Profile of Human Milk Phospholipids at Different Lactation Stages with UPLC/Q-TOF-MS: Characterization, Distribution, and Differences.

Human milk phospholipids are important for the regular growth and development of infants. Ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) was employed to qualitatively and quantitatively analyze 277 phospholipid molecular species in 112 human milk samples to obtain a detailed profile of human milk phospholipids along the lactation stage. MS/MS fragmentation patterns of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine were characterized in detail. Phosphatidylcholine is the most dominant group, followed by sphingomyelin. PC(18:0/18:2), SM(d18:1/24:1), PE(18:0/18:0), PS(18:0/20:4), and PI(18:0/18:2) showed the highest average concentration among all of the phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol molecular species, respectively. The fatty acids attached to the phospholipid molecules were mainly palmitic, stearic, oleic, and linoleic acids, and the plasmalogens decreased along the lactation stage. The increase of sphingomyelins and phosphatidylethanolamines and the decrease of phosphatidylcholines are the key changes from colostrum to transitional milk; the increase of lysophosphatidylcholines and lysophosphatidylethanolamines and the continuous decrease of phosphatidylcholines are the vital changes from transitional milk to mature milk.

Phosphatidylinositol 4,5-Bisphosphate Sensing Lipid Raft via Inter-Leaflet Coupling Regulated by Acyl Chain Length of Sphingomyelin.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is an important molecule located at the inner leaflet of cell membrane, where it serves as anchoring sites for a cohort of membrane-associated molecules and as a broad-reaching signaling intermediate. The lipid raft is thought as the major platform recruiting proteins for signal transduction and also known to mediate PIP2 accumulation across the membrane. While the significance of this cross-membrane coupling is increasingly appreciated, it remains unclear whether and how PIP2 senses the dynamic change of the ordered lipid domains over the packed hydrophobic core of the bilayer. Herein, by means of molecular dynamic simulation, we reveal that inner PIP2 molecules can sense the outer lipid domain via inter-leaflet coupling, and the coupling manner is dictated by the acyl chain length of sphingomyelin (SM) partitioned to the lipid domain. Shorter SM promotes membrane domain registration, whereby PIP2 accumulates beneath the domain across the membrane. In contrast, the anti-registration is thermodynamically preferred if the lipid domain has longer SM due to the hydrophobic mismatch between the corresponding acyl chains in SM and PIP2. In this case, PIP2 is expelled by the domain with a higher diffusivity. These results provide molecular insights into the regulatory mechanism of correlation between the outer lipid domain and inner PIP2, both of which are critical components for cell signal transduction.

Cofilin-Membrane Interactions: Electrostatic Effects in Phosphoinositide Lipid Binding.

The actin cytoskeleton interacts with the cell membrane primarily through the indirect interactions of actin-binding proteins such as cofilin-1. The molecular mechanisms underlying the specific interactions of cofilin-1 with membrane lipids are still unclear. Here, we performed coarse-grain molecular dynamics simulations of cofilin-1 with complex lipid bilayers to analyze the specificity of protein-lipid interactions. We observed the maximal interactions with phosphoinositide (PIP) lipids, especially PIP2 and PIP3 lipids. A good match was observed between the residues predicted to interact and previous experimental studies. The clustering of PIP lipids around the membrane bound protein leads to an overall lipid demixing and gives rise to persistent membrane curvature. Further, through a series of control simulations, we observe that both electrostatics and geometry are critical for specificity of lipid binding. Our current study is a step towards understanding the physico-chemical basis of cofilin-PIP lipid interactions.

Gordonia zhenghanii sp. nov. and Gordonia liuliyuniae sp. nov., isolated from bat faeces.

