Category Archives: Journal club

Quantum dots for Immunofluorescence — Rapha-z-lab

An important post on quantum dots:

Guest post by Dave Mason In modern cell biology and light microscopy, immunofluorescence is a workhorse experiment. The same way antibodies can recognise foreign pathogens in an animal, so the specificity of antibodies can be used to label specific targets within the cell. When antibodies are bound to a fluorophore of your choice, and in […]

via Quantum dots for Immunofluorescence — Rapha-z-lab

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

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.

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Roger Tsien – the scientist that colored our research

Roger Tsien died a few days ago, at the relatively young age of 64. He was a UCSD scientist, a Nobel laureate and he was one of the first to see the significance and usefulness of GFP.

I’ve never met him. But, I guess, this blogs owes him its existence.

I don’t want to discuss his body of work, his achievements, or awards he won (e.g. the Nobel award). Many wrote nice things about him, such as here, here or here and all over the internet, with nice pictures of fluorescent proteins used in research.

I thought it will be nice to look back at his first GFP paper.

title1

His goal in this paper was to investigate the formation of the fluorophore of GFP. Specifically, he asked:

“What is the mechanism of fluorophore formation? How does fluorescence relate to protein structure? Can its fluorescence properties be tailored and improved-in particular, to provide a second distinguishable color for comparison of independent proteins and gene expression events?”

Already here he looked to utilize GFP – to improve it, to change it, so it can be useful for fluorescent studies in biology.

He used random mutagenesis of the GFP cDNA to screen for mutants with altered brightness and emission. A simple yet powerful method, still used today, to find new FPs with exciting and useful properties.

Here is an ex/em spectral analysis of some of the mutants:

spectra

One mutant, I167T, proved to be almost twice as bright as the WT GFP protein.

But the most exciting was the finding of a blue FP (Y66H):

blue mutant

To sum up in his words:

“The availability of several forms of GFP with such different excitation and emission maxima [the most distinguishable pair being mutant P4 (Y66H) vs. mutant Pll (I167T)] should facilitate two-color assessment of differential gene expression, developmental fate, or protein trafficking. It may also be possible to use these GFP variants analogously to fluorescein and rhodamine to tag interacting proteins or subunits whose association could then be monitored dynamically in intact cells by fluorescence resonance energy transfer (19, 20). Such fluorescence labeling via gene fusion would be site-specific and would eliminate the present need to purify and label proteins in vitro and microinject them into cells.”

He saw the future, and it was bright green.

 
ResearchBlogging.orgHeim, R., Prasher, D., & Tsien, R. (1994). Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proceedings of the National Academy of Sciences, 91 (26), 12501-12504 DOI: 10.1073/pnas.91.26.12501

Design guidlines for tandem fluorescent timers

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.

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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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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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