Novel GFP-fused protein probes for detecting phosphatidylinositol-4-phosphate in the plasma membrane

Phosphatidylinositol-4-phosphate (PI4P) plays a crucial role in cellular functions, including protein trafficking, and is mainly located in the cytoplasmic surface of intracellular membranes, which include the trans-Golgi network (TGN) and the plasma membrane. However, many PI4P-binding domains of membrane-associated proteins are localized only to the TGN because of the requirement of a second binding protein such as ADP-ribosylation factor 1 (ARF1) in order to be stably localized to the specific membrane. In this study, we developed new probes that were capable of detecting PI4P at the plasma membrane using the known TGN-targeting PI4P-binding domains. The PI4P-specific binding pleckstrin homology (PH) domain of various proteins including CERT, OSBP, OSH1, and FAPP1 was combined with the N-terminal moderately hydrophobic domain of the short-form of Aplysia phosphodiesterase 4 (S(N30)), which aids in plasma membrane association but cannot alone facilitate this association. As a result, we found that the addition of S(N30) to the N-terminus of the GFP-fused PH domain of OSBP (S(N30)-GFP-OSBP-PH), OSH1 (S(N30)-GFP-OSH1-PH), or FAPP1 (S(N30)-GFP-FAPP1-PH) could induce plasma membrane localization, as well as retain TGN localization. The plasma membrane localization of S(N30)-GFP-FAPP1-PH is mediated by PI4P binding only, whereas those of S(N30)-GFP-OSBP-PH and S(N30)-GFP-OSH1-PH are mediated by either PI4P or PI(4,5)P₂ binding. Taken together, we developed new probes that detect PI4P at the plasma membrane using a combination of a moderately hydrophobic domain with the known TGN-targeting PI4P-specific binding PH domain.     

Suppression of ADAM8 attenuates angiotensin II-induced cardiac fibrosis and endothelial-mesenchymal transition via inhibiting TGF-β1/Smad2/Smad3 pathways

Endothelial-to-mesenchymal transition (EndMT) is involved in cardiac fibrosis induced by angiotensin II (Ang II). A disintegrin and metalloproteinase 8 (ADAM8), a member of ADAMs family, participates in cell adhesion, proteolysis and various signaling. However, its effects on the development of cardiac fibrosis remain completely unknown. This study aimed to reveal whether ADAM8 aggravates cardiac fibrosis induced by Ang II in vivo and in vitro. The C57BL/6J mice or cardiac endothelial cells were subjected to Ang II infusion to induce fibrosis. The results showed that systolic blood pressure and diastolic blood pressure were significantly increased under Ang II infusion, and ADAM8 was up-regulated. ADAM8 inhibition attenuated Ang II-induced cardiac dysfunction. ADAM8 knockdown suppressed Ang II-induced cardiac fibrosis as evidenced by the down-regulation of CTGF, collagen I, and collagen III. In addition, the endothelial marker (VE-cadherin) was decreased, whilst mesenchymal markers (α-SMA and FSP1) were increased following Ang II infusion. However, ADAM8 repression inhibited Ang II-induced EndMT. Moreover, ADAM8 silencing repressed the activation of TGF-β1/Smad2/Smad3 pathways. Consistent with the results in vivo, we also found the inhibitory effects of ADAM8 inhibition on EndMT in vitro. All data suggest that ADAM8 promotes Ang II-induced cardiac fibrosis and EndMT via activating TGF-β1/Smad2/Smad3 pathways.     

Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin

Twinfilin is a ubiquitous actin monomer-binding protein that regulates actin filament turnover in yeast and mammalian cells. To elucidate the mechanism by which twinfilin contributes to actin filament dynamics, we carried out an analysis of yeast twinfilin, and we show here that twinfilin is an abundant protein that localizes to cortical actin patches in wild-type yeast cells. Native gel assays demonstrate that twinfilin binds ADP-actin monomers with higher affinity than ATP-actin monomers. A mutant twinfilin that does not interact with actin monomers in vitro no longer localizes to cortical actin patches when expressed in yeast, suggesting that the ability to interact with actin monomers may be essential for the localization of twinfilin. The localization of twinfilin to the cortical actin cytoskeleton is also disrupted in yeast strains where either the CAP1 or CAP2 gene, encoding for the alpha and beta subunits of capping protein, is deleted. Purified twinfilin and capping protein form a complex on native gels. Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and its actin monomer-sequestering activity is inhibited by PI(4,5)P2. Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells.     

