Youngdae Yoon

Curvature sensing by the epsin N-terminal homology domain measured on cylindrical lipid membrane tethers.

The protein epsin is believed to play important roles in clathrin-mediated endocytosis, including generation of the high membrane curvature necessary for vesicle formation. Here we assess the basis for this hypothesis by systematically quantifying the curvature dependence of the area density of epsin N-terminal homology (ENTH) domain on cylindrical membranes of controlled curvature. In cylindrical tethers pulled from micropipet-aspirated giant unilamellar vesicles, repartitioning of membrane-bound ENTH from vesicles onto highly curved membranes was observed by fluorescence microscopy. First-order thermodynamic theory used to analyze our data yielded the first measurement of Leiblers thermodynamic curvature-composition coupling coefficient to be reported for an endocytic accessory protein. Our results highlight the possibility that epsin contributes to cellular membrane curvature sensing and generation, and we believe that our method will provide useful contributions toward the goal of relating molecular descriptions of interactions to macroscopic membrane remodeling in cells and identifying and characterizing roles for proteins in these processes.

In situ quantitative imaging of cellular lipids using molecular sensors.

Membrane lipids are dynamic molecules that play important roles in cell signaling and regulation but an in situ imaging method for quantitatively tracking lipids in living cells is lacking at present. Here we report a new chemical method of quantitative lipid imaging using sensors engineered by labeling proteins with an environmentally sensitive fluorophore. A prototype sensor for phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2)– a key signaling lipid in diverse cellular processes–was generated by covalently attaching a single 2-dimethylamino-6-acyl-naphthalene group to the N-terminal α-helix of the engineered epsin1 ENTH domain– a protein that selectively binds PtdIns(4,5)P2. The sensor allows robust and sensitive in situ quantitative imaging in mammalian cells, providing new insight into the spatiotemporal dynamics and fluctuation of this key signaling lipid. Application of the sensor to immune cells reveals the presence of a local threshold PtdIns(4,5)P2 concentration required for triggering phagocytosis. This sensor strategy is generally applicable to in situ quantification of other cellular lipids.

Molecular Basis of the Potent Membrane-remodeling Activity of the Epsin 1 N-terminal Homology Domain

The mechanisms by which cytosolic proteins reversibly bind the membrane and induce the curvature for membrane trafficking and remodeling remain elusive. The epsin N-terminal homology (ENTH) domain has potent vesicle tubulation activity despite a lack of intrinsic molecular curvature. EPR revealed that the N-terminal α-helix penetrates the phosphatidylinositol 4,5-bisphosphate-containing membrane at a unique oblique angle and concomitantly interacts closely with helices from neighboring molecules in an antiparallel orientation. The quantitative fluorescence microscopy showed that the formation of highly ordered ENTH domain complexes beyond a critical size is essential for its vesicle tubulation activity. The mutations that interfere with the formation of large ENTH domain complexes abrogated the vesicle tubulation activity. Furthermore, the same mutations in the intact epsin 1 abolished its endocytic activity in mammalian cells. Collectively, these results show that the ENTH domain facilitates the cellular membrane budding and fission by a novel mechanism that is distinct from that proposed for BAR domains.

Characterization of phospholipase C gamma enzymes with gain-of-function mutations.

Phospholipase Cγ isozymes (PLCγ1 and PLCγ2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLCγ2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLCγ1 and PLCγ2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLCγ2 without increasing Rac binding. Importantly, the activation of the ALI14-PLCγ2 and corresponding PLCγ1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state.

