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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Published: June 4th, 2019

DOI:

10.3791/58545

1Biological Sciences Department Hunter College, City University of New York, 2Program in Molecular, Cellular, and Developmental Biology, Graduate Center, City University of New York

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA's life cycle.

Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Cell biology studies that visualize dynamic events with spatial and temporal resolution have been made possible by the development of fluorescence-based live cell imaging techniques. Presently, in vivo mRNA visualization is achieved via technologies that are based on RNA aptamer-protein interactions, RNA aptamer-induced fluorescence of organic dyes and nucleic acid probe annealing1,2,3. They all offer high specificity, sensitivity and signal-to-background ratio. However, RNA aptamer-centered approaches require extensive genetic manipulation, where a transgene is engi....

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1. Design of MBs for Live Cell Imaging

  1. Fold the target RNA sequence to predict the mRNA target’s secondary structure using the “RNA form” from the mfold server (http://unafold.rna.albany.edu/?q=mfold/RNA-Folding-Form).
    1. Paste/upload the target sequence in FASTA format, select 5 or 10% sub-optimality (structures with a free energy of folding within 5 or 10% of the MFE value, respectively), and adjust the maximum number of computed foldings accordingly (e.g.......

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Using PinMol, several MBs can be designed for one mRNA target (Figure 1B-C). After synthesis and purification, the selected MBs are characterized and compared using in vitro analysis.

Figure 1
Figure 1: Technique and tissue description for live cell imaging of endogenous mRNAs. .......

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Live visualization of endogenous mRNA trafficking in Drosophila egg chambers relies on the use of specific, efficient, and nuclease-resistant MBs, which can now be easily designed with PinMol software. MBs are specific probes designed to detect unique sequences within a target mRNA (preferably regions free of secondary structure), making possible highly resolved detection of a transcript. The only limitation when adopting this technique/protocol for other tissues/cell types is the efficiency of MB deliv.......

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We thank Salvatore A.E. Marras (Public Health Research Institute Center, Rutgers University) for the synthesis, labeling and purification of molecular beacons, and Daniel St Johnston (The Gurdon Institute, University of Cambridge) for the oskar-MS2/MCP-GFP transgenic fly stock. This work was supported by a National Science Foundation CAREER Award 1149738 and a Professional Staff Congress-CUNY Award to DPB.

....

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Name Company Catalog Number Comments
Spectrofluorometer Fluoromax-4 Horiba-Jobin Yvon n/a Photon counting spectrofluorometer
Quartz cuvette Fireflysci (former Precision Cells Inc.) 701MFL
Dumont #5 tweezer World Precision Instruments 501985 Thin tweezers are very important to separate out the individual egg chambers
Halocarbon oil 700 Sigma-Aldrich H8898
Cover slip No.1 22 x 40mm VWR 48393-048
Dissecting microscope Leica MZ6 Leica Microsystems Inc. n/a
CO2 fruit fly anesthesia pad Genesee Scienific 59-114
Tris-HCL pH7.5 Sigma-Aldrich 1185-53-1
Magnesium chloride Sigma-Aldrich 7791-18-6
NaCl Sigma-Aldrich 7647-14-5
Spinning disc confocal microscope Leica DMI-4000B inverted microscope equipped with Yokogawa CSU 10 spinning disc Leica Microsystems Inc. n/a
Hamamatsu C9100-13 ImagEM EMCCD camera Hamamatsu n/a
PatchMan NP 2 Micromanipulator Eppendorf Inc. 920000037
FemtoJet Microinjector Eppendorf Inc. 920010504
Injection needle: Femtotips II Eppendorf Inc. 930000043
Loading tip: 20ul Microloader Eppendorf Inc. 930001007
Micro Cover glasses no. 1 or 1.5, 22x40mm VWR 48393-026; 48393-172
Dry yeast Any grocery store n/a
Computer, > 20 GB RAM Although processing can be carried out on most computers, higher capabilities will increase the speed of the processing

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