In vivo quantitative studies of dynamic intracellular processes using fluorescence correlation spectroscopy.
In vivo quantitative studies of dynamic intracellular processes using fluorescence correlation spectroscopy.
复制标题
DOI：
10.1529/biophysj.105.077891
复制
发表时间：
2006-07
影响因子：
3.4
通讯作者：
Zifu Wang;J. Shah;M. Berns;D. Cleveland
中科院分区：
文献类型：
--
作者：
Zifu Wang;J. Shah;M. Berns;D. Cleveland
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 microm2/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 microm2/s, whose size is most consistent with free Cdc20. This free pool accumulates to 50% of total Cdc20 (approximately 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 reveal 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.
