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
In eukaryotes, the DNA is packages tightly in nucleosomes, which are composed primarily out of histone proteins. There are four major types of histones (1,2,3 & 4). Extensive work has been done on how histones facilitate and regulate transcription. It turns out that there are multiple post-translational modifications on histones, such as methylation and acetylation that are linked to transcription regulation. The majority of the studies use a method called chromatin immunoprecipitation (ChIP) to study these modifications. In essence, an antibody specific for a certain modification is used to affinity-purify only modified histones, along with any DNA region they are associated with. Thus, one could get a map of the specific modified histone along the chromosomes and correlate this locations with transcription activity, ChIP maps of other transcription related proteins etc…
There are two problems with this approach. The first, since the cells are fixed, the time resolution is limited to several minutes, at best. Second, the results are an average of the entire cell population, and therefore factors considered linked may not actually be present in the same cell, same genomic location at the same time.
So, Timothy Stasevich et al. tried a different approach by using a novel method to image histone modifications in live cells.
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
The Nobel prize in chemistry 2014 was awarded to Eric Betzig, Stefan W. Hell & William E. Moerner for the development of Super resolution fluorescent microscopy!
See here for details on the history of this discovery.
As readers of this blog know, Super-resolution microscopy has made a revolution in the field of fluorescent microscopy in a very short time.
This is a justified award.
Fluorescent sensors are important tools that can allow real-time, live, single molecule imaging of microscopic millisecond scale events. It is even better if these sensors are genetically encoded sensors (i.e. fluorescent proteins). We have already encountered the pH sensors pHluorin and pHTomato and the Ca2+ sensor GCaMP. There have been a few others, such as HyPer that detects H2O2 or ArcLight and ElectrikPk which are voltage sensors.
Now, the group of Loren Looger from HHMI Janelia developed a sensor for a very important molecule: L-glutamate. Continue reading