Genetic Engineering & Biotechnology News

NOV1 2018

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12 | NOVEMBER 1, 2018 | Genetic Engineering & Biotechnology News | GENengnews.com believe they're the first to use fluorescence lifetime imaging microscopy (FLIM) with fluorescence reso- nance energy transfer (FRET) microscopy to track mRNA as it travels through the pores. FLIM-FRET microscopy allows scientists to look at dynamic pro- cesses within cells at high spatial (nanometer) and temporal (nanosecond) resolution. The team used FLIM-FRET and live-cell imaging to follow single mRNAs through the pores, and it focused on the interactions of the pore with TAP, an export receptor complex. Using FLIM-FRET, they discovered that TAP plays an important role in the fi- nal stages of mRNA's passage toward the cytoplasm, at which point a helicase motor protein assists in pulling the mRNA out of the pore and separating it from the TAP protein. The TAP protein is then re- cycled so that it can look for new mRNA. "FLIM-FRET isn't a new technique, but it hasn't been used too much in studies of the nucleus, and specifically not in the nuclear pore," says Dr. Shav- Tal. "But because the nuclear pore complex is a big structure, you get lots of high-resolution information about every pore." The team used FLIM-FRET to observe hun- dreds of nuclear pores simultaneously and reveal how mRNA enters and leaves the nucleus. Nuclear pores were known to be bidirectional gates with lots of cargo travelling through the pores in any given timeframe. "People had this idea that nuclear pores either specialized in import or export," notes Dr. Shav-Tal. "Our study showed that every pore is capable of mRNA export." The Frontier of Single-Cell Imaging "Metabolomics and proteomics at the single-cell level are still at the beginning of their emergence," says Dr. Vertes. Technologies such as PCR have helped researchers to amplify tiny amounts of RNA extracted from a single cell. However, no similar technologies ex- ist for amplifying cell proteins and metabo- lites—his primary area of study. "We are still working on instrumental solutions to deal with tiny amounts of me- tabolites," he says. As well as improving sen- sitivity, researchers working with metabolites must achieve high throughputs and detect rare cells, such as cancer stem cells, which are present in a tumor at levels of one in a million. He adds, "So we're not only trying to analyze something small, we're also trying to find a needle in a haystack." Dr. Vertes turned to fluorescence micros- copy to help him analyze rare cells. "We built a dual-channel microscope that images cells in brightfield and fluorescence mode simultane- ously, and combined this with a laser-ablation technique developed in our lab," he says. This technique had been applied to multiple cells, but he tweaked it for use with single cells. The laser-ablation electrospray ionization (LAESI) system uses a sharpened optical fiber to deliver mid-infrared laser light directly to individual cells. The laser light ablates the cells and ionizes the ablation plume, which can be analyzed by mass spectrometry. At the same time, a dual-channel microscope visualizes both the cell morphology and fluorescent labels. Dr. Vertes has used the system to distin- guish infected and uninfected cells. "There are efforts in other places to combine meth- ods for single-cell analysis with fluorescence microscopy, but I think we have an edge combining it with the laser-based system," he asserts. Inside Single Cells Continued from page 11 Drug Discovery Using Peptones to Achieve Diverse and Demanding Bioproduction Goals As bioproduction requirements advance, it is critical to have consistent, high-quality media and supplements that continue to meet evolving industry needs. Peptones have been successfully used in bioproduction applications for more than 30 years to meet diverse and demanding production requirements. Their unique nutritional profiles and usage flexibility make peptones ideal components for creating a robust bioprocess. Join us for this exciting new GEN webinar that will demonstrate the benefits of peptones and how they can be used to enhance process performance and consistently yield a high-quality product. A live Q&A session will follow the presentation, offering you a chance to pose questions to our expert panelist. Free Registration! www.genengnews.com/Peptone View It Now On Demand DURATION: 60 minutes COST: Complimentary Speaker Stacy Holdread Staff Scientist BD Life Sciences Produced with support from Webinars At George Washington University, scientists led by Akos Vertes, Ph.D., have used laser-ablation electrospray ionization mass spectrometry to conduct in situ metabolic profiling of living plant systems. In these brightfield and brightfield/ fluorescence microscopy images, from a poster co-authored by Dr. Vertes, soybean cells of different types (epidermal, infec ted, and uninfected) are shown. Both the epidermal and infected cell types were sampled as single cells while, due to the small size of the uninfected c e l l s , s m a l l c e l l c l u s t e r s we r e a n a l y z e d . Corresponding mass spectra are provided for each cell type.

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