This protocol describes in detail the technique used in Trcek et al to visualize and quantify the spatial organization of RNAs in embryos of the fruit fly Drosophila. The enrichment of specific RNAs in the posterior region of the embryo gives rise to the germ line (the cells that will eventually become the reproductive cells of the animal).
mRNA quantification using single-molecule FISH in Drosophila embryos.
Trcek T, Lionnet T, Shroff H, Lehmann R.
(2017) Nature Protocols 12(7):1326-1348.
The fruit fly (Drosophila) is one of the most important animals used to understand brain circuits. This is because one can use extensive fly genetics tools to perturb specific neurons and understand their role in various behaviors. However, knowing which genes each neuron expresses in order to achieve the required tasks has remained difficult. We developed a technique to image mRNAs (the gene intermediate between DNA and protein) in entire brains of fruit flies that provides this missing tool. First, we process the fixed tissue so that fluorescent probes efficiently bind to mRNAs in the entire brain. These probes can then be imaged seamlessly thanks to a clearing treatment that makes the tissue optically transparent. Combined with a light sheet microscope capable of light sheet illumination, the technique allows counting individual molecules of mRNA to detect when, where and how many mRNAs are expressed throughout the brain.
Quantitative mRNA Imaging Throughout the Entire Drosophila Brain
We built on the Janelia Fluor™ dyes to design organic fluors that can be turned on with blue light. The dyes penetrate living cells and can be conjugated in vivo to target proteins (HaloTag). Because the fluors are bright and we can easily control their concentration using blue light, they are perfectly suited for single molecule tracking in vivo.
Bright photoactivatable fluorophores for single-molecule Imaging.
A new imaging technique that captures on camera the nascent peptides being produced by individual mRNA molecules in living cells. This technology opens the door to investigating when and where translation occurs.
Real-time Quantification of single RNA translation dynamics in living cells.
Morisaki T, Lyon K, DeLuca KF, DeLuca JG, English BP, Zhang Z, Lavis LD, Grimm JB, Viswanathan S, Looger LL, Lionnet T, Stasevich T. (2016) Science Jun 17; 352 (6292): 1425-9
We observed that the enzyme that synthesizes mRNA from DNA (Pol II) accumulates around genes when they are induced. Dozens of Pol II form these clusters for a few seconds before dissociating. The longer these clusters last, the more RNA is produced. The factors that trigger these elusive clusters remain to be discovered!
RNA Polymerase II cluster dynamics predicts mRNA ouput in living cells.
A novel design for the optics at the center of the Multifocus Microscope (MFM): a microscope capable of acquiring the full volume of a sample in a single camera shot.
Multifocus microscopy with precise color multi-phase diffractive optics applied in functional neuronal imaging.
Abrahamsson S, Ilic R, Wisniewski J, Mehl B, Yu L, Chen L, Davanco M, Oudjedi L, Fiche J-B, Hajj B, Jin X, Pulupa J, Cho C, Mir M, El Beheiry M, Darzacq X, Nollmann M, Dahan M, Wu C, Lionnet T, Liddle AJ, Bargmann CI (2016) Biomedical Optics Express 7 (3) 855 PMC
By co-tracking individual molecules of mRNAs and ribosomes (the molecules synthesizing proteins from mRNA), we were able to identify locations of the cell where RNA is made into proteins.
Mapping translation ‘hot spots’ in live cells by tracking single molecules of mRNA and ribosomes.
We harnessed the CRISPR system to label genomic loci in fixed cells. The CasFISH technique can provide results in as little as 15 minutes, a significant improvement on the traditional DNA-FISH based methods.
CASFISH : CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells.
In Drosophila embryos, mRNA molecules from specific genes aggregate into granules at the posterior pole (the region of the embryo that will eventually give rise to gametes). Within each of these small small (~100 nanometers) granules, the positions of mRNAs is not random; rather, mRNAs from the same species accumulate together at preferred locations.
Drosophila germ granules are structured and contain homotypic mRNA clusters.
We used the response of the Actin gene to serum as a model for rapid transcription dynamics and interrogated the influence of different upstream factors on transcription kinetics and robustness.
Cellular Levels of Signaling Factors Are Sensed by β-actin Alleles to Modulate Transcriptional Pulse Intensity.
We labeled mRNAs with 2 colors which allowed us to distinguish whether each mRNA molecule in a cell had been translated (read by the protein synthesizing machinery).
An RNA biosensor for imaging the first round of translation from single cells to living animals.
We designed and characterized novel cell permeable dyes – Janelia Fluor™ : dyes that label proteins in living cells with fluorophores brighter and more stable than existing ones (or fluorescent proteins).
A general method to improve fluorophores for live-cell and single-molecule microscopy.
We measured the dynamics of individual molecules of transcription factors to quantitatively address how these proteins find their preferred sequences within the billions of basepairs that constitute the genome. This helps understand how they can selectively activate their target genes.
Single-molecule dynamics of enhanceosome assembly in embryonic stem cells.
We used multiplex RNA-FISH to visualize where in the cell the RNA strands that form the influenza virus genome come together. This helped us uncover a new step in the influenza infection cycle.
Colocalization of different influenza viral RNA segments in the cytoplasm before viral budding as shown by single-molecule sensitivity FISH analysis.
- Corresponding Author
Identifying the RNA sequence recognized by the protein ZBP1 with biochemistry enables identifying the protein’s target genes.
Spatial arrangement of conserved recognition elements identifies RNA regulatory networks.
The first demonstration in a mammalian organism that the MS2 reporter (a technology that renders mRNAs fluorescent) can be used on endogenous genes without detrimental effects.
A transgenic mouse for in vivo detection of endogenous labeled mRNA.
By counting the number of mRNAs in yeast cells, we concluded that transcription of so-called housekeeping genes (genes continuously expressed by the cell) was not coordinated, even within genes that shared the same biological function.
Transcription of functionally related genes is not coordinated.
We measured how fast a helicase could unwind a DNA hairpin as a function of the magnitude of an assisting force pulling the DNA apart (helicases are proteins that unzip DNA). This helped us demonstrate that the helicase exploits natural opening fluctuations of the fork, rather than actively destabilizing the double helix.
Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism.
Lionnet T, Spiering MM, Benkovic SJ, Bensimon D, Croquette V (2007). Proc. Natl. Acad. Sci. 104 (50): 19790-5 PMC
We predicted the effect of the DNA sequence on the double helix twist-stretch coupling (the amount that the DNA lengthens upon twisting). Our results show that this mechanical property depends strongly one the DNA sequence, suggesting it would be possible to read the chemical sequence of the DNA by purely mechanical means.
Sequence dependent twist-stretch coupling in DNA.
We twisted a single strand of DNA and measured its length with nanometer accuracy. This allowed us to discover a counterintuitive property: the double helix unwinds when stretched, in contrast with our everyday intuition of a braided rope. This effect could have important implications in the way DNA binding proteins recognize their target sequence.
Wringing out DNA.
This is the first direct observation of the UvrD helicase at the single molecule level (helicases are proteins that unzip the DNA). We discovered that UvrD switches DNA strands, a property that might help it repair DNA more efficiently.
Single-molecule assay reveals strand switching and enhanced processivity of UvrD.