Ethanol inhibits Kv7.2/7.3 channel open probability by reducing the PI(4,5)P2 sensitivity of Kv7.2 subunit

Ethanol often causes critical health problems by altering the neuro-nal activities of the central and peripheral nerve systems. One of the cellular targets of ethanol is the plasma membrane proteins including ion channels and receptors. Recently, we reported that ethanol elevates membrane excitability in sympathetic neurons by inhibiting Kv7.2/7.3 channels in a cell type-specific manner. Even though our studies revealed that the inhibitory effects of ethanol on the Kv7.2/7.3 channel was diminished by the increase of plasma membrane phosphatidylinositol 4,5-bisphosphate (PI (4,5)P₂), the molecular mechanism of ethanol on Kv7.2/7.3 channel inhibition remains unclear. By investigating the kinetics of Kv7.2/7.3 current in high K⁺ solution, we found that ethanol inhibited Kv7.2/7.3 channels through a mechanism distinct from that of tetraethylammonium (TEA) which enters into the pore and blocks the gate of the channels. Using a non-stationary noise analysis (NSNA), we demonstrated that the inhibitory effect of ethanol is the result of reduction of open probability (P_O) of the Kv7.2/7.3 channel, but not of a single channel current (i) or channel number (N). Finally, ethanol selectively facilitated the kinetics of Kv7.2 current suppression by voltage-sensing phosphatase (VSP)-induced PI(4,5)P₂ depletion, while it slowed down Kv7.2 current recovery from the VSP-induced inhibition. Together our results suggest that ethanol regulates neuronal activity through the reduction of open probability and PI(4,5)P₂ sensitivity of Kv7.2/7.3 channels.     

An unexpected role for PI4,5P2 in EGF receptor endosomal trafficking

In mammalian cells, three Cdc25 phosphatases A, B, C coordinate cell cycle progression through activating dephosphorylation of Cyclin-dependent kinases. Whereas Cdc25B is believed to trigger entry into mitosis, Cdc25C is thought to act at a later stage of mitosis and in the nucleus. We report that a fraction of Cdc25C localises to centrosomes in a cell cycle-dependent fashion, as of late S phase and throughout G2 and mitosis. Moreover, Cdc25C colocalises with Cyclin B1 at centrosomes in G2 and in prophase and Fluorescence Recovery after Photobleaching experiments reveal that they are both in dynamic exchange between the centrosome and the cytoplasm. The centrosomal localisation of Cdc25C is essentially mediated by its catalytic C-terminal domain, but does not require catalytic activity. In fact phosphatase-dead and substrate-binding hotspot mutants of Cdc25C accumulate at centrosomes together with phosphoTyr15-Cdk1 and behave as dominant negative forms that impair entry into mitosis. Taken together, our data suggest an unexpected function for Cdc25C at the G2/M transition, in dephosphorylation of Cdk1. We propose that Cdc25C may participate in amplification of Cdk1-Cyclin B1 activity following initial activation by Cdc25B, and that this process is initiated at the centrosome, then further propagated throughout the cytoplasm thanks to the dynamic behavior of both Cdc25C and Cyclin B1.     

