We aim to develop new probes and methods that will allow direct measurements of the spatio- temporal behavior of proteins in living cells. Using multifunctional dendrimers decorated with two dyes, a fluorescent donor array and a fluorogenic quencher dye, we will develop very high brightness probes that are activated by small fusion protein tags. Because the binding of these modules produces an intermittent signal, we will integrate these probes into a new superresolution imaging method that enables molecular scale localization and tracking of all tagged proteins in a sample region by stochastic activation at equilibrium. We will validate these new probes and methods in studies of ?2 adrenergic receptor and cytoskeletal dynamics. Collectively, this program will develop, validate, and deliver new imaging tools and methods to track positions and dynamics of tagged proteins at nanometer length scales. Collectively, these methods are anticipated to deliver at least an order of magnitude improvement in the single molecule brightness, the localization accuracy, and the timescale of dynamic single molecule measurements in living cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM086237-02
Application #
7943995
Study Section
Special Emphasis Panel (ZRG1-BST-Q (51))
Program Officer
Deatherage, James F
Project Start
2009-09-30
Project End
2011-11-30
Budget Start
2010-09-01
Budget End
2011-11-30
Support Year
2
Fiscal Year
2010
Total Cost
$166,592
Indirect Cost
Name
Carnegie-Mellon University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
052184116
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Saurabh, Saumya; Zhang, Ming; Mann, Victor R et al. (2015) Kinetically Tunable Photostability of Fluorogen-Activating Peptide-Fluorogen Complexes. Chemphyschem 16:2974-80
Wang, Yi; Telmer, Cheryl A; Schmidt, Brigitte F et al. (2015) Fluorogen activating protein-affibody probes: modular, no-wash measurement of epidermal growth factor receptors. Bioconjug Chem 26:137-44
Yan, Qi; Schwartz, Samantha L; Maji, Suvrajit et al. (2014) Localization microscopy using noncovalent fluorogen activation by genetically encoded fluorogen-activating proteins. Chemphyschem 15:687-695
Tan, Cheemeng; Saurabh, Saumya; Bruchez, Marcel P et al. (2013) Molecular crowding shapes gene expression in synthetic cellular nanosystems. Nat Nanotechnol 8:602-8
Maji, Suvrajit; Bruchez, Marcel P (2012) Inferring biological structures from super-resolution single molecule images using generative models. PLoS One 7:e36973
Yushchenko, Dmytro A; Zhang, Ming; Yan, Qi et al. (2012) Genetically targetable and color-switching fluorescent probe. Chembiochem 13:1564-8
Saurabh, Saumya; Maji, Suvrajit; Bruchez, Marcel P (2012) Evaluation of sCMOS cameras for detection and localization of single Cy5 molecules. Opt Express 20:7338-49
Saunders, Matthew J; Szent-Gyorgyi, Christopher; Fisher, Gregory W et al. (2012) Fluorogen activating proteins in flow cytometry for the study of surface molecules and receptors. Methods 57:308-17
Micheva, Kristina D; Bruchez, Marcel P (2012) The gain in brain: novel imaging techniques and multiplexed proteomic imaging of brain tissue ultrastructure. Curr Opin Neurobiol 22:94-100
Bruchez, Marcel P (2011) Quantum dots find their stride in single molecule tracking. Curr Opin Chem Biol 15:775-80

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