Single molecule FISH is currently the best method to get accurate measurements of mRNA levels at single molecule, single cell level in cell culture or tissue slices with a spatial resolution of ~200 nanometer (or less). One of the drawbacks of this method is the deterioration of the fluorescent signal (bleaching) of the organic dyes that are used to label the probes. Andrew Smith’s lab from University of Illinois now show how FISH can work with quantum dots instead of organic dyes. This provides better fluorophore stability and also the possibility to have more colors with less overlap of the emission spectra.
One of the greatest breakthroughs of the past decade was the development of the next generation sequencing. Sequencing of DNA of course. It is relatively easy to sequence DNA – the polymerase is doing it for you – simply add fluorescently labeled nucleotides. For RNA sequencing, we simply convert it into DNA. We now even have a method for in situ sequencing of RNA. But proteins pose a challenge. Now, maybe, this challenge can be overcome with a new-old method to sequence peptides.
Previously, on the story of MS2 labeling of mRNA in yeast: Roy Parker published a short letter to the editor, indicating that the MS2 system might cause accumulation of 3′ fragments. We wrote a response, showing that it is not always the case for endogenously expressed mRNAs, but it is exaggerated when over-expressed (Part 1)*. Later, Karsten Weis’s group confirmed Parker’s initial observation but their report still had some questions unanswered, and no solution to the problem; I was unhappy (Part 2). Now, Evelina Tutucci and Maria Vera together with Jeet Biswas (all from Rob Singer’s lab) seem to have resolved the issue and solved the problem, with the development of the MBS version 6. Continue reading
Posted in FISH, Gene expression, Journal club, MS2-like systems, stress response
Tagged mRNA decay, mRNA localization, MS2, quantitative microscopy, Singer lab, single molecule, yeast
About a year and a half ago I wrote here about new uses of CRISPR/Cas9 as an imaging tool. In particular, I was excited about the possibility to use enzyme-dead Cas9 (dCas9) as an RNA binding protein for live imaging of mRNA. Unfortunately, in my hands this did not work (the dCas9 has exited the nucleus with non-targeting guide RNA at the same rate as with the specific guide RNA).
Last week, a new CRISPR tool was published in Nature, from Feng Zhang’s lab.
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.
Almost 4 years ago, I wrote a post on tandem fluorescent timers (tFTs). The idea is to have two different fluorescent proteins fused together to the protein of interest. In the paper from 4 years ago, it was superfolder GFP (sfGFP) and mCherry. sfGFP matures very fast (within minutes) and mCherry matures more slowly (t1/2 ~40min). The ratio beween green to red fluorescent signal indicates the percentage of new vs old proteins, thus acts as a “timer”. This latests paper on tFTs from the same group of Michael Knop’s lab, found that analyzing tFTs might be more complicated due to some possible problems of this system.
Posted in Green protein, Journal club, protein degradation, timers
Tagged clover, GFP, GFPm, GFPmut3, maturation, mCherry, mNeonGreen, mutations, quantitative microscopy, superfolder, tFT, yeast
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.