Lipid signaling in Drosophila photoreceptors

Drosophila photoreceptors are sensory neurons whose primary function is the transduction of photons into an electrical signal for forward transmission to the brain. Photoreceptors are polarized cells whose apical domain is organized into finger like projections of plasma membrane, microvilli that contain the molecular machinery required for sensory transduction. The development of this apical domain requires intense polarized membrane transport during development and it is maintained by post developmental membrane turnover. Sensory transduction in these cells involves a high rate of G-protein coupled phosphatidylinositol 4,5 bisphosphate [PI(4,5)P₂] hydrolysis ending with the activation of ion channels that are members of the TRP superfamily. Defects in this lipid-signaling cascade often result in retinal degeneration, which is a consequence of the loss of apical membrane homeostasis. In this review we discuss the various membrane transport challenges of photoreceptors and their regulation by ongoing lipid signaling cascades in these cells. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

Role of the lysine-rich cluster of the C2 domain in the phosphatidylserine-dependent activation of PKCalpha

The C2 domain of PKCalpha is a Ca(2+)-dependent membrane-targeting module involved in the plasma membrane localization of the enzyme. Recent findings have shown an additional area located in the beta3-beta4 strands, named the lysine-rich cluster, which has been demonstrated to be involved in the PtdIns(4,5)P(2)-dependent activation of the enzyme. Nevertheless, whether other anionic phospholipids can bind to this region and contribute to the regulation of the enzyme's function is not clear. To study other possible roles for this cluster, we generated double and triple mutants that substituted the lysine by alanine residues, and studied their binding and activation properties in a Ca(2+)/phosphatidylserine-dependent manner and compared them with the wild-type protein. It was found that some of the mutants exerted a constitutive activation independently of membrane binding. Furthermore, the constructs were fused to green fluorescent protein and were expressed in fibroblast cells. It was shown that none of the mutants was able to translocate to the plasma membrane, even in saturating conditions of Ca(2+) and diacylglycerol, suggesting that the interactions performed by this lysine-rich cluster are a key event in the subcellular localization of PKCalpha. Taken together, the results obtained showed that these lysine residues might be involved in two functions: one to establish an intramolecular interaction that keeps the enzyme in an inactive conformation; and the second, once the enzyme has been partially activated, to establish further interactions with diacylglycerol and/or acidic phospholipids, leading to the full activation of PKCalpha.     

Synthesis of 2α-substituted-14-epi-previtamin D3 and its genomic activity

We synthesized and isolated 2α-substituted analogs of 14-epi-previtamin D₃ after thermal isomerization at 80 °C for the first time. The VDR binding affinity and transactivation activity of osteocalcin promoter in HOS cells were evaluated, and the 2α-methyl-substituted analog was found to have greater genomic activity than 14-epi-previtamin D₃.     

TGF-β superfamily signaling is essential for tooth and hair morphogenesis and differentiation

Members of the transforming growth factor β (TGF-β) superfamily of signaling molecules are involved in the regulation of many developmental processes that involve the interaction between mesenchymal and epithelial tissues. Smad7 is a potent inhibitor of many members of the TGF-β family, notably TGF-β and activin. In this study, we show that embryonic overexpression of Smad7 in stratified epithelia using a keratin 5 promoter, results in severe morphogenetic defects in skin and teeth and leads to embryonic and perinatal lethality. To further analyze the functions of Smad7 in epithelial tissues of adult mice, we used an expression system that allowed a controlled overexpression of Smad7 in terms of both space and time. Skin defects in adult mice overexpressing Smad7 were characterized by hyper-proliferation and missing expression of early markers of keratinocyte differentiation. Upon Smad7-mediated blockade of TGF-β superfamily signaling, ameloblasts failed to produce an enamel layer in incisor teeth. In addition, TGF-β blockade in adult mice altered the pattern of thymic T cell differentiation and the number of thymic T cells was significantly reduced. This study shows that TGF-β superfamily signaling is essential for development of hair, tooth and T-cells as well as differentiation and proliferation control in adult tissues.     

Linking integrin to IP(3) signaling is important for ovulation in Caenorhabditis elegans

Signals from germ and myoepithelial sheath cells initiate ovulation in Caenorhabditis elegans. The coordinated dilation and contraction of spermatheca lead to subsequent fertilization of oocyte. Either the dominant negative mutant pat-3 beta integrin or disruption of talin expression block ovulation. Talin loss-of-function uncovers roles in cell contractility and migration in C. elegans. Role of beta pat-3 integrins in development and function of Caenorhabditis elegans muscles and gonads, suggesting that the interaction between the cell and the extracellular matrix (ECM) is also important for ovulation. Here, we report that integrin plays an essential role in fertility via IP(3) signaling. Sterility caused by RNAi of pat-3 and ECM molecules was suppressed by increased IP(3) signaling. Our data suggest that the cell-ECM interaction controls ovulation via IP(3) signaling.     