Vitamin E isoforms directly bind PKCα and differentially regulate activation of PKCα

Vitamin E isoforms have opposing regulatory effects on leucocyte recruitment during inflammation. Furthermore, in vitro, vitamin E isoforms have opposing effects on leucocyte migration across endothelial cells by regulating VCAM (vascular cell-adhesion molecule)-1 activation of endothelial cell PKCα (protein kinase Cα). However, it is not known whether tocopherols directly regulate cofactor-dependent or oxidative activation of PKCα. We report in the present paper that cofactor-dependent activation of recombinant PKCα was increased by γ-tocopherol and was inhibited by α-tocopherol. Oxidative activation of PKCα was inhibited by α-tocopherol at a 10-fold lower concentration than γ-tocopherol. In binding studies, NBD (7-nitrobenz-2-oxa-1,3-diazole)-tagged α-tocopherol directly bound to full-length PKCα or the PKCα-C1a domain, but not PKCζ. NBD-tagged α-tocopherol binding to PKCα or the PKCα-C1a domain was blocked by diacylglycerol, α-tocopherol, γ-tocopherol and retinol, but not by cholesterol or PS (phosphatidylserine). Tocopherols enhanced PKCα-C2 domain binding to PS-containing lipid vesicles. In contrast, the PKCα-C2 domain did not bind to lipid vesicles containing tocopherol without PS. The PKCα-C1b domain did not bind to vesicles containing tocopherol and PS. In summary, α-tocopherol and γ-tocopherol bind the diacylglycerol-binding site on PKCα-C1a and can enhance PKCα-C2 binding to PS-containing vesicles. Thus the tocopherols can function as agonists or antagonists for differential regulation of PKCα.

Phosphatidylinositol 4,5-Bisphosphate (PtdIns(4,5)P2) Specifically Induces Membrane Penetration and Deformation by Bin/Amphiphysin/Rvs (BAR) Domains

Background: The roles of anionic lipids in the actions of N-BAR domains are not fully understood. Results: PtdIns(4,5)P2 specifically induces membrane penetration and self-association of N-BAR domains. Conclusion: PtdIns(4,5)P2 is an important regulator of the membrane deforming activity of N-BAR domains. Significance: This study provides new insight into how PtdIns(4,5)P2 in the plasma membrane regulates the endocytic function of N-BAR domain proteins. Cellular proteins containing Bin/amphiphysin/Rvs (BAR) domains play a key role in clathrin-mediated endocytosis. Despite extensive structural and functional studies of BAR domains, it is still unknown how exactly these domains interact with the plasma membrane containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and whether they function by a universal mechanism or by different mechanisms. Here we report that PtdIns(4,5)P2 specifically induces partial membrane penetration of the N-terminal amphiphilic α-helix (H0) of two representative N-BAR domains from Drosophila amphiphysin (dAmp-BAR) and rat endophilin A1 (EndoA1-BAR). Our quantitative fluorescence imaging analysis shows that PtdIns(4,5)P2-dependent membrane penetration of H0 is important for self-association of membrane-bound dAmp-BAR and EndoA1-BAR and their membrane deformation activity. EndoA1-BAR behaves differently from dAmp-BAR because the former has an additional amphiphilic α-helix that penetrates the membrane in a PtdIns(4,5)P2-independent manner. Depletion of PtdIns(4,5)P2 from the plasma membrane of HEK293 cells abrogated the membrane deforming activity of EndoA1-BAR and dAmp-BAR. Collectively, these studies suggest that the local PtdIns(4,5)P2 concentration in the plasma membrane may regulate the membrane interaction and deformation by N-BAR domain-containing proteins during clathrin-mediated endocytosis.

SH2 Domains Serve as Lipid-Binding Modules for pTyr-Signaling Proteins

The Src-homology 2 (SH2) domain is a protein interaction domain that directs myriad phosphotyrosine (pY)-signaling pathways. Genome-wide screening of human SH2 domains reveals that ∼90% of SH2 domains bind plasma membrane lipids and many have high phosphoinositide specificity. They bind lipids using surface cationic patches separate from pY-binding pockets, thus binding lipids and the pY motif independently. The patches form grooves for specific lipid headgroup recognition or flat surfaces for non-specific membrane binding and both types of interaction are important for cellular function and regulation of SH2 domain-containing proteins. Cellular studies with ZAP70 showed that multiple lipids bind its C-terminal SH2 domain in a spatiotemporally specific manner and thereby exert exquisite spatiotemporal control over its protein binding and signaling activities in T cells. Collectively, this study reveals how lipids control SH2 domain-mediated cellular protein-protein interaction networks and suggest a new strategy for therapeutic modulation of pY-signaling pathways.

