Phosphoinositides (PtdInss) play key roles in cell polarization and motility. With a series of biosensors based on Fö rster resonance energy transfer, we examined the distribution and metabolism of PtdInss and diacylglycerol (DAG) in stochastically migrating Madin-Darby canine kidney (MDCK) cells. The concentrations of phosphatidylinositol (4,5)-bisphosphate, phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ), phosphatidylinositol (3,4)-bisphosphate, and DAG were higher at the plasma membrane in the front of the cell than at the plasma membrane of the rear of the cell. The difference in the concentrations of PtdInss was estimated to be less than twofold between the front and rear of the migrating MDCK cells. To decode the spatial activities of PtdIns metabolic enzymes from the obtained concentration maps of PtdInss, we developed a one-dimensional reaction diffusion model of PtdIns metabolism. In this model, the activities of phosphatidylinositol monophosphate 5-kinase, phosphatidylinositol 3-kinase, phospholipase C, and PIP 3 5-phosphatases were higher at the plasma membrane of the front than at the plasma membrane of the rear of the cell. This result suggests that, although the difference in the steady-state level of PtdInss is less than twofold, PtdInss were more rapidly turned over at the front than the rear of the migrating MDCK cells. INTRODUCTIONCell migration is an important event during early development, inflammatory responses to infection, and wound healing, and it is an important pathological event during tumor invasion and metastasis. Despite morphological and functional differences, different migratory cells share a conserved set of polarity signals. Kinases for phosphoinositides (PtdInss), Rho GTPases, and the actin and microtubule cytoskeletons play key roles in signaling polarity in cells ranging from Dictyostelium discoideum (Charest and Firtel, 2006) to neurons (Luo, 2000;Aoki et al., 2007) and neutrophils (Van Keymeulen et al., 2006;Wong et al., 2006).PtdInss are a family of phospholipids containing myoinositol as their head group (reviewed in Takenawa and Itoh, 2001). Despite a relatively low abundance in biological membranes, PtdInss have been reported to regulate a myriad of cellular processes. Among them, phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P 2 ] is the major PtdInss at the inner leaflet of the plasma membrane. On growth factor stimulation, intracellular second messengers such as inositol (1,4,5)-trisphosphate (IP 3 ), diacylglycerol (DAG), and phosphatidylinositol (3,4,5)-triphosphate (PIP 3 ) are generated from PI(4,5)P 2 .The discovery that the pleckstrin homology (PH) domain in the signaling proteins recognizes specific phosphoinositides revealed that stimulated proteins translocate to specific regions of the membrane to participate in signaling events via interaction with these lipids (reviewed in Di Paolo and De Camilli, 2006). A series of experiments indicated that the PH domains from different proteins recognize different PtdInss and led to the development of a novel t...

Rac1 has a crucial role in epidermal growth factor (EGF)-induced membrane ruffling, lamellipodial protrusion, and cell migration. Several guanine nucleotide exchange factors (GEFs) including Sos1, Sos2, Tiam1 and Vav2 have been shown to transduce the growth signal from the EGF receptor to Rac1. To clarify the role of each GEF, we time-lapse imaged the EGF-induced activity change of Rac1 in A431 cells transfected with siRNA targeting each Rac1 GEF. Because knockdown of these GEFs suppressed EGF-induced Rac1 activation only partially, we looked for another Rac1 GEF downstream of the EGF receptor and found that Asef, a Rac1-Cdc42 GEF bound to the tumor suppressor APC, also contributed to EGF-induced Rac1 activation. Intriguingly, EGF stimulation induced phosphorylation of Tyr94 within the APC-binding region of Asef in a manner dependent on Src-family tyrosine kinases. The suppression of EGF-induced Rac1 activation in siRNA-treated cells was restored by wild-type Asef, but not by the Tyr94Phe mutant of Asef. This observation strongly argues for the positive role of Tyr94 phosphorylation in EGF-induced Asef activation following the activation of Rac1.

Phosphatidic acid (PA) is one of the major phospholipids in the plasma membrane. Although it has been reported that PA plays key roles in cell survival and morphology, it remains unknown when and where PA is produced in the living cell. Based on the principle of Förster resonance energy transfer (FRET), we generated PA biosensor, and named Pii (phosphatidic acid indicator). In these biosensors, the lipid-binding domain of DOCK2 is sandwiched with the cyan fluorescent protein and yellow fluorescent protein and is tagged with the plasma membrane-targeting sequence of K-Ras. The addition of synthetic PA, or the activation of phospholipase D or diacylglycerol kinase at the plasma membrane, changed the level of FRET in Pii-expressing cells, demonstrating the response of Pii to PA. The biosensor also detected divergent PA content among various cell lines as well as within one cell line. Interestingly, the growth factor-induced increment in PA content correlated negatively with the basal PA content before stimulation, suggesting the presence of an upper threshold in the PA concentration at the plasma membrane. The biosensor also revealed uneven PA distribution within the cell, i.e. the basal level and growth factorinduced accumulation of PA was higher at the cell-free edges than at the cell-cell contact region. An insufficient increase in PA may account for ineffective Ras activation at areas of cell-cell contact. In conclusion, the PA biosensor Pii is a versatile tool for examining heterogeneity in the content and distribution of PA in single cells as well as among different cells.Like that of other phospholipids, the regulation of phosphatidic acid (PA) 3 homeostasis is not simple, in part because PA can be a precursor or product of a number of metabolic pathways, and in part because several enzymes are involved in the production of PA. Two major precursors of PA are phosphatidylcholine and diacylglycerol (DAG), which are the substrates of phospholipase D (PLD) and DAG kinases, respectively (1). The produced PA is then dephosphorylated by PA phosphatases to yield DAG, or hydrolyzed by phospholipase A to yield lyso-PA. In mammalian cells, there are two major PLDs, PLD1 and PLD2, which are distinct from each other in terms of both overexpression and knock-down phenotype (2). In addition, PLD1 and PLD2 differ strikingly in subcellular localization (3). Such observations have suggested that the PA generation in different subcellular membrane compartments is regulated by different PLDs and that the PA in different subcellular compartment regulates different cellular functions. Similarly, mammalian DAG kinases comprise an extended family with 10 members classified into five different subtypes with different regulatory domains (4).Biochemical methods such as the radioisotopic labeling of phosphate or fatty acids have been used to measure cellular PA content. However, spatial information cannot be obtained though this approach. To overcome this problem, the yeast t-SNARE Spo20 (5), mammalian Raf1 kinase (6), the Rac1 exchang...

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