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Fluorescence correlation spectroscopy helps clarify cell mitosis

BioPhotonics
Jun 2006
Lynn M. Savage

Understanding the processes at the heart of the cellular functions regulating mitosis, such as signal transduction, requires precise quantitation of the cell’s molecules. According to researcher Zifu Wang at the University of California, Irvine, the best way to achieve this is through fluorescence correlation spectroscopy.

Wang and his colleagues at Irvine and at the University of California, San Diego, in La Jolla have used the technique to determine the amount of Cdc20 in living cells undergoing mitosis. Cdc20 is a protein that aids mitosis by activating anaphase-promoting complex/cyclosome (APC/C), a large, multiprotein compound that is responsible for segregating the daughter strands of a dividing chromosome.

Using an inverted microscope and a 63x, 1.4-NA objective from Carl Zeiss and a Ti:sapphire laser from Coherent Inc., the investigators performed fluorescence correlation spectroscopy with two-photon excitation. They collected the emission from cells modified to express enhanced cyan fluorescent protein, using a photomultiplier tube detector from Hamamatsu.

The spectroscopic technique resolves mixtures of fluorescent particles by showing the differences in their diffusion constants, which change with their molecular weights.

They report in the April 7 edition of Biophysical Journal’s BioFast that, before mitosis begins, Cdc20 is complexed with APC/C at a concentration of 76 ±24 nM and that this compound diffuses within the cytoplasm at 1.8 μm2/s. During mitosis, about half of the Cdc20 dissociates from the complex, at 12 pM/s, and gathers into a pool that diffuses at 19.5 ±5.0 μm2/s. Ultimately, after mitosis concludes, the dissociated Cdc20 disappears entirely.

The researchers hope to use the technique to determine whether Cdc20 is an essential component of APC/C or an independent, temporary facilitator of its role in mitosis. They also note that the Cdc20-APC/C complex must be deactivated so that other mitotic processes may occur, such as cyclin B stabilization before segregation of the chromosome, and that fluorescence correlation spectroscopy could help them discover how that happens.


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