Determining protein polarization proteome-wide using physical dissection of individual Stentor coeruleus cells

Cellular components are non-randomly arranged with respect to the shape and polarity of the whole cell.^1-4 Patterning within cells can extend down to the level of individual proteins and mRNA.^5,6 But how much of the proteome is actually localized with respect to cell polarity axes? Proteomics combined with cellular fractionation^7-11 has shown that most proteins localize to one or more organelles but does not tell us how many proteins have a polarized localization with respect to the large-scale polarity axes of the intact cell. Genome-wide localization studies in yeast^12-15 found that only a few percent of proteins have a localized position relative to the cell polarity axis defined by sites of polarized cell growth. Here, we describe an approach for analyzing protein distribution within a cell with a visibly obvious global patterning-the giant ciliate Stentor coeruleus.^16,17 Ciliates, including Stentor, have highly polarized cell shapes with visible surface patterning.^1,18 A Stentor cell is roughly 2 mm long, allowing a "proteomic dissection" in which microsurgery is used to separate cellular fragments along the anterior-posterior axis, followed by comparative proteomic analysis. In our analysis, 25% of the proteome, including signaling proteins, centrin/SFI proteins, and GAS2 orthologs, shows a polarized location along the cell's anterior-posterior axis. We conclude that a large proportion of all proteins are polarized with respect to global cell polarity axes and that proteomic dissection provides a simple and effective approach for spatial proteomics.