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.
(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).
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
Posted in conferences & courses, epi, FISH, Gene expression, MS2-like systems, Optogenetics, Organelles, stress response, Transport & Trafficking, virology
Tagged ASAPbio, ascb15, bioRxiv, Mammalian cell, mRNA export, mRNA localization, PP7, QCBNet, quantitative microscopy, single molecule, yeast
The ASCB meeting brings scientists from all levels to talk about cell biology, which is actually almost anything “biology”. But there’s also a full program dedicated to other matters, like science careers, science publishing, science communications and science policy. This is also a great venue for companies to show their products, and for organizations/institutions to recruit new members. If I remember the numbers correctly, there were over 550 oral presentations and over 2,700 posters. I overheard someone saying there were ~6000 people attending the meeting. I typically go to RNA meetings that are mostly in the lower 100’s of participants. So, to me, that’s a large meeting.