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.

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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 Pb2+ 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 Pb2+ with higher affinity than its natural cofactor, Ca2+. To gain insight into the coordination geometry of protein-bound Pb2+, we determined the crystal structures of apo and Pb2+-bound C2α at 1.9 Å and 1.5 Å resolution, respectively. A comparison of these structures revealed that the metal-binding site is not pre-organized and that rotation of the oxygen-donating sidechains is required for the metal coordination to occur. Remarkably, we found that holodirected and hemidirected coordination geometries for the two Pb2+ ions coexist within a single protein molecule. Using protein-to-membrane Förster resonance energy transfer (FRET) spectroscopy, we demonstrated that Pb2+ displaces Ca2+ from C2α in the presence of lipid membranes through the high-affinity interaction with the membrane-unbound C2α. In addition, Pb2+ associates with phosphatidylserine-containing membranes and thereby competes with C2α for the membrane-binding sites. This process can contribute to the inhibitory effect of Pb2+ on the PKCα activity.

SUMMARY 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 is 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, these studies reveal how lipids control SH2 domain-mediated cellular protein-protein interaction networks and suggest a new strategy for therapeutic modulation of pY signaling pathways.

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 ␣-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.Cell membranes undergo dynamic structural changes and remodeling during movement, division, and vesicle trafficking (1, 2). In particular, vesicle budding and fusion constantly take place in various cell membranes to maintain communication and transport between membrane-bound compartments (3). Dynamic membrane remodeling involves changes in local membrane curvature (or deformation) that are orchestrated by membrane lipids, integral membrane proteins, and cytoskeletal proteins (4, 5). Recently, several groups of cytosolic proteins that reversibly bind membranes and induce and/or detect different types of membrane curvatures during membrane remodeling have been identified. In particular, cytosolic proteins that are involved in different stages of clathrin-mediated endocytosis have received the most attention (6, 7), and many of them contain either an Ap180 N-terminal homology (ANTH) 2 /epsin N-terminal homology (ENTH) (8, 9) or a Bin-amphiphysin-Rvs (BAR) domain (9 -12). Although ENTH (13) and BAR domains (14 -17) have been reported to have in vitro vesicle-tubulating activities, the exact mechanisms by which these domains induce membrane deformation and larger scale membrane remodeling, especially under physiological conditions, are yet to be elucidated. For BAR domains, their unique intrinsic molecular curvatures have been postulated to be important for membrane deformation through a scaffolding mechanism (4). Also, recent studies have shown that F-BAR domains from FBP17 and CIP4 form highly ordered self-assembly in two-dimensional (18) and three-dimensional (19) crystals and that disruption of intermolecular interactions abrogates their membrane deformation activities. Despite remarkable success in structural characterization of various BAR and ENTH domains, questions still remain as to whether individual domains function by a universal mechanism or by different mechanisms, whether the intact proteins harborin...

-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLC␥2 without increasing Rac binding. Importantly, the activation of the ALI14-PLC␥2 and corresponding PLC␥1 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 have opposing regulatory effects on leukocyte recruitment during inflammation. Furthermore, in vitro, vitamin E isoforms have opposing effects on leukocyte migration across endothelial cells by regulating vascular cell adhesion molecule (VCAM)-1 activation of endothelial cell protein kinase Cα (PKCα). However, it is not known whether tocopherols directly regulate co-factor-dependent or oxidative activation of PKCα. We report herein that co-factor-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-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 phosphatidylserine (PS). 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.

During cytokinesis in plants, -Golgi network-derived vesicles accumulate at the center of dividing cells and undergo various structural changes to give rise to the planar cell plate. However, how this conversion occurs at the molecular level remains elusive. In this study, we report that SH3 Domain-Containing Protein 2 (SH3P2) in plays a crucial role in converting vesicles to the planar cell plate. RNAi plants showed cytokinesis-defective phenotypes and produced aggregations of vesicles at the leading edge of the cell plate. SH3P2 localized to the leading edge of the cell plate, particularly the constricted or curved regions of the cell plate. The BAR domain of SH3P2 induced tubulation of vesicles. SH3P2 formed a complex with dynamin-related protein 1A (DRP1A) and affected DRP1A accumulation to the cell plate. Based on these results, we propose that SH3P2 functions together with DRP1A to convert the fused vesicles to tubular structures during cytokinesis.