Pb2+ as modulator of protein-membrane interactions

Lead is a potent environmental toxin that mimics the effects of divalent metal ions, such as zinc and calcium, in the context of specific molecular targets and signaling processes. The molecular mechanism of lead toxicity remains poorly understood. The objective of this work was to characterize the effect of Pb(2+) on the structure and membrane-binding properties of C2α. C2α is a peripheral membrane-binding domain of Protein Kinase Cα (PKCα), which is a well-documented molecular target of lead. Using NMR and isothermal titration calorimetry (ITC) techniques, we established that C2α binds Pb(2+) with higher affinity than its natural cofactor, Ca(2+). To gain insight into the coordination geometry of protein-bound Pb(2+), we determined the crystal structures of apo and Pb(2+)-bound C2α at 1.9 and 1.5 Å resolution, respectively. A comparison of these structures revealed that the metal-binding site is not preorganized and that rotation of the oxygen-donating side chains is required for the metal coordination to occur. Remarkably, we found that holodirected and hemidirected coordination geometries for the two Pb(2+) ions coexist within a single protein molecule. Using protein-to-membrane Förster resonance energy transfer (FRET) spectroscopy, we demonstrated that Pb(2+) displaces Ca(2+) from C2α in the presence of lipid membranes through the high-affinity interaction with the membrane-unbound C2α. In addition, Pb(2+) associates with phosphatidylserine-containing membranes and thereby competes with C2α for the membrane-binding sites. This process can contribute to the inhibitory effect of Pb(2+) on the PKCα activity.

Simultaneous in situ quantification of two cellular lipid pools using orthogonal fluorescent sensors

Lipids regulate a wide range of biological activities. Since their local concentrations are tightly controlled in a spatiotemporally specific manner, the simultaneous quantification of multiple lipids is essential for elucidation of the complex mechanisms of biological regulation. Here, we report a new method for the simultaneous in situ quantification of two lipid pools in mammalian cells using orthogonal fluorescent sensors. The sensors were prepared by incorporating two environmentally sensitive fluorophores with minimal spectral overlap separately into engineered lipid-binding proteins. Dual ratiometric analysis of imaging data allowed accurate, spatiotemporally resolved quantification of two different lipids on the same leaflet of the plasma membrane or a single lipid on two opposite leaflets of the plasma membrane of live mammalian cells. This new imaging technology should serve as a powerful tool for systems-level investigation of lipid-mediated cell signaling and regulation.

Phytotoxicity of arsenic compounds on crop plant seedlings

The effects of inorganic and organic arsenic on the germination and seedling growth of 10 crop plants were investigated to elucidate the relationship between toxicity and the arsenic chemical states. Two types of soils, soil A and B, were also tested to determine how physicochemical properties of soils were related to toxicity of arsenic and the sensitivity of the plants. All tested plant species, except mung bean and cucumber, showed inhibition of germination by two types of inorganic arsenic, arsenite, and arsenate, while the organic arsenic compound, dimethylarsinic acid (DMA), had no inhibitory effects on plants in soil A. In contrast, the growth of seedlings of all 10 plant species was sensitive to the presence of arsenic. The sensitivity of the plants toward inorganic arsenic compounds showed similar trends but differed for DMA. Overall, seedling growth was a more sensitive endpoint to arsenic toxicity than germination, and the relative toxicity of arsenic compounds on plants was arsenite > DMA > arsenate. Interestingly, the sensitivity of wheat varied significantly when the soil was changed, and the DMA was most toxic rather than arsenite in soil B. Thus, the systematic study employed here provides insights into the mechanisms of arsenic toxicity in different plant species and the role of physicochemical properties of soils.