Imaging mRNA turnover in P-bodies and its application to brain ischemia Injured cells adapt to an insult by removing their mRNAs for storage or destruction. Both these processes occur in the same cytoplasmic foci referred to as P-bodies. The storage and destruction of transcripts cause the translation block and their relative intensity determines the fate of the cell. The ability to separately visualize these processes directly in P-bodies on a cell-by-cell basis is essential for the assessment and future control of cellular response to injury. However, in spite of the enabling nature of such an in situ technology, at present no method can selectively label both the decay and the storage of mRNA transcripts in P-bodies using standard fixed tissue sections. In this project we will resolve this limitation and will introduce the first histotechnology which separately visualizes mRNA decay and storage in P-bodies. We will then apply the new assay to study mRNA turnover in focal brain ischemia in rat.
Specific Aims of the proposal are: 1. To develop the first histotechnology for in situ labeling of mRNA turnover in P-bodies. The assay will be applicable to fixed tissue sections. The approach will encompass visualization of the activity of mRNA decay in P-bodies by using the new random DNA nanomer probes. It will permit the co-labeling of P-body core proteins for the assessment of mRNA storage occurring in P-bodies. 2. To test and validate this newly developed in situ labeling technique in sections of experimental stroke. To optimize its specificity, sensitivity and reliability of detection. By making use of fixed tissue sections, the assay will enable the analysis of the archived pathology samples. The assay will be broadly applicable in biomedicine because it will allow visualization of previously undetectable molecular pathways in a convenient histotechnology format. Its application to brain ischemia will provide useful information about the dynamics of ischemic cell injury.
Statement The purpose of this project is to introduce an enabling technology with broad application in fluorescence microscopy. It will contribute to the development of more efficient therapies and advance biomedical research of cancer, stroke, heart disease and many others.
| Didenko, Vladimir V (2017) Zebra Tail Amplification: Accelerated Detection of Apoptotic Blunt-Ended DNA Breaks by In Situ Ligation. Methods Mol Biol 1644:167-177 |
