LETTERS Phosphatidylserine is polarized and required for proper Cdc42 localization and for development of cell polarity Gregory D. Fairn 1 , Martin Hermansson 2 , Pentti Somerharju 2 and Sergio Grinstein 1,3 Polarity is key to the function of eukaryotic cells. On the establishment of a polarity axis, cells can vectorially target secretion, generating an asymmetric distribution of plasma membrane proteins. From Saccharomyces cerevisiae to mammals, the small GTPase Cdc42 is a pivotal regulator of polarity. We used a fluorescent probe to visualize the distribution of phosphatidylserine in live S. cerevisiae. Remarkably, phosphatidylserine was polarized in the plasma membrane, accumulating in bud necks, the bud cortex and the tips of mating projections. Polarization required vectorial delivery of phosphatidylserine-containing secretory vesicles, and phosphatidylserine was largely excluded from endocytic vesicles, contributing to its polarized retention. Mutants lacking phosphatidylserine synthase had impaired polarization of the Cdc42 complex, leading to a delay in bud emergence, and defective mating. The addition of lysophosphatidylserine resulted in resynthesis and polarization of phosphatidylserine, as well as repolarization of Cdc42. The results indicate that phosphatidylserine—and presumably its polarization—are required for optimal Cdc42 targeting and activation during cell division and mating. Phospholipids, including phosphatidylserine, are asymmetrically distributed between and across membranes. Phosphatidylserine comprises only 3–10% of the phospholipids in eukaryotic cells and is enriched in the plasma membrane, where it is found almost exclusively in the inner leaflet 1 . In mammalian cells the plasmalemmal cytosolic leaflet is composed of ≈20% phosphatidylserine, and the percentage is even higher in yeast 2 . Furthermore, in yeast, phosphatidylserine levels show peak concentrations during bud emergence, followed by a decrease through the remainder of the cell cycle. This contrasts with the behaviour of phosphatidylcholine and phosphatidylethanolamine, which increase linearly in abundance as the cycle progresses. This differential behaviour is reflected in the cellular phosphatidyl- choline/phosphatidylserine ratio, which is ≈2 : 1 at the time of bud emergence, whereas at later stages of the cycle it approaches 6:1 (ref. 3). 1 Program in Cell Biology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. 2 Institute of Biomedicine, Department of Biochemistry and Developmental Biology, Haartmaninkatu 8, 00014 University of Helsinki, Finland. 3 Correspondence should be addressed to S.G. (e-mail: sergio.grinstein@sickkids.ca) Received 3 August 2011; accepted 24 August 2011; published online 2 October 2011; DOI: 10.1038/ncb2351 We generated a fusion of GFP with the discoidin-like C2 domain of lactadherin 4 (GFP–Lact-C2) to visualize the intracellular distribution of phosphatidylserine in S. cerevisiae. At all stages of the cell cycle, the fluorescence signal of GFP–Lact-C2 was largely confined to the plasma membrane. However, the probe showed differential accumulation in distinct regions of the membrane depending on the stage of the cycle (Fig. 1a,d). Phosphatidylserine was concentrated at incipient bud sites (stage i in Fig. 1) and in small buds (stage ii). As the cells progress through the cycle and the bud becomes larger, increased signal is seen at the bud neck and the bud itself is enriched in phosphatidylserine when compared with the mother cell (stages iii and iv). In contrast, the distribution of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P 2 ), monitored using a tandem PH (pleckstrin homology) domain of PLCδ fused to GFP, showed only modest polarization during the cycle (Fig. 1b,d). PtdIns(4,5)P 2 was enriched ≈30% in the bud cortex and neck when compared with the remainder of the mother cell. In comparison, GFP–Lact-C2 was increased by >200% in the bud cortex and >300% at the bud neck. Hereafter, phosphatidylserine polarization was deemed to occur only when the GFP–Lact-C2 fluorescence intensity at the bud was at least twofold greater than that of the mother. We also used the plasmalemmal protein GFP–Ras2 as a marker to ensure that the observed differences in fluorescence intensity were not caused by increased membrane density 5 . As shown in Fig. 1c, Ras2 is rather evenly distributed throughout the membrane at all stages of the cycle. These results demonstrate that phosphatidylserine enrichment at sites of bud formation is not a general property of plasma membrane lipids or lipid-modified plasmalemmal proteins. S. cerevisiae also undergo polarization during the formation of projections in response to mating factors. Stimulation of the receptor Ste2 by mating factor α leads to recruitment of Far1, which in turn recruits Cdc24, promoting the activation of Cdc42 (refs 6,7). As during bud formation, Cdc42 activation generates a polarity axis leading to the directed secretion of vesicles to the tips of mating projections. Figure 1e shows wild-type cells expressing GFP–Lact-C2, exposed to mating factor α for 3 h. The tips of mating projections were clearly enriched in phosphatidylserine. As in vegetative yeast, GFP–Ras2 distributed 1424 NATURE CELL BIOLOGY VOLUME 13 | NUMBER 12 | DECEMBER 2011 © 20 11 M acmillan Publishers Limited. All rights reserved.