Identification of an intracellular microdomain of the P2X7 receptor that is crucial in basolateral membrane targeting in epithelial cells

We investigated membrane targeting of the P2X₇ receptor (P2X₇R) in polarized epithelial cells using immunofluorescent confocal imaging. The wild-type receptor was targeted to the basolateral membrane, independently of adaptor protein μ1B. Deletion of the majority of the intracellular C-terminus, or the last 26 residues (P570-Y595), conferred targeting of the protein to the apical membrane. Alanine substitution in the microdomain P582-Q587 caused similar apical membrane targeting without major effect on the receptor function and surface expression. Our results show basolateral membrane targeting of the P2X₇R in epithelial cells and that the intracellular C-terminal microdomain P582-Q587 is crucial in this process.

Structured summary

MINT-8055849:Beta-catenin (uniprotkb:B6V8E6) and P2X7R (uniprotkb:Q64663) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

Time-dependent inhibition of inositol-1,4,5-trisphosphate-5-phosphatase by calmidazolium chloride in rat GH3 cells

The calmodulin inhibitor calmidazolium chloride inhibited the activity of soluble and particulate Ins(1,4,5)P₃-5-phosphatase from GH₃ cells, with an IC₅₀ value of 100 μM following a 10-min preincubation with enzyme. The inhibition was time-dependent and could not be reversed by washing of the particulate fraction. It is concluded that although the inhibitory effect of calmidazolium chloride cannot be related per se to inhibition of calmodulin function, effects of this compound unrelated to actions upon calmodulin function may be found when concentrations that are only moderately supramaximal are used.     

The arf6 GAP centaurin alpha-1 is a neuronal actin-binding protein which also functions via GAP-independent activity to regulate the actin cytoskeleton

Centaurin alpha-1 is a high-affinity PtdIns(3,4,5)P3-binding protein enriched in brain. Sequence analysis indicates centaurin alpha-1 contains two pleckstrin homology domains, ankyrin repeats and an Arf GAP homology domain, placing it in the AZAP family of phosphoinositide-regulated Arf GAPs. Other members of this family are involved in actin cytoskeletal and focal adhesion organization. Recently, it was reported that centaurin alpha-1 expression diminishes cortical actin and decreases Arf6GTP levels consistent with it functioning as an Arf6 GAP in vivo. In the current report, we show that centaurin alpha-1 binds Arfs in vitro and colocalizes with Arf6 and Arf5 in vivo, further supporting an interaction with Arfs. Centaurin alpha-1 expression produces dramatic effects on the actin cytoskeleton, decreasing stress fibers, diminishing cortical actin, and enhancing membrane ruffles and filopodia. Expression of centaurin alpha-1 also enhances cell spreading and disrupts focal adhesion protein localization. The effects of centaurin alpha-1 on stress fibers and cell spreading are reminiscent of those of Arf6GTP. Consistent with this, we show that many of the centaurin alpha-1-induced effects on the actin cytoskeleton and actin-dependent activities do not require GAP activity. Thus, centaurin alpha-1 likely functions via both GAP-dependent and GAP-independent mechanisms to regulate the actin cytoskeleton. Furthermore, we demonstrate that in vitro, centaurin alpha-1 binds F-actin directly, with actin binding activity localized to the PtdIns(3,4,5)P3-binding PH domain. Our data suggest that centaurin alpha-1 may be a component of the neuronal PI 3-kinase cascade that leads to regulation of the neuronal actin cytoskeleton.     

