Category Archives: cytoskeleton

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

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In the right place at the right time: visualizing and understanding mRNA localization

The title of this post is also the title of a review paper that I co-authored  with Adina Buxbaum, a recently graduated PhD student from Rob Singer’s lab. The review was published last week in Nature Reviews Molecular Cell biology.

In this paper we review some of the old and new methods to visualize mRNA. These include mostly FISH and MS2-like systems, which I’ve discussed extensively in this blog. There is also a short section (“box”) on quantitative analysis tools for mRNA localization imaging.

We then discuss the current knowledge on the mechanisms of mRNA localization and how it relates to the biology in two very distinct model systems – unicellular organisms (budding yeast) and the extremely polarized neuronal cell.  We also discuss examples in other organisms from bacteria through fly to frog and mammals.

I’m biased, of course, but I think this turned out to be a balanced, comprehensive, yet not too detailed review paper that will benefit both beginners which are unfamiliar with the RNA localization field, as well as experts which are used to a single method or a single model organism.
ResearchBlogging.orgBuxbaum, A., Haimovich, G., & Singer, R. (2014). In the right place at the right time: visualizing and understanding mRNA localization Nature Reviews Molecular Cell Biology DOI: 10.1038/nrm3918

Don’t eat this FISH-STIC

single molecule FISH (smFISH) is a great way to detect single RNA molecules in fixed cells. The “traditional” FISH uses fluorescently labeled oligonucleotides which directly hybridize with the target RNA sequence. The two most common approaches are the use of 1-5 50-mer oligos, that are labeled with 5 fluorophores, or the use of more than 20 20-mer oligos labeled at one or both ends.
A more recent approach is nicknamed “Christmas tree”, in which 2-3 rounds of hybridizations are done, with only the last round uses fluorescently labeled probed. I’ve expanded on that when I discussed RNAScope.  Other people have independently developed similar protocols (e.g. branched DNA FISH (bDNA), which was recently used to get smFISH transcriptomics in human cells).

Now, a new paper came out with a very similar method whish they call FISH-STIC (Fluorescence In Situ Hybridization with Sequential Tethered and Intertwined ODN Complexes).

However, their images do not compare to the quality shown by the RNAScope or bDNA papers. Maybe its just bad imaging and not the FISH itself, but their images look blurry and doesn’t seem to be up to modern FISH standards. I used 20-mer FISH and can get nicer images.

They also get a few high-intensity spots which the call “mRNA-independent” (since they can also see it outside the cell). To me it means that their protocol was just not optimized – either the washes, or the oligo sequences which should not make complexes like that.

They also seem to have higher background fluorescence compared to RNAScope.

Simultaneous detection of Actb and Actg mRNAs with FISH STIC probes. Source: Sinnamon & Czaplinski (2014) RNA 20:260-266.

Simultaneous detection of Actb and Actg mRNAs with FISH STIC probes. Source: Sinnamon & Czaplinski (2014) RNA 20:260-266.

On the other hand, their beta-actin FISH-STIC looks better than the RNAScope-FISH. beta-actin mRNA is found in 500-2000 copies per cell, so it is not easy to get smFISH. Still, 20-mer FISH that I do looks better than both.

20-mer FISH for beta-actin mRNA in immortalized MEFs. Source: myself.

20-mer FISH for beta-actin mRNA in immortalized MEFs. Source: myself.

Notice also in my image the transcription sites (TS) are clrearly visible, whereas I did not see any TS in their images, which is weird (unless they chose cells without TS on purpose. Don’t know why, since smFISH is a powerful tool to quantify transcription 1, 2).

They did not test FISH-STIC on a less prevalent mRNA, so I cannot comment on how it would look like compared to RNAScope or 20-mer FISH.

Anyway, the methods seem similar, but I think that the RNAScope demonstrates better results than the FISH-STIC. However, I haven’t tried this approach myself. I continue to do FISH with 20-mer probes and so far pleased with my results.

ResearchBlogging.orgSinnamon JR, & Czaplinski K (2014). RNA detection in situ with FISH-STICs. RNA (New York, N.Y.), 20, 260-266 PMID: 24345395
Battich N, Stoeger T, & Pelkmans L (2013). Image-based transcriptomics in thousands of single human cells at single-molecule resolution. Nature methods, 10 (11), 1127-33 PMID: 24097269

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