Fluorescence Speckle Microscopy (FSM) is a new technique we have discovered for visualizing movement, assembly, and turnover of macromolecular assemblies in living cells. When a small fraction of subunits have fluorescent label, these polymers are seen to acquire a fluorescent speckle distribution along their lattice during their assembly. The highest speckle contrast is achieved for fractions of about 1% or less where the fluorescence of each speckle corresponds to a variable number of only a few fluorophores per resolvable unit (about 0.25 um) in the microscope. We have shown that speckles containing 1 or 2 fluorophores can be detected and recorded using a wide-field fluorescence light microscope, and digital imaging with a low noise cooled CCD camera. We are at very early stages in the development of SM and there are numerous improvements needed to take FSM much more powerful and open up new applications. These include improving the sensitivity, stability and resolution in 4-D (3D plus time) of FSM, combining FSM with other modes of light microscopy, determining new ways of measuring the kinetics of speckle motions and life-times from FSM images, and testing the developments in important collaborative projects. To achieve these goals, we have the following Specific Aims: 1) Assemble a stable, precision, multi-mode, multi-wavelength digital microscope and computer system for 4-D for FSM; 2) Use in vitro and living cell models for microtubles and actin filament assembly to determine the optimal fraction of fluorescently labeled subunits for FSM using fluorescent dyes and GFP. Apply ways of produce multiple fluorophores per subunit that are reduced in the problems of quenching and photobleaching, and explore their advantages; 3) trajectories and lifetimes in 2D and 3D from stacks of time-lapse FSM images; and 4) Test new FSM developments in biomedical projects investigating the motility and dynamics of microtuble and actin filament arrays in mitotic and migrating cells as well as in assaying microtuble rotation by microtuble motor proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM060678-01
Application #
6044467
Study Section
Special Emphasis Panel (ZRG1-SSS-I (02))
Program Officer
Deatherage, James F
Project Start
2000-01-01
Project End
2003-12-31
Budget Start
2000-01-01
Budget End
2000-12-31
Support Year
1
Fiscal Year
2000
Total Cost
$475,309
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Gatlin, Jesse C; Matov, Alexandre; Groen, Aaron C et al. (2009) Spindle fusion requires dynein-mediated sliding of oppositely oriented microtubules. Curr Biol 19:287-96
Wan, Xiaohu; O'Quinn, Ryan P; Pierce, Heather L et al. (2009) Protein architecture of the human kinetochore microtubule attachment site. Cell 137:672-84
Gardner, Melissa K; Haase, Julian; Mythreye, Karthikeyan et al. (2008) The microtubule-based motor Kar3 and plus end-binding protein Bim1 provide structural support for the anaphase spindle. J Cell Biol 180:91-100
Joglekar, Ajit P; Salmon, E D; Bloom, Kerry S (2008) Counting kinetochore protein numbers in budding yeast using genetically encoded fluorescent proteins. Methods Cell Biol 85:127-51
Dorn, Jonas F; Danuser, Gaudenz; Yang, Ge (2008) Computational processing and analysis of dynamic fluorescence image data. Methods Cell Biol 85:497-538
Yang, Ge; Cameron, Lisa A; Maddox, Paul S et al. (2008) Regional variation of microtubule flux reveals microtubule organization in the metaphase meiotic spindle. J Cell Biol 182:631-9
Cameron, Lisa A; Yang, Ge; Cimini, Daniela et al. (2006) Kinesin 5-independent poleward flux of kinetochore microtubules in PtK1 cells. J Cell Biol 173:173-9
Molk, Jeffrey N; Salmon, E D; Bloom, Kerry (2006) Nuclear congression is driven by cytoplasmic microtubule plus end interactions in S. cerevisiae. J Cell Biol 172:27-39
Joglekar, Ajit P; Bouck, David C; Molk, Jeffrey N et al. (2006) Molecular architecture of a kinetochore-microtubule attachment site. Nat Cell Biol 8:581-5
Tirnauer, Jennifer S; Salmon, E D; Mitchison, Timothy J (2004) Microtubule plus-end dynamics in Xenopus egg extract spindles. Mol Biol Cell 15:1776-84

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