In vivo quantitative studies of dynamic intracellular processes using fluorescence correlation spectroscopy

Area of Science:

• Cell Biology
• Biophysics
• Molecular Biology

Background:

• Quantitative analysis of dynamic intracellular processes in live cells is crucial for understanding cell signaling and cycle regulation.
• Cdc20 is a key protein in the mitotic checkpoint, regulating cell cycle transitions.

Purpose of the Study:

• To quantitatively study the dynamic intracellular processes of Cdc20 throughout the cell cycle using fluorescence correlation spectroscopy.
• To investigate the association and dissociation of Cdc20 with the APC/C complex during mitosis.

Main Methods:

• Fluorescence Correlation Spectroscopy (FCS) for in vivo quantitative studies.
• Photon counting histograms to analyze molecular complexes.
• Measurement of diffusion constants and concentrations of Cdc20.

Main Results:

• Cdc20 exists as a large complex (>1 Mda) with APC/C during interphase.
• During mitosis, Cdc20 dissociates from APC/C into a soluble pool at a rate of 12 pM/s.
• This free Cdc20 pool accumulates up to 50% during mitotic checkpoint activation and disappears during mitotic exit at 31 pM/s.

Conclusions:

• The dynamic changes in Cdc20's biochemical assembly states correlate with APC/CCdc20 activity during mitosis.
• Fluorescence correlation spectroscopy is a powerful tool for in vivo quantitative studies of dynamic intracellular processes.
• The study provides insights into the mechanisms regulating APC/CCdc20 activity throughout the cell cycle.