~1000-fold better than light microscopy (yet lower than electron microscopy).  In essence, AFM uses a tiny cantilever with a very sharp tip that travels over the sample. The tip is deflected from the sample based on forces exerted from the surface (e.g. mechanical contact force, electrostatic forces, magnetic forces, Van der Waals force).  The deflection is registered by a laser light.

Principle of AFM. Source: Wikipedia

These vesicles supposedly contain the plasmid DNA that enables the lateral inheritance of the new characteristic (drug resistance & fluorescent protein).

Malaria parasites-derived

Malaria parasites-derived “exosome-like” vesicles as seen by Atomic Force Microscopy. Upper row: fraction that does not contain the vesicles. Lower row: fraction that does contain vesicles. Source: Regev-Rudzki et al. Cell 153(5): 1120-1133

The authors put a lot of effort in proving that the information – DNA plasmid – is transferred via these vesicles.  They perform DNA FISH and PCR to show that the “acceptor” parasites contain these genes. Alas, they never show that the vesicles that they isolated also contain this DNA. This should have been simple to do: they already have the isolated exosomes and just need to do PCR on them. I do not know why this was not requested by the reviewers.

Their last figure, which is intended to give a broader biological meaning to their findings, suggests that this form of communication is required for production of gametocytes (a sexual differentiation step required for intake by the mosquito). They show that the gametocytes contain both fluorescent markers, and are produced at greater numbers when parasites are co-cultured.

Malaria gametocytes from co-cultures express both fluorescent proteins. Source: Regev-Rudzki et al. Cell 153(5): 1120-1133.

Malaria gametocytes from co-cultures express both fluorescent proteins. Source: Regev-Rudzki et al. Cell 153(5): 1120-1133.

However, they do not show that application of the isolated vesicles can induce this sexual differentiation. More so, the exosomes may contain other factors (proteins, RNAs) that can induce sexual differentiation, and be unrelated to the DNA transfer observed.

In any case, an interesting paper, that may have major clinical applications in the future.

ResearchBlogging.orgRegev-Rudzki N, Wilson DW, Carvalho TG, Sisquella X, Coleman BM, Rug M, Bursac D, Angrisano F, Gee M, Hill AF, Baum J, & Cowman AF (2013). Cell-Cell Communication between Malaria-Infected Red Blood Cells via Exosome-like Vesicles. Cell, 153 (5), 1120-33 PMID: 23683579