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Counting Cytokinesis Proteins Globally and Locally in Fission Yeast
Jian-Qiu Wu1 and
Thomas D. Pollard1,2*
We used fluorescence microscopy to measure global and localconcentrations of 28 cytoskeletal and signaling proteins fusedto yellow fluorescent protein (YFP) in the fission yeast Schizosaccharomycespombe. Native promoters controlled the expression of these functionalYFP fusion proteins. Fluorescence measured by microscopy orflow cytometry was directly proportional to protein concentrationmeasured by quantitative immunoblotting. Global cytoplasmicconcentrations ranged from 0.04 (formin Cdc12p) to 63 micromolar(actin). Proteins concentrated up to 100 times in contractilerings and 7500 times in spindle pole bodies at certain timesin the cell cycle. This approach can be used to measure theglobal and local concentrations of any fusion protein.
1 Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520–8103, USA. 2 Departments of Cell Biology and Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520–8103, USA.
* To whom correspondence should be addressed. E-mail: thomas.pollard{at}yale.edu
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