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
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
Transcription factors (TFs) have a fundamental role is regulating gene expression. The basic model, based on numerous biochemical analyses, has determined where TFs bind (usually at specific sites at or near promoters), when they bind the DNA (at a resolution of minutes/hours) and what do they do there (induce/repress transcription. Duh!). However, much is yet unknown. One aspect that is fairly unknown is the dynamics of how TFs search for their binding sites, bind them and later dissociate, particularly at the single molecule level. To explore this, the Transcription Imaging Consortium (TIC) at Janelia Research Center (JRC) (it used to be Janelia Farm, but the “farm” part was removed from the name. oh well) applied sophisticated imaging techniques to measure the dynamics of two TFs, SOX2 and OCT4 in the nuclei of live embryonic stem (ES)cells. Their results were published in Cell almost a year ago.
Posted in epi, FCS, Gene expression, HaloTag, Journal club, multi-focus microscopy, TIRF
Tagged HaloTag, HHMI Janelia, Mammalian cell, quantitative microscopy, single molecule, transcription