Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is known to regulate a wide range of molecular targets and cellular processes, from ion channels to actin polymerization [1] [2] [3] [4] [5] [6]. Recent studies have used the phospholipase C-delta1 (PLC-delta1) pleckstrin-homology (PH) domain fused to green fluorescent protein (GFP) as a detector for PI(4,5)P(2) in vivo [7] [8] [9] [10]. Although these studies demonstrated that PI(4,5)P(2) is concentrated in the plasma membrane, its association with actin-containing structures was not reported. In the present study, fluorescence imaging of living NIH-3T3 fibroblasts expressing the PLC-delta1 PH domain linked to enhanced green fluorescent protein (PH-EGFP) reveals intense, non-uniform fluorescence in distinct structures at the cell periphery. Corresponding fluorescence and phase-contrast imaging over time shows that these fluorescent structures correlate with dynamic, phase-dense features identified as ruffles and with microvillus-like protrusions from the cell's dorsal surface. Imaging of fixed and permeabilized cells shows co-localization of PH-EGFP with F-actin in ruffles, but not with vinculin in focal adhesions. The selective concentration of the PH-EGFP fusion protein in highly dynamic regions of the plasma membrane that are rich in F-actin supports the hypothesis that localized synthesis and lateral segregation of PI(4,5)P(2) spatially restricts actin polymerization and thereby affects cell spreading and retraction.

Fibroblasts migrate into and repopulate connective tissue wounds. At the wound edge, fibroblasts differentiate into myofibroblasts, and they promote wound closure. Regulated fibroblast-to-myofibroblast differentiation is critical for regenerative healing. Previous studies have focused on the role in fibroblasts of urokinase plasmingen activator/urokinase plasmingen activator receptor (uPA/uPAR), an extracellular protease system that promotes matrix remodeling, growth factor activation, and cell migration. Whereas fibroblasts have substantial uPA activity and uPAR expression, we discovered that cultured myofibroblasts eventually lost cell surface uPA/uPAR. This led us to investigate the relevance of uPA/uPAR activity to myofibroblast differentiation. We found that fibroblasts expressed increased amounts of full-length cell surface uPAR (D1D2D3) compared with myofibroblasts, which had reduced expression of D1D2D3 but increased expression of the truncated form of uPAR (D2D3) on their cell surface. Retaining full-length uPAR was found to be essential for regulating myofibroblast differentiation, because 1) protease inhibitors that prevented uPAR cleavage also prevented myofibroblast differentiation, and 2) overexpression of cDNA for a noncleavable form of uPAR inhibited myofibroblast differentiation. These data support a novel hypothesis that maintaining full-length uPAR on the cell surface regulates the fibroblast to myofibroblast transition and that down-regulation of uPAR is necessary for myofibroblast differentiation. INTRODUCTIONMyofibroblast differentiation from fibroblasts is a critical component of the healing process. Regenerative healing (without scarring) results from the successful execution of what have been characterized as three distinct phases of wound healing. In the first phase, fibroblasts that migrate into the wound secrete proteases, extracellular matrix (ECM) molecules, and growth factors. In the second phase, fibroblasts differentiate into nonmotile, wound-contracting myofibroblasts that also secrete ECM proteins and remodel the ECM (Jester et al., 1995;Mohan et al., 2003;Netto et al., 2005). In the third phase, after wound closure, myofibroblasts usually disappear by apoptosis (Desmouliere et al., 1995). Pathological states such as hypertrophic scars, liver cirrhosis, idiopathic lung fibrosis, and glomerulosclerosis are characterized by the persistence of myofibroblasts, which contribute to disease progression by overproduction of ECM and by excessive contraction (Desmouliere et al., 2003;Gabbiani, 2003).To better understand the molecular basis for the fibroblast to myofibroblast transition, we have focused on the role of the urokinase plasmingen activator (uPA) pathway during wound healing. uPA is an extracellular serine protease that binds to its receptor, uPAR, and generates plasmin from plasminogen at the cell-matrix interface. Plasmin is a broadspectrum protease that not only cleaves fibrin and other ECM proteins but also promotes cell migration by activating matrix-sequestered metallopr...

Nuclear phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate, fluctuate throughout the cell cycle and are linked to proliferation and differentiation. Here we report that phospholipase C-␦ 1 accumulates in the nucleus at the G 1 /S boundary and in G 0 phases of the cell cycle. Furthermore, as wild-type protein accumulated in the nucleus, nuclear phosphatidylinositol 4,5-bisphosphate levels were elevated 3-5-fold, whereas total levels were decreased compared with asynchronous cultures. To test whether phosphatidylinositol 4,5-bisphosphate binding is important during this process, we introduced a R40D point mutation within the pleckstrin homology domain of phospholipase C-␦ 1 , which disables high affinity phosphatidylinositol 4,5-bisphosphate binding, and found that nuclear translocation was significantly reduced at G 1 /S and in G 0 . These results demonstrate a cell cycle-dependent compartmentalization of phospholipase C-␦ 1 and support the idea that relative levels of phosphoinositides modulate the portioning of phosphoinositide-binding proteins between the nucleus and other compartments.A distinct phosphoinositide cycle is present in nucleus, and growing evidence suggests its metabolism is important for DNA repair, mRNA export, and gene transcription (1-5). Moreover, changes in nuclear phosphoinositide levels are correlated to cell cycle progression and independently regulated from the phosphoinositide cycle at the plasma membrane (6, 7). Because phospholipase C (PLC) 1 is a key regulator of phosphoinositide metabolism at the plasma membrane, understanding what controls the localization of this enzyme to the nucleus and how its activity there affects nuclear phosphoinositide metabolism is important.In mammals, PLC is a 13-member family of phosphodiesterases (␤, ␥, ␦, ⑀, and subtypes) essential to a wide range of cellular responses, including exocytosis, endocytosis, gene transcription, cytoskeletal remodeling, and membrane trafficking (8 -11). PLC-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) generates inositol 1,4,5-trisphosphate and diacylglycerol critical second messengers that mobilize cellular calcium and activate protein kinase C, respectively (8). PLC␦ 1 has been shown to be activated by capacitative calcium entry (12), transglutaminase II (13), a form of Rho GTPase-activating protein (14), free fatty acids such as arachidonic acid (15), and phosphatidylserine (16). Upon activation, PLC␦ 1 binds the inositol head group of PIP 2 with high affinity and specificity through its non-catalytic PH domain (17)(18)(19). This interaction facilitates association with the plasma membrane, particularly with ruffles, where PIP 2 is enriched (20). When a point mutation in the PH domain (R40D) is introduced, however, the ability of PLC␦ 1 to target the plasma membrane is greatly reduced, resulting in a higher cytosolic concentration (18).Several PLC isoforms are detected in the nuclear compartment, primarily due to alternative spliced variation (21). PLC␦ 1 , however, is the only...