Tag Archives: Mammalian cell

Intercellular mRNA transfer through membrane nanotubes – behind the scenes.

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.

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New CRIPSR-based RNA imaging tool

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.

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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

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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The wild ride of the exosomes

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.

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