Tag Archives: personal experience

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

The Bio-protocol experience

A few months ago, I joined Bio-protocol as an associate editor. The first  protocol I edited is now published, so I thought I’ll write about this experience.

logo2

Bio-protocol is an open access, peer-reviewed e-journal which specializes, you guessed it, in publishing life-science protocols. Submission is almost exclusively by formal invitation and it is free of charge (i.e. no submission or publishing fees).

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

Corrigendum

It takes a lot of work to publish a paper. There’s the research itself, of course. Then there’s the writing and preparation of the figures for the paper, that are submitted to the journal for publication. Typically, most of the people who contributed to the research are involved in that, so the draft is circulated between at least 2 people, usually many more. There is one author (typically the 1st author, or the PI who is leading the research) who also needs to combine all the comments & changes into one coherent text.

The paper then goes through several rounds of  changes, following reviewers’ comments, editor’s comments, final proof-reading etc…

The accepted manuscript (MS) should be “perfect”. But mistakes can happen. Mistakes did happen for three of my recent papers.

For my Cell paper, I have prepared a nice schematic model to summarize the main highlights. It was nice, but we decided it is worth spending money on a professional artist to make a nicer one. Somehow, my scheme was uploaded for the accepted MS, instead of the more artistic one, and this is the one that was eventually  published  (you can see the artistic version in the blog post about the paper). I found that out after the fact, but since it was just a model, and the only difference was the art, we decided that publishing a correction just wasn’t worth the trouble.

Then, we published a paper in PLOS One. Only after it was published, I noticed that something happened to figure 4. Here it is:

Can you find the error?

Can you find the error?

Here, we had no choice but to publish a correction.

Recently we published a review paper, in Nature Reviews Molecular Cell biology. We worked very hard with lots of proof-reading and integrating each other’s changes to make sure it is perfect. Then, a few weeks ago, I was looking for some papers which I knew we referenced in that review. Strangely enough, those papers were mis-quoted in our review. Maybe its silly to publish a corrigendum just for a tiny mistake in references. But I think that it is important to keep science as mistake-free as possible, even with those tiny seemingly unimportant mistakes. So we published a corrigendum.

Here is also the place to thank the editors for handling these corrections pleasantly and efficiently.

I’m looking forward for my next corrigendum. What will that be?

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.

Continue reading

Bad imaging day

This week I finally went live, i.e. started live imaging.

The first session, on Tuesday, was ok.

Then today…

I came early to start up the systems. The most important thing is the heating chamber. Heating is important since the temperature can affect the quality of the imaging. How? The metal tray (where you place your sample) expands when heated. The lenses in the objective also. The immersion oil changes viscosity. So does the cell media. So when imaging mammalian cells at 37C, it takes time for all components to equalize their temp on 37C.

3 hours later, I was ready to image. I started the camera, the computer, the lasers. The only thing left to do before starting is align the laser so it will go directly up through the objective (at 90 degree to the sample). The laser sometimes drifts, or users change it and so one has to check before starting.

The laser was waaayyy off the target. I tried my best. Then, over the next  4-5 hours(!), three other people tried until our physicists succeeded, in a way (it’s centered but the laser looked dispersed, not sharp).

Problem is, the temp in the chamber isn’t equalized now (since we kept opening the chamber, switching objectives, removed the metal plate holder etc…). I tried imaging without waiting, but the z-axis kept drifting. So now I have to wait another half-hour or hour to let everything heat up properly and it’s already 6pm.

Guess I won’t be doing lots of imaging today…

If you chew that mRNA, you must make a new one!

Gene expression is very complex.  My paper, which was published in Cell today, just shows that it is more complicated than previously realized.

Traditionally, eukaryotic gene expression is divided into five steps:

Continue reading