Four mesophilic actinobacteria (HY002T, HY442, HY366T and HY285) isolated from the faeces of bats collected in southern China were found to be strictly aerobic, non-motile, rod-shaped, oxidase-negative, Gram-stain-positive and catalase-positive. Strains HY002T and HY366T contained meso-diaminopimelic acid as the diagnostic diamino acid and MK-9(H2) the sole respiratory quinone. Arabinose, galactose and ribose were detected in the whole-cell hydrolysates of both type strains. The main cellular fatty acids (> 10.0%) of all strains were C16 : 0, C18 : 1 ω9c, 10-methyl-C18 : 0 and summed feature 3. Strains HY002T and HY366T contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidyl inositol mannosides as the major polar lipids. The phylogenetic/phylogenomic analyses based on 16S rRNA gene and genomic sequence comparison revealed that the four strains belong to the genus Gordonia, most closely related to G. neofelifaecis NRRL B-59395T(98.2-98.3% sequence similarity) on the EzBioCloud database. The G+C contents of strains HY002T and HY366T based on genomic DNA were 66.5 and 66.9%, respectively. The DNA-DNA relatedness values between the two types strains and members of the genus Gordonia were far below 70 % (18.6-23.1 %). All genotypic and phenotypic data indicated that the four strains are representatives of two novel separate species, for which the names Gordonia zhenghanii sp. nov. and Gordonia liuliyuniae sp. nov. are proposed, with HY002T (=CGMCC 4 7757T=JCM 34 878T) and HY366T (=CGMCC 1 19146T=JCM 34 879T) as the respective type strains.

Streptomyces solincola sp. nov., isolated from soil in Malaysia.

A urease-producing Gram-stain-positive actinobacterium, designated strain T5T, was isolated from a soil sample collected at a highway hillslope in Selangor, Malaysia. The strain was found to produce pale yellowish-pink aerial mycelia with smooth long chain spores and extensively branched light yellowish-pink substrate mycelia on oatmeal agar. Strain T5T grew at 15-37 °C, pH 6-11, and tolerated up to 9 % (w/v) NaCl, with optimal growth occurring at 28 °C, pH 6-9 and without NaCl. The whole-cell sugar hydrolysate of strain T5T contained galactose, glucose and ribose. The ll-diaminopimelic acid isomer was detected in the cell wall. Diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol were found to be the predominant polar lipids. The main fatty acids were anteiso-C17 : 0, iso-C16 : 0, anteiso-C15 : 0 and iso-C14 : 0. Comparative analysis of the 16S rRNA gene sequences indicated that strain T5T belonged to Streptomyces of the family Streptomycetaceae with the highest 16S rRNA gene sequence similarity to Streptomyces lichenis LCR6-01T (99.0 %). The overall genome relatedness indices revealed that the closest related species was S. lichenis LCR6-01T with 89.4 % average nucleotide identity and 33.7 % digital DNA-DNA hybridization. Phylogeny analyses showed that strain T5T was closely related to Streptomyces fradiae, Streptomyces lavendofoliae, Streptomyces lichenis, Streptomyces roseolilacinus and Streptomyces somaliensis. Based on these polyphasic data, strain T5T represents a novel species, for which the name Streptomyces solincola sp. nov. is proposed. The type strain is T5T (=TBRC 5137T= DSM 42166T).

Occultella gossypii sp. nov., an alkali-resistant isolate from soil sampled in a cotton field.

