The overall goal of this research is to further develop, test and implement our original experimental technique to study dynamic processes in living cells. Because the cytosol is a densely crowded and dynamically heterogeneous environment, measurements that establish a connection between spatial scale and mobility can provide insight into its organization and mechanisms of cytoskeletal activity. Fourier imaging correlation spectroscopy (FICS) is an attemative to direct imaging methods and is based on selectively detecting fluorescence fluctuations from N labeled sub-cellular species at a specified (and adjustable) spatial scale. Our recent FICS studies of the mitochondriat reticulum in live osteosarcoma cells show that the length-scale-dependence of the organelle's mobility follows a systematic and complex pattern, which likely reflects the relevant length- and time-scales of cytoskeletal motor protein activity. When actin or tubuiin components of the cytoskeleton are selectively removed, the organelle's dynamics become sequentially simplified, in the absence of both cytosketal components, the dynamics of the reticulum become primarily diffusive and may be modelled as a collection of friction points interconnected by elastic springs. The main advantage of FICS is that the signal is enhanced (relative to that from a single particle) by a factor of N and thereby affords superior temporal resolution and signal sensitivity in comparison to fluorescence microscopy. In this proposal, we develop Molecular FICS, a variation of the method that is sensitive to the motions and structural changes of individual proteins and other macromolecules in the celt. The combination of Molecular FICS with the previously established organelle-sensitive version of the method will allow us to study how macromolecular species are transported through their intracellular compartments.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067891-03
Application #
6891238
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Deatherage, James F
Project Start
2003-05-01
Project End
2007-04-30
Budget Start
2005-05-01
Budget End
2006-04-30
Support Year
3
Fiscal Year
2005
Total Cost
$245,441
Indirect Cost
Name
University of Oregon
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
948117312
City
Eugene
State
OR
Country
United States
Zip Code
97403
Lee, Wonbae; Jose, Davis; Phelps, Carey et al. (2013) A single-molecule view of the assembly pathway, subunit stoichiometry, and unwinding activity of the bacteriophage T4 primosome (helicase-primase) complex. Biochemistry 52:3157-70
Widom, Julia R; Lee, Wonbae; Perdomo-Ortiz, Alejandro et al. (2013) Temperature-dependent conformations of a membrane supported zinc porphyrin tweezer by 2D fluorescence spectroscopy. J Phys Chem A 117:6171-84
Senning, Eric N; Marcus, Andrew H (2010) Actin polymerization driven mitochondrial transport in mating S. cerevisiae. Proc Natl Acad Sci U S A 107:721-5
Senning, Eric N; Marcus, Andrew H (2010) Subcellular dynamics and protein conformation fluctuations measured by Fourier imaging correlation spectroscopy. Annu Rev Phys Chem 61:111-28
Lott, Geoffrey A; Senning, Eric N; Fink, Michael C et al. (2009) I. Conformational dynamics of biological macromolecules by polarization-modulated Fourier imaging correlation spectroscopy. J Phys Chem B 113:6847-53
Senning, Eric N; Lott, Geoffrey A; Fink, Michael C et al. (2009) II. Kinetic pathways of switching optical conformations in DsRed by 2D Fourier imaging correlation spectroscopy. J Phys Chem B 113:6854-60
Senning, Eric N; Lott, Geoffrey A; Marcus, Andrew H (2008) Fourier imaging correlation spectroscopy for cellular structure-function. Methods Cell Biol 90:117-37
Goulding, Morgan B; Canman, Julie C; Senning, Eric N et al. (2007) Control of nuclear centration in the C. elegans zygote by receptor-independent Galpha signaling and myosin II. J Cell Biol 178:1177-91
Tekavec, Patrick F; Lott, Geoffrey A; Marcus, Andrew H (2007) Fluorescence-detected two-dimensional electronic coherence spectroscopy by acousto-optic phase modulation. J Chem Phys 127:214307
Fink, Michael C; Adair, Kenneth V; Guenza, Marina G et al. (2006) Translational diffusion of fluorescent proteins by molecular fourier imaging correlation spectroscopy. Biophys J 91:3482-98

Showing the most recent 10 out of 11 publications