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.
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.
Posted in Fluorescent microscopy, Gene expression, Journal club, MS2-like systems, Organelles, signaling, stress response, Transport & Trafficking
Tagged ER, GFP, HaloTag, JFRC, Mammalian cell, MS2, neurons, PP7, quantitative microscopy, Singer lab, single molecule, spaghetti monster, Suntag, translation
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.
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:
Posted in FISH, Gene expression, Journal club, MS2-like systems
Tagged FISH-Quant, mRNA decay, MS2, my pics, personal experience, quantitative microscopy, Singer lab, yeast
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.
Posted in CRISPR/Cas9, FISH, Gene expression, Genetics, Journal club, Whole tissue imaging
Tagged CASFISH, CRISPR, DNA FISH, HaloTag, Mammalian cell, quantitative microscopy, RCas9, Singer lab, stress granules
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:
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.