The goal of our application is to design and develop an experimental approach that will allow us to visualize transcription in real time within living tissues. The technology will combine expertise in optics, protein chemistry and molecular biology. Our approach will address key technical hurdles such as, i) fashioning an imaging beam capable of penetrating tissue and capturing the fluorescence signal emanating from it, ii) designing an RNA that will become tagged with a fluorescent marker when expressed in living tissue, iii) selecting a fluorescent tag with an emission wavelength that acts transparently in tissue and can be excited by two-photon absorption. We intend to visualize transcription of the beta-actin gene in tissues, since it has been well-characterized and constitutively expressed in all cells. We have engineered transgenic mice that harbor multiple MS2 stem-loop structures within genomic copies of the beta-actin gene. The stem-loops have a strong affinity for a capsid protein which is fused to a fluorescent reporter. Using two-photon microscopy we can observe the transcription in muscle and brain tissue, and furthermore our fluctuation analysis can tell us how many polymerases are engaged on the gene at any given time. The data we collect will allow us to determine the initiation, elongation, and termination rates of beta-actin transcription in each cell within the tissue. The methods devised by our approach will have broad applications and serve as a novel strategy in quantifying the levels of gene expression in tissues and more importantly to derive a model for gene expression in general that will be the basis for understanding how genes are regulated in different tissues, and among cells within the same tissue. Studying dynamics of a single gene in real time will lead to experiments directly testing hypotheses concerning the stochastic nature of gene activity during cell differentiation and homeostasis as well as deriving an approach to study disease genes.
The purpose of this application is to develop a new approach to imaging gene expression in living tissues. For this we will develop new probes for imaging in the far red and use them in two- photon microscopy for the imaging of transcription in a mouse that expresses a tagged actin gene. This work will allow for the first time, the ability to describ the mechanisms controlling genes in their native environment and the basis for understanding how certain genes may cause disease.
|Park, Hye Yoon; Lim, Hyungsik; Yoon, Young J et al. (2014) Visualization of dynamics of single endogenous mRNA labeled in live mouse. Science 343:422-4|
|Piatkevich, Kiryl D; English, Brian P; Malashkevich, Vladimir N et al. (2014) Photoswitchable red fluorescent protein with a large Stokes shift. Chem Biol 21:1402-14|
|Shcherbakova, Daria M; Verkhusha, Vladislav V (2014) Chromophore chemistry of fluorescent proteins controlled by light. Curr Opin Chem Biol 20:60-8|
|Preibisch, Stephan; Amat, Fernando; Stamataki, Evangelia et al. (2014) Efficient Bayesian-based multiview deconvolution. Nat Methods 11:645-8|
|Wu, Bin; Chen, Jiahao; Singer, Robert H (2014) Background free imaging of single mRNAs in live cells using split fluorescent proteins. Sci Rep 4:3615|
|Shcherbakova, Daria M; Sengupta, Prabuddha; Lippincott-Schwartz, Jennifer et al. (2014) Photocontrollable fluorescent proteins for superresolution imaging. Annu Rev Biophys 43:303-29|
|Piatkevich, Kiryl D; Malashkevich, Vladimir N; Morozova, Kateryna S et al. (2013) Extended Stokes shift in fluorescent proteins: chromophore-protein interactions in a near-infrared TagRFP675 variant. Sci Rep 3:1847|
|Piatkevich, Kiryl D; Subach, Fedor V; Verkhusha, Vladislav V (2013) Far-red light photoactivatable near-infrared fluorescent proteins engineered from a bacterial phytochrome. Nat Commun 4:2153|
|Shcherbakova, Daria M; Verkhusha, Vladislav V (2013) Near-infrared fluorescent proteins for multicolor in vivo imaging. Nat Methods 10:751-4|
|Filonov, Grigory S; Verkhusha, Vladislav V (2013) A near-infrared BiFC reporter for in vivo imaging of protein-protein interactions. Chem Biol 20:1078-86|