The unique features of fluorescence fluctuation spectroscopy (FFS) make this technique attractive for cellular applications. FFS requires no external perturbation to determine kinetic and molecular properties with submicron spatial resolution and single molecule sensitivity. Especially, the application of FFS to proteins tagged with green fluorescent protein (GFP) has the potential to revolutionize in vivo studies. The long-term objective of the proposed research lies in the concurrent development and application of fluctuation techniques, so that their full potential for in vivo studies is realized. The impact of this new technology will be felt in many biological areas with applications ranging from basic research in cell biology to pharmaceutical drug screening. Fluorescence correlation spectroscopy (FCS) uses the autocorrelation function to determine kinetic parameters, such as the diffusion coefficient. In addition to regular FCS two other fluctuation techniques, the photon counting histogram (PCH) and scanning FCS will be used. PCH, a recently developed technique, determines the molecular brightness, which will be used to address the association of proteins. Scanning FCS has the potential to detect immobilized proteins. Together, the fluctuation techniques provide complementary information necessary for a successful characterization of biochemical processes in vivo. To assess the true potential of these techniques in vivo a thorough characterization will be performed and the statistical accuracy of each technique will receive special emphasis. The properties of EGFP and the autofluorescence in the different cellular compartments will be characterized in order to understand the quantitative range of FFS measurements in living cells. The fluctuation techniques will be applied to study Retinoid X Receptor (RXR), a nuclear receptor, in vivo. Nuclear receptors are ligand controlled transcription regulators and RXR has been identified as the key regulator of hormone receptors. The oligomerization state of RXR, which ranges from tetramer to monomer, serves as a control mechanism for its activation. The resolution of the oligomenzation state of RXR by fluctuation techniques will be at the focus of this in vivo study. In addition, the binding of RXR to DNA will be studied.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM064589-03
Application #
6701748
Study Section
Special Emphasis Panel (ZRG1-SSS-U (01))
Program Officer
Lewis, Catherine D
Project Start
2002-02-01
Project End
2005-01-31
Budget Start
2004-02-01
Budget End
2005-01-31
Support Year
3
Fiscal Year
2004
Total Cost
$144,387
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Hennen, Jared; Hur, Kwang-Ho; Karuka, Siddarth Reddy et al. (2018) Protein oligomerization and mobility within the nuclear envelope evaluated by the time-shifted mean-segmented Q factor. Methods :
Chen, Yan; Sun, Hui-Qiao; Eichorst, John P et al. (2018) Comobility of GABARAP and Phosphatidylinositol 4-Kinase 2A on Cytoplasmic Vesicles. Biochemistry 57:3556-3559
Hennen, Jared; Saunders, Cosmo A; Mueller, Joachim D et al. (2018) Fluorescence fluctuation spectroscopy reveals differential SUN protein oligomerization in living cells. Mol Biol Cell 29:1003-1011
Eichorst, John P; Chen, Yan; Mueller, Joachim D et al. (2018) Distinct Pathway of Human T-Cell Leukemia Virus Type 1 Gag Punctum Biogenesis Provides New Insights into Enveloped Virus Assembly. MBio 9:
Hennen, Jared; Angert, Isaac; Hur, Kwang-Ho et al. (2018) Investigating LINC Complex Protein Homo-oligomerization in the Nuclear Envelopes of Living Cells Using Fluorescence Fluctuation Spectroscopy. Methods Mol Biol 1840:121-135
Hennen, Jared; Hur, Kwang-Ho; Saunders, Cosmo A et al. (2017) Quantitative Brightness Analysis of Protein Oligomerization in the Nuclear Envelope. Biophys J 113:138-147
Li, Jinhui; Barylko, Barbara; Eichorst, John P et al. (2016) Association of Endophilin B1 with Cytoplasmic Vesicles. Biophys J 111:565-576
Hur, Kwang-Ho; Chen, Yan; Mueller, Joachim D (2016) Characterization of Ternary Protein Systems In Vivo with Tricolor Heterospecies Partition Analysis. Biophys J 110:1158-67
Smith, Elizabeth M; Hennen, Jared; Chen, Yan et al. (2015) Z-scan fluorescence profile deconvolution of cytosolic and membrane-associated protein populations. Anal Biochem 480:11-20
Hur, Kwang-Ho; Mueller, Joachim D (2015) Quantitative Brightness Analysis of Fluorescence Intensity Fluctuations in E. Coli. PLoS One 10:e0130063

Showing the most recent 10 out of 47 publications