{"id":39195,"date":"2013-02-25T18:31:32","date_gmt":"2013-02-25T23:31:32","guid":{"rendered":"http:\/\/planetsave.com\/?p=34806"},"modified":"2013-02-25T18:31:32","modified_gmt":"2013-02-25T23:31:32","slug":"new-imaging-technique-allows-real-time-tracking-of-single-rna-molecules-in-living-cells-videos","status":"publish","type":"post","link":"https:\/\/planetsave.com\/articles\/new-imaging-technique-allows-real-time-tracking-of-single-rna-molecules-in-living-cells-videos\/","title":{"rendered":"New Imaging Technique Allows Real-Time Tracking of Single RNA Molecules in Living Cells [VIDEOS]"},"content":{"rendered":"
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Image Credit: \u03b2-actin, PDB<\/a> rendering based on 1atn – Emw<\/a> – CC -By – SA 3.0<\/figcaption><\/figure>\n

Presenting at the annual Science conference in Boston last week, Dr, Robert H. Singer of Albert Einstein College of New York, captivated the packed room of scientists and science journalists with motion images of real-time messenger RNA<\/em>\u00a0(mRNA) activity in living (mouse brain) cells. The visualization is a first for single molecule imaging.<\/p>\n

Up until this development, it had been extremely challenging to track or monitor this key component of a cell’s transcriptional machinery (messenger RNA is the first molecular step in gene transcription), much less visualize it in real-time.<\/p>\n

<\/p>\n

But thanks to a\u00a0single molecule labeling<\/em> technique perfected by Dr. Singer’s research team, visualizing these critical molecules — and the cell structures that they populate — has now become possible, to spectacular result.<\/p>\n

Single Molecule Labeling & Real-Time Tracking<\/h2>\n

The labelling technique involves short “hairpin” (shaped) RNA structures that bind\u00a0fluorescent protein tags and can be inserted into any RNA\u00a0molecule\u00a0one wishes to follow in vivo<\/em>. The result of this operation is a fluorescent molecule that can be tracked as it moves around the cell.<\/p>\n

Final imaging of the mRNA was accomplished through multi-photon<\/em> microscopy. Combining the two allows biologists to “capture” gene-expression of these RNA transcripts and follow them as they move — ultimately connecting with the ribosomes<\/em> where they will be translated into one of the thousands of proteins that keep our cells growing and functioning properly.<\/p>\n

The Technique in Finer Detail – The ‘Halo’ Effect<\/h2>\n

Dr. Singer’s team developed 24 different “cassettes” of these hairpin molecules which are formed of smaller pieces of RNA (short hairpin RNAs, or shRNAs)<\/span>. These structures “hook” (bind) to special fluorescent proteins which then bind to the mRNA. The particular form of RNA targeted here is called \u03b2-actin\u00a0 which codes for the \u03b2-actin protein.<\/p>\n

Since these smaller RNA molecules naturally bind a dimer (two molecule) form, the team can attach up to 48 (2 x 24) protein cassettes to the targeted mRNA molecule.This is readily accomplished because these mRNAs have what Dr. Singer calls “light bulb sockets” into which the hairpin cassettes fit quite nicely. This all creates a fluorescent “halo” around the mRNA, allowing it to be tracked as it moves from the cell nucleus to the cytoplasm in what’s known as nucleocytoplasmic<\/i> transport.<\/span><\/p>\n

The New Labelling Technique Leads to New Cell Discoveries<\/h2>\n

One of the more startling discoveries brought about by this new labelling\/imaging technique is that expression and diffusion of these mRNAs throughout the cell can occur within a few seconds. The tracking technique also revealed that the “fate” of a given mRNA is determined at “birth”, that is, at the moment it is transcribed from its parent gene.<\/span><\/p>\n

The new imaging technique also allows scientists to develop more accurate “expression profiles” of targeted genes so as to calculate how much RNA is produced over a given time period and how much this expression fluctuates. Earlier quantification techniques for profiling gene\/RNA expression levels have lacked accuracy and are considered flawed now by many.<\/p>\n

In general, the technique allows tracking of molecular activity in real-time with unprecedented accuracy, and thus allows us to understand better the fundamental processes occurring within our cells and tissue. Eventually, this technique could be used on other molecules, such as transcription factors <\/em>that control when a given gene gets “switched on”.<\/p>\n

Quoting from the published abstract:<\/p>\n

\n

‘We simultaneously followed transcription from the \u03b2-actin alleles in real time and observed transcriptional bursting in response to serum stimulation with precise temporal resolution. We tracked single endogenous\u00a0labelled\u00a0mRNA particles being transported in primary hippocampal neurons. The MBS cassette also enabled high-sensitivity fluorescence in situ<\/i> hybridization (FISH), allowing detection and localization of single \u03b2-actin mRNA molecules in various mouse tissues.’<\/p>\n<\/blockquote>\n

In his presentation at the annual Science\/AAAS conference — New Frontiers in Single Molecule Detection and Single Cell Analysis’ — Dr. Singer presented a microscopic “video” showing single mRNAs migrating through the hippocampus<\/em> (in a mouse brain) which is a brain structure responsible for coordinating active memory (see below for video links).<\/p>\n

The technique is detailed in the research paper ‘A transgenic mouse for in vivo detection of endogenous labeled mRNA<\/a>‘, which was previously published in the journal Nature Methods <\/i>in mid-January of this year.<\/p>\n

Dr.Singer works in the Department of Anatomy and Structural Biology at the Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine, New York City.<\/p>\n

Watch these short videos of this important advance in imaging of single molecule migration (links will take you to a quick time movie file page):<\/strong><\/p>\n

Video # 1<\/a><\/strong> – Live-cell imaging of a serum-induced primary fibroblast<\/a>. MCP-GFP-labelled\u00a0mRNA particles can be detected moving around the cell; the two transcription sites of the primary cell appear as two bright spots in the nucleus. Note that the transcription sites intensity is intentionally saturated to allow visualizing the dimmer single particles.<\/p>\n

Video #2\u00a0<\/a> <\/strong>–\u00a0Individual mRNP (messenger ribonucleic protein) moving along a neuronal process.<\/p>\n

Video #3<\/strong>\u00a0<\/a> – Branching mRNP motion along a neuronal process.<\/p>\n

Video Credits: Lionnet et al via Nature Methods<\/a><\/em><\/p>\n","protected":false},"excerpt":{"rendered":"

Presenting at the annual Science conference in Boston last week, Dr, Robert H. Singer of Albert Einstein College of New York, captivated the packed room of scientists and science journalists with motion images of real-time messenger RNA\u00a0(mRNA) activity in living (mouse brain) cells. The visualization is a first for single molecule imaging. 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