A non-spore-forming, motile and alkali-resistant actinobacterium, designated N2-46T, was isolated from an alkaline soil sample collected from a cotton field in the Xinjiang region of PR China. Strain N2-46T formed creamy colonies on tryptone soy agar and managed to survive in extreme alkaline conditions at a pH value of 11. Strain N2-46T displayed the highest 16S rRNA gene similarity of 99.65 % to Haloactinobacterium kanbiaonis HY164T, followed by Occultella aeris F300T (99.61%) and Occultella glacieicola T3246-1T (98.54 %). 16S rRNA-directed phylogenetic analysis showed that strain N2-46T was embedded in a subclade with O. aeris F300T with a bootstrap value of 71.8 %. The phylogenetic tree based on core genes of genome sequences showed that strain N2-46T formed a unique subclade next to H. kanbiaonis HY164T and O. aeris F300T with a bootstrap value of 100 %. Digital DNA-DNA hybridization and the average nucleotide identity analyses showed that strain N2-46T displayed the highest values of 67.1 % (63.2-70.7 %) and 91.82 % with H. kanbiaonis HY164T, respectively. Comparative genomic analysis indicated that strain N2-46T and its three closest neighbours exhibited comparable distribution patterns in heavy metal resistance genes and biosynthetic gene clusters, while displaying distinctions probably related to ecological adaptation. MK-8(H4) was identified as the predominant isoprenoid quinone. The main fatty acids were identified as iso-C14 : 0 and anteiso-C15 : 0. Polar lipids are composed of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, mono and diacylated phosphatidylinositol dimannosides, as well as several uncharacterized polar lipid, glycolipid, and phospholipids. Genotypic and physiological analyses support the view that strain N2-46T (=JCM 34413T=CGMCC 1.18819T) should be classified as a novel species of the genus Occultella, for which the name Occultella gossypii sp. nov. is proposed.

Streptomyces barringtoniae sp. nov., isolated from rhizosphere of plant with antioxidative potential.

A novel actinomycete strain, JA03T, belonging to the genus Streptomyces, was isolated from the rhizosphere of Barringtonia racemosa (L.) Spreng. It was characterized taxonomically using a polyphasic approach. It grew at 25-37 °C, at pH 5-10 and with 6 % (w/v) NaCl. It contained ll-diaminopimelic acid in the cell-wall peptidoglycan. Ribose and glucose were detected in its whole-cell hydrolysate. The predominant cellular fatty acids were iso-C16 : 0, anteiso-C15 : 0, C16 : 0, iso-C14 : 0 and iso-C15 : 0. Detected polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides, unidentified phospholipids and unidentified amino lipids. Based on the results of 16S rRNA gene sequence analyses, strain JA03T showed highest similarity to Streptomyces filipinensis NBRC 12860T (98.76 %), Streptomyces fodineus TW1S1T (98.69 %) and Streptomyces shenzhennensis 172115T (98.68 %). Strain JA03T has a genome size of 9 092 851 bp with DNA G+C content of 71.28 mol%. The average nucleotide identity (ANI)-blast and ANI-MUMmer values of strain JA03T and related type strains were 79.6-89.2 and 86.7-92.5 %, respectively, and the digital DNA-DNA hybridization values were 27.3-46.4 %. Ethyl acetate extract of JA03T exhibited total phenolic content (33.4±0.6 µg mg-1 gallic acid equivalent), ferric reducing power value (70.8±1.8 µg mg-1 ascorbic acid equivalent) and 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity (IC50=67.0±21.1 µg ml-1). Intracellular reactive oxygen species and NO production in RAW264.7 macrophage cells induced by H2O2 and lipopolysaccharide were inhibited with IC50 of 67.40 and 16.95 µg ml-1, respectively. Based on the taxonomic results, it has been concluded that strain JA03T represents a novel species of the genus Streptomyces for which the name Streptomyces barringtoniae sp. nov., is proposed. The type strain is JA03T (=LMG 32415T=TISTR 2999T).

Actinotalea soli sp. nov., isolated from Kubuqi Desert soil.

