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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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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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:

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Imaging with CRISPR/Cas9

The hottest buzz-word in biology today is CRISPR: an adaptive immune system in bacteria and archea. At its basis is a nuclease, named Cas9, which is targeted to DNA by a short single-guide RNA (sgRNA). This turned out to be a very useful system for genome engineering in any organism due to its specificity (provided by the sgRNA) and its simplicity (all you need is to express the Cas9 and sgRNA in the cell). However, this system can also be used for other purposes. One such use is modulation of gene expression, for example by targeting a nuclease dead Cas9 (dCas9) fused to a transcription activator or repressor to promoter regions. Another such use is for imaging.

Here, I’ll described how Cas9 can be used to visualize specific DNA loci or specific RNA transcripts in fixed and live cells.

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My 2nd grade science project

My daughter’s school has a tradition for 2nd grade, to celebrate 100 days of school.

So my daughter had to prepare something relating to the number “100”. At 1st grade they celebrated 50 days of school and my wife made a cake shaped like the number 50. So this year my daughter decided to spare her mother and not bring a cake (as several other kids did).

We pitched several ideas, and she chose my idea of using the microscope to enlarge 100x certain objects (hence, the relevance to this blog 🙂 ).

A few years ago I bought a microscope at AmScope to use at home with the kids. It is a 40x-400x compound microscope, and it has a digital camera that you can connect with a USB to your laptop. This allowed us to take really nice images of the objects we examined.

I brought E. coli and yeast samples from the lab and we imaged them 100x and 400x. We then imaged a single hair from her head. She got really excited about that and also took a hair from her dog, to compare.

She then brought me salt which gave a pretty picture of the cube-shaped crystal. Last, we imaged a leaf of strawberry and mold that grew on a cucumber.

We made a nice poster:

final poster

 

This was a great experience for both of us. She was really excited and we imaged for over an hour, late in the evening. She’s interested in science, particularly medicine. She’s rational, she’s smart and clever. She will do great things when she grows up.

 

ASCB15 – part 3

(part 1, part 2)

I ended part 2 Monday night. It was an exciting day with many excellent talks, but the best talk (mine, of course!) was due the next day.

Tuesday started with the seminar on engineering cells and tissues. There was the mandatory CRISPR talk as the great new thing in bio-engineering these days. Jennifer Doudna talked about the discovery, then went on to discuss new experiments (using Halo-tag to track Cas9 in live cell nuclei to study movement & binding kinetics) and improved technologies (transfect cells with pre-assembled Cas9-gRNA for quick editing & less off-target cleavage).

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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