Fluorescence correlation spectroscopy helps clarify cell mitosis
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
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
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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