In vivo quantitative studies of dynamic intracellular processes using fluorescence correlation spectroscopy
Zifu Wang 1*, Jagesh V. Shah 2, Michael W. Berns 3 and Don W. Cleveland 4
1 University of California at Irvine
2 Harvard Medical School
3 University of California, Irvine
4 University of California at San Diego
* To whom correspondence should be addressed. E-mail: zifuw{at}uci.edu.
Submitted on November 16, 2005
Revised on January 15, 2006
Accepted on 10 March 2006
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Abstract |
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It has been a significant challenge to quantitatively study the dynamic intracellular processes in live cells. These studies are essential for a thorough understanding of the underlying mechanisms regulating the signaling pathways and the transitions between cell cycle stages. Our studies of Cdc20, an important mitotic checkpoint protein, throughout the cell cycle demonstrate that fluorescence correlation spectroscopy is a powerful tool for in vivo quantitative studies of dynamic intracellular processes. In this study, Cdc20 is found to be present primarily in a large complex (> 1 Mda) during interphase with a diffusion constant of 1.8±0.1 µm2/s and a concentration of 76±24 nM, consistent with its association with the APC/C. During mitosis, however, a proportion of Cdc20 dissociates from APC/C at a rate of 12 pM/s into a soluble pool with a diffusion constant of 19.5±5.0 µm2/s, whose size is most consistent with free Cdc20. This free pool accumulates to 50% of total Cdc20 (~40 nM) during chronic activation of the mitotic checkpoint, but disappears during mitotic exit at a rate of 31 pM/s. The observed changes in the biochemical assembly states of Cdc20 closely correlate to the known temporal pattern of the activity of APC/CCdc20 in mitosis. Photon counting histograms revealed that both complexes contain only a single molecule of Cdc20. The underlying mechanisms of the activities of APC/CCdc20 throughout the cell cycle are discussed in light of our experimental observations.
Key Words:
Cdc20, Fluorescence Correlation Spectroscopy, Photon counting histograms, anaphase-promoting complex/cyclosome (APC/C), diffusion, mitotic checkpoint
