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1. Mechanisms of Eukaryotic Transcription by MPM imaging of Drosophila Polytene Nuclei
2. RNA Aptamer-based Probes for Intracellular Targeting

John T. Lis, Professor of Molecular Biology and Genetics, Cornell University.
Funding: NIH-GM25232 (04/01/78-03/31/14), T32-GM00727 (07/01/75-06/30/11).

Project 1. Extensive biochemical, genetic and structural studies have identified many essential factors that regulate transcription and have deciphered their roles during distinct stages of Pol II transcription. Live-cell imaging can provide such an analysis and reveal significant insights into gene regulatory mechanisms; however, progress in imaging transcriptional events at specific endogenous genes has been limited due to difficulties in resolving individual gene loci and detecting the small number of protein molecules functioning within active transcription units. Drosophila polytene nuclei contain extended, interphase-like giant chromosomes produced by endo-eduplication processes. Polytene chromosomes have been invaluable in providing the first links between genetic and physical maps and in allowing the mapping of cloned sequences. Examining transcription factors “in action” within polytene nuclei may provide the unique opportunity to visualize gene regulation dynamics in vivo. Through the use of MPM imaging of polytene nuclei in living Drosophila salivary gland cells, we aim to investigate the functions and dynamics of transcription factors at specific endogenous genes in vivo and in real time. We have succeeded in imaging the naturally-amplified Drosophila polytene nuclei in living salivary gland tissues using MPM and have been able to recognize individual genetic loci in living cells and to study the recruitment and exchange dynamics of transcription factors at these native genes (Yao et al., 2006). After heat shock, we have visualized the recruitment of RNA polymerase II (Pol II) to native hsp70 gene loci 87A and 87C in real time. Assays based on FRAP show a rapid exchange of HSF at chromosomal loci under non-heat-shock conditions but a very slow exchange after heat shock. Our results provide evidence that activated HSF is stably bound to DNA in vivo and that turnover or disassembly of transcription activator is not required for rounds of hsp70 transcription.

This collaborative project drives our core R&D in several areas of instrumentation development. Although polytene nuclei provide natural amplification of protein factors, detection of red fluorescence proteins still suffers from lower detector sensitivity compared to the green emission. This problem cannot be overcome by increasing expression levels because the proteins need to function at a physiology-relevant concentration and our application of improved detectors has been critical to this project. We will also apply our new fast lifetime imaging scheme for better resolving protein interactions using FRET combinations. Simultaneous imaging of many factors in the same cell will be particularly helpful in understanding the interplays between individual factors during transcription. Finally, our development of arbitrary point scanning hardware with pixel level intensity control to facilitate photoactivation experiments.

Project 2. The goal of this project is to develop protein targeting aptamers that have a binding arm with a high affinity for a second aptamer (Figure 1A). The second aptamer itself will have a fluorophore binding site which both binds and stabilizes the fluorophore making it increase in brightness.  This scheme will be used to target specific proteins in vivo. The GFP binding aptamers from the Kotlikoff collaboration will be used as our initial targeting aptamer for two reasons: (1) They make an ideal test system since we can detect binding by the decrease in GFP signal, and (2) many cell lines are available that express GFP fusion proteins.  Our initial fluor-binding aptamer will be based on the sulforhodamine B (SRB) aptamers we and others have already characterized (Babendure et al 2003; Werner etal, 2009). By screening non-fluorescent Rhodamine-like molecules we have found several that exhibit a large increase in fluorescence upon binding to the SRB aptamer.  The best so far, patent blue violet (PBV), a commercial food colorant, is basically non-fluorescent unbound, but becomes fluorescent upon binding (Figure 1B).  PBV is cell-permeant and we will test intracellular targeting by transfection of GFP-expressing cells with aptamer coding DNA and incubate with the non-florescent PBV.

Figure 1. A. Scheme for aptamer construction for tagging a targted protein. B. PBV bound to aptamer increases its fluorescence by more than a factor of 100 based on counts/molecule measurements using FCS.

References

Yao, J., Munson, K. M., Webb, W. W., and Lis, J. T. (2006). Dynamics of heat shock factor association with native gene loci in living cells. Nature 442, 1050-1053.

J.R. Babendure, S.R. Adams and R.Y. Tsien, (2003) Aptamers Switch on Fluorescence of Triphenylmethane Dyes, J. Am. Chem. Soc. 125 (2003) 14716-14717.

A. Werner, P.V. Konarev, D.I. Svergun, U. Hahn, (2009) Characterization of a fluorophore binding RNA aptamer by Fluorescence Correlation Spectroscopy and Small Angle X-Ray Scattering, Analytical Biochemistry, in press.

 

 



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