Calcium Promotes the Formation of Syntaxin 1 Mesoscale Domains through Phosphatidylinositol 4,5-Bisphosphate

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is a minor component of total plasma membrane lipids, but it has a substantial role in the regulation of many cellular functions, including exo- and endocytosis. Recently, it was shown that PI(4,5)P₂ and syntaxin 1, a SNARE protein that catalyzes regulated exocytosis, form domains in the plasma membrane that constitute recognition sites for vesicle docking. Also, calcium was shown to promote syntaxin 1 clustering in the plasma membrane, but the molecular mechanism was unknown. Here, using a combination of superresolution stimulated emission depletion microscopy, FRET, and atomic force microscopy, we show that Ca²⁺ acts as a charge bridge that specifically and reversibly connects multiple syntaxin 1/PI(4,5)P₂ complexes into larger mesoscale domains. This transient reorganization of the plasma membrane by physiological Ca²⁺ concentrations is likely to be important for Ca²⁺-regulated secretion.     

The interactions between integrin α5β1 of liver cancer cells and fibronectin of fibroblasts promote tumor growth and angiogenesis

Hepatocellular carcinoma (HCC) progression is closely related to pathological fibrosis, which involves heterotypic intercellular interactions (HIIs) between liver cancer cells and fibroblasts. Here, we studied them in a direct coculture model, and identified fibronectin from fibroblasts and integrin-α₅β₁ from liver cancer cells as the primary responsible molecules utilizing CRISPR/Cas9 gene-editing technology. Coculture led to the formation of 3D multilayer microstructures, and obvious fibronectin remodeling was caused by upregulated integrin-α₅β₁, which greatly promoted cell growth in 3D microstructures. Integrin-α₅ was more sensitive and specific than integrin-β₁ in this process. Subsequent mechanistic exploration revealed the activation of integrin-Src-FAK, AKT and ERK signaling pathways. Importantly, the growth-promoting effect of HIIs was verified in a xenograft tumor model, in which more blood vessels were observed in bigger tumors derived from the coculture group than that derived from monocultured groups. Hence, we conducted triculture by introducing human umbilical vein endothelial cells, which aligned to and differentiated along multilayer microstructures in an integrin-α₅β₁ dependent manner. Furthermore, fibronectin, integrin-α₅, and integrin-β₁ were upregulated in 52 HCC tumors, and fibronectin was related to microvascular invasion. Our findings identify fibronectin, integrin-α₅, and integrin-β₁ as tumor microenvironment-related targets and provide a basis for combination targeted therapeutic strategies for future HCC treatment.     

Phosphatidylinositol binding of Saccharomyces cerevisiae Pdr16p represents an essential feature of this lipid transfer protein to provide protection against azole antifungals

Pdr16p is considered a factor of clinical azole resistance in fungal pathogens. The most distinct phenotype of yeast cells lacking Pdr16p is their increased susceptibility to azole and morpholine antifungals. Pdr16p (also known as Sfh3p) of Saccharomyces cerevisiae belongs to the Sec14 family of phosphatidylinositol transfer proteins. It facilitates transfer of phosphatidylinositol (PI) between membrane compartments in in vitro systems. We generated Pdr16p^{E235A, K267A} mutant defective in PI binding. This PI binding deficient mutant is not able to fulfill the role of Pdr16p in protection against azole and morpholine antifungals, providing evidence that PI binding is critical for Pdr16 function in modulation of sterol metabolism in response to these two types of antifungal drugs. A novel feature of Pdr16p, and especially of Pdr16p^{E235A, K267A} mutant, to bind sterol molecules, is observed.     

Regulation of Drosophila TRPC channels by protein and lipid interactions

The Drosophila TRPC channels TRP and TRPL are the founding members of the TRP superfamily of ion channels, proteins likely to be important components of calcium influx pathways. The activation of these channels in the context of fly phototransduction is one of the few in vivo models for TRPC channel activation and has served as a paradigm for understanding TRPC function. TRP and TRPL are activated by G-protein coupled PI(4,5)P₂ hydrolysis through a mechanism in which IP₃ receptor mediated calcium release seems dispensable. Recent analysis has provided compelling evidence that the accurate turnover of PI(4,5)P₂ generated lipid messengers in essential for regulating TRP and TRPL activity. TRP channels also appear to exist in the context of a macromolecular complex containing key components involved in activation such as phospholipase Cβ and protein kinase C. This complex may be important for activation. The role of these protein and lipid elements in regulating TRP and TRPL activity is discussed in this review.