A Gram-stain positive, facultatively anaerobic, motile rod-shaped strain, BY-33T, was isolated from a soil sample obtained from the Kubuqi Desert, PR China. Phylogenetic analysis based on 16S rRNA gene sequence similarity revealed that strain BY-33T was most closely related to the genus Actinotalea, including Actinotalea ferrariae CF5-4T (98.2 % similarity), 'Actinotalea subterranea' HO-Ch2T (98.0 %), Actinotalea solisilvae THG-T121T (97.6 %), 'Actinotalea bogoriensis' 69B4T (97.5 %), Actinotalea fermentans MT (97.3 %) and 'Actinotalea carbonis' T26T (97.0 %). The strain grew at 0‒37 °C (optimum, 28-30 °C) and pH 6.0-11.0 (optimum, pH 9.0-10.0) and with 0‒8.0 % (w/v) NaCl (optimum, 3.0%) on tryptic soy agar. It had catalase activity, but no oxidase activity. The polar lipids of strain BY-33T contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, and phosphatidylinositol mannosides. The major respiratory quinone of strain BY-33T was MK-10 (H4). Its major fatty acids were anteiso-C15 : 0, anteiso-C15 : 1 A and C16 : 0. The genomic DNA G+C content of strain BY-33T was 73.0 mol% based on total genome calculations. The average nucleotide identity scores between the genomic sequences of strain BY-33T and the other species of the genus Actinotalea were found to be low (ANIm <85.0 %, ANIb <77.0 % and OrthoANIu <78.0 %). Furthermore, the digital DNA-DNA hybridization and average amino acid identity values between strain BY-33T and the closely related species ranged from 20.5 to 21.0% and from 62.2 to 72.2 %, respectively. Based on the results of phylogenetic, phenotypic, genotypic and chemotaxonomic analyses, it is concluded that strain BY-33T represents a novel species within the genus Actinotalea, for which the name Actinotalea soli sp. nov. is proposed. The type strain is BY-33T (=CGMCC 1.17460T=KCTC 49362T).

Isomer Selective Comprehensive Lipidomics Analysis of Phosphoinositides in Biological Samples by Liquid Chromatography with Data Independent Acquisition Tandem Mass Spectrometry.

Phosphoinositides (PIPx) play central roles in membrane dynamics and signal transduction of key functions like cellular growth, proliferation, differentiation, migration, and adhesion. They are highly regulated through a network of distinct phosphatidylinositol phosphates consisting of seven groups and three regioisomers in two groups (PIP and PIP2), which arise from phosphorylation at 3', 4', and 5' positions of the inositol ring. Numerous studies have revealed the importance of both fatty acyl chains, degree of phosphorylation, and phosphorylation positions under physiological and pathological states. However, a comprehensive analytical method that allows differentiation of all regioisomeric forms with different acyl side chains and degrees of phosphorylation is still lacking. Here, we present an integrated comprehensive workflow of PIPx analysis utilizing a chiral polysaccharide stationary phase coupled with electrospray ionization high-resolution mass spectrometry with a data independent acquisition technique using the SWATH technology. Correspondingly, a targeted data mining strategy in the untargeted comprehensively acquired MS and MS/MS data was developed. This powerful highly selective method gives a full picture of PIPx profiles in biological samples. Herein, we present for the first time the full PIPx profiles of NIST SRM1950 plasma, Pichia pastoris lipid extract, and HeLa cell extract, including profile changes upon treatment with potential PI3K inhibitor wortmannin. We also illustrate using this inhibitor that measurements of the PIPx profile averaged over the distinct regioisomers by analytical procedures, which cannot differentiate between the individual PIPx isomers, can easily lead to biased conclusions.

Spatial Analysis of Phosphatidylinositol Molecular Species in Pork Chop Tissues Using Matrix-assisted Laser Desorption/ionization-Mass Spectrometry Imaging.

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a powerful technique for visualizing lipids in biological tissues. Phosphatidylinositol (PI), a phospholipid in pork, is a major source of inositol in animal-derived foods believed to be protective against diseases related to pregnancy and cancer. However, the distribution of PI molecular species in pork is not well understood. Here, we performed MALDI-MSI analysis to investigate the distribution and composition of PI molecular species in pork chop comprising Longissimus thoracis et lumborum muscle (loin), intermuscular fat tissue, transparent tissue, and spinalis muscle. Twelve diacyl-PI molecular species were identified using liquid chromatography-electrospray ionization-tandem mass spectrometry (MS/MS) and MALDI-MS/MS analysis and visualized using MALDI-MