Fluorescence Correlation Spectroscopy (FCS) provides insights into mRNA binding proteins
Proteins are critical for countless roles in our bodies including structure, function, and regulation of our tissues and organs. To create proteins from the genes in our DNA, an intermediate molecule, mRNA, is needed. mRNA is made from DNA in the nucleus and then transported out of the nucleus where it is translated into protein.
Dr. Finn Cilius Nielsen and his team at the Center for Genomic Medicine, Denmark, work to understand the molecules which control mRNAs. Dr. Nielsen’s lab recently published an article using superresolution microscopy, fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS, respectively) to better understand these molecules.
Dr. Finn Cilius Nielsen with his lab members Àngels Mateu-Regué (left) and Lelde Kalnina (center).
Àngels Mateu-Regué at the microscope.
We spoke with Àngels Mateu-Regué, a graduate student in Dr. Nielsen’s lab and lead author on the publication, about their findings and use of this technique.
Can you briefly describe your area of research and the findings in your article?
mRNAs are coated with a wide-range of proteins, forming complexes known as messenger ribonucleoprotein particles or “mRNPs”. These proteins regulate mRNA transport, translation, stability, etc. It had been hypothesized that mRNAs could be transported in groups, thus coordinating transport and translation of different mRNAs in space and time.
To answer these questions, we used a combination of superresolution microscopy and fluorescence correlation spectroscopy (FCS). Using these techniques, we could see that mRNAs exist as single molecules in cells and that the association between different RNA-binding proteins happen via mRNA-dependent interactions. Therefore, coordinate protein synthesis is unlikely to be a result of assembly of different mRNAs in the same mRNP granule.
Structured Illumination Microscopy (SIM) image of HeLa cells obtained by indirect immunofluorescence staining. The RNA-binding proteins IMP1 (green) and YBX1 (red) are associated in the cytoplasm. Nuclear pore is depicted by the nuclear protein NUP153 (cyan) and cell nucleus is stained with DAPI (blue).
A zoomed in area from the Structured Illumination Microscopy (SIM) image of HeLa cells. mRNP complexes are shown to be formed in the cytoplasmic side of the nuclear pore.
How did fluorescence correlation spectroscopy (FCS) contribute to your findings?
We characterized the dynamic behavior of the mRNA-protein complexes using two GFP-tagged RNA binding proteins present in these particles: IMP1 and YBX1. Through the analysis of their autocorrelation curves, we learned that mRNP granule dynamics is not described by a simple diffusion model. We could conclude that mRNP granules diffuse in at least two different motions in live cells. Data visualization and quantification of diffusion was very easy to perform. GFP alone, in contrast, gave a very fast and simple 3D diffusion compared to the two other factors.
Additionally, we were also able to count the number of proteins bound to a complex by comparing the brightness of monomeric GFP with the brightness of the complex.
How did you use Fluorescence Cross-Correlation Spectroscopy (FCCS)?
We wanted to study the nature of the interaction between the above mentioned proteins: IMP1 and YBX1. Specifically, we wanted to see whether the interaction was direct or RNA-mediated (both proteins bound to the same mRNA). This time, we used a mCherry-tagged YBX1 and we co-transfected cells with GFP-IMP1 or a GFP-IMP1 mutant that is not able to bind to RNA. It was very clear to see that when we used wild type GFP-IMP1 and mCherry-YBX1, fluorescence signals coming from GFP or mCherry were synchronous, and that resulted in a positive cross-correlation curve. On the other hand, when we used the GFP-IMP1 mutant, we could clearly see that the cross-correlation curve was completely flat, indicating that the interaction between the two proteins is RNA-dependent.
Fluorescence Cross-Correlation Spectroscopy (FCCS) cytoplasmic measurement of HeLa cells co-transfected with GFP-IMP1 (green autocorrelation curve) and mCherry-YBX1 (red autocorrelation curve) showing in vivo interaction between the two RNA-binding proteins (positive cross-correlation curve). When a RNA-binding impaired IMP1 mutant is used (IMP1_KH1-4mut), cross-correlation with YBX1 is negative (right graph).
What are the main advantages of FCS and FCCS compared to other commonly used techniques?
The possibilities of FCS are enormous in terms of measuring how proteins diffuse in live cells. Having such a direct, fast and easy measurement about protein dynamics and stoichiometry in a single cell in vivo is already a major advantage of the technique. Using the same equipment, FCS and FCCS allow a precise and well defined biochemical characterization of macromolecular complexes combined with imaging in live cells at the same time.
One could think about endless possibilities – not only measuring protein diffusion in normal conditions but also how the diffusion (or biological activity) of a particular protein changes, for instance upon the addition of drugs, etc.
Regarding FCCS, the advantage of protein-protein interaction studies inside the cells (their native environment) is by itself of major importance. The analysis with this technique is performed directly in intact live cells, in which the real protein-protein interactions can be captured. This is in contrast to extensively used protein-protein interaction methods, such as immunoprecipitation.
Àngels Mateu-Regué analyzing fluorescence correlation spectroscopy (FCS) data.
FCS / FCCS can be seen as an intimidating fluorescence microscopy modality. What would you recommend to a researcher new to these techniques?
After trying FCS and FCCS, our understanding and our view about the technique has radically changed. We could not do anything other than say to other researchers: go for it!
As a start, we would recommend to read a few broad spectrum FCS and FCCS reviews or explanatory material. It is very important to understand and incorporate the theory behind the technique in order to understand the outcome and be able to troubleshoot in your experiments later. In the same line, we would also suggest that you get some training if possible, in order to familiarize with the technique and try it out with very simple things. For example, these simple things could be an antibody coupled with an Alexa Fluor fluorophore diluted in PBS, a cell lysate (or even a cell) expressing GFP alone. It might be easier for researchers who already work with fluorophores and fluorescent proteins.
Learn More
- Read the full article from Dr. Nielsen’s group: “Single mRNP Analysis Reveals that Small Cytoplasmic mRNP Granules Represent mRNA Singletons.” Link
FCS Reviews:
- Wikipedia: Fluorescence Correlation Spectroscopy Link
- Nature Protocols: Imaging fluorescence (cross-) correlation spectroscopy in live cells and organisms Link
ZEISS Technology with FCS capabilities:
- ZEISS LSM 980 laser scanning confocal microscope Link