My paper was recently published. I suggest that you read it before reading this post (it is an open access paper). In this paper we show that full-length mRNA molecules can be transferred between mammalian cells through membrane nanotube-like extensions that connect the cells.
Posted in Cell-Cell communication, epi, FISH, Gene expression, membranes, MS2-like systems, Transport & Trafficking
Tagged HHMI Janelia, Mammalian cell, membrane nanotubes, mRNA localization, MS2, my pics, personal experience, Singer lab, single molecule
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
Posted in cytoskeleton, epi, HaloTag, Journal club, membranes, Organelles, Super resolution, Transport & Trafficking
Tagged GFP, HaloTag, Kinesin, Mammalian cell, mitochondria, peroxisomes, STORM, super-resolution
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, HHMI Janelia, 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.
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.
Almost exactly a year ago, I wrote a post regarding my concerns with SmartFlare, supposedly a novel method for live imaging of RNA in cells.
In a nutshell, SmartFlare are gold nanoparticles covered in oligos specific to a certain mRNA of interest. Supposedly, cells internalize these particles and, once the mRNA hybridize to the oligo, a complementary fluorecently labeled oligo is being unquenchhed and “flares”, indicating the present of said mRNA.
You can read about my concerns in that older post, but apparently I wasn’t the only one concerned about their validity.
Raphaël Lévy from U. of Liverpool (UK) was concerned as well. He endeavored into an open science project to try and answer his concerns (which is why I allow myself to openly review his paper).