The development of Total Internal Reflection Fluorescence Microscopy, or TIRFM has been a tremendous advance in the study of dynamics events near the cell membrane. The main advantage of TIRFM is the unrivaled axial localization (compared to other types of fluorescence microscopes), whereby only fluorophores within a few hundreds of nanometers of the coverslip-water interface are excited and visualized. We propose to design, build, test and characterize a novel fluorescence microscope that will provide increased spatial-temporal information about the molecular function and regulation of phosphoinositide- anchored protein complexes. This novel microscope system will combine Total Internal Reflection Fluorescence (TIRF) microscopy with Epi-fluorescence and Structured Illumination Microscopy, thus T-E-S- M. TIRF will be used to acquire highly localized (depth <500 nm) information about membrane-associated (plasma membrane, pits, endosomes, vesicles) fluorescent molecules. TIRF provides diffraction-limited lateral resolution, with depth (axial) information encoded in the fluorescence intensities. Structured-light epi- illumination will be used to acquire and compute optical-sections with axial resolution (-500-700 nm) covering the same spatial domain of the TIRF. The computed optical section images will be provide depth- independent intensity information on these molecules without contamination from out-of-focus fluorescence. The relative axial position of these membrane-associated molecules is computed from the exponential relationship of the two sources of intensity information. Computer simulations of TIRF imaging and wide-field, epi-fluorescence imaging of GFP-fusion molecules (such as clathrin, EEA1, GLUT4, Rabs) and tagged ligands (transferrin, EGF), demonstrate that the axial localizations of 10 nm are achievable with known signal levels and system characteristics. The TESM will be able to follow molecular dynamics at speeds of as fast as 25 Hz. The 3-D localization information that will be available from the TESM will provide superior data for image analysis of vesicle mobility, fusion, fission, of the co-localization of vesicles, receptors and other molecules, and vesicles with the different components of the cytoskeleton, and where in the TIRF domain these interaction occur.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Program Projects (P01)
Project #
5P01DK060564-09
Application #
8068302
Study Section
Special Emphasis Panel (ZDK1)
Project Start
Project End
Budget Start
2010-05-01
Budget End
2011-04-30
Support Year
9
Fiscal Year
2010
Total Cost
$141,794
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Type
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
Ly, Socheata; Navaroli, Deanna M; Didiot, Marie-C├ęcile et al. (2017) Visualization of self-delivering hydrophobically modified siRNA cellular internalization. Nucleic Acids Res 45:15-25
Rohatgi, R A; Janusis, J; Leonard, D et al. (2015) Beclin 1 regulates growth factor receptor signaling in breast cancer. Oncogene 34:5352-62
Stockler, Sylvia; Corvera, Silvia; Lambright, David et al. (2014) Single point mutation in Rabenosyn-5 in a female with intractable seizures and evidence of defective endocytotic trafficking. Orphanet J Rare Dis 9:141
Malaby, Andrew W; van den Berg, Bert; Lambright, David G (2013) Structural basis for membrane recruitment and allosteric activation of cytohesin family Arf GTPase exchange factors. Proc Natl Acad Sci U S A 110:14213-8
Davey, Jonathan R; Humphrey, Sean J; Junutula, Jagath R et al. (2012) TBC1D13 is a RAB35 specific GAP that plays an important role in GLUT4 trafficking in adipocytes. Traffic 13:1429-41
Li, Jian; Malaby, Andrew W; Famulok, Michael et al. (2012) Grp1 plays a key role in linking insulin signaling to glut4 recycling. Dev Cell 22:1286-98
Young, James L; Mora, Alfonso; Cerny, Anna et al. (2012) CD14 deficiency impacts glucose homeostasis in mice through altered adrenal tone. PLoS One 7:e29688
Navaroli, Deanna M; Bellve, Karl D; Standley, Clive et al. (2012) Rabenosyn-5 defines the fate of the transferrin receptor following clathrin-mediated endocytosis. Proc Natl Acad Sci U S A 109:E471-80
Tan, Shi-Xiong; Ng, Yvonne; Burchfield, James G et al. (2012) The Rab GTPase-activating protein TBC1D4/AS160 contains an atypical phosphotyrosine-binding domain that interacts with plasma membrane phospholipids to facilitate GLUT4 trafficking in adipocytes. Mol Cell Biol 32:4946-59
Xie, Xiangyang; Gong, Zhenwei; Mansuy-Aubert, Virginie et al. (2011) C2 domain-containing phosphoprotein CDP138 regulates GLUT4 insertion into the plasma membrane. Cell Metab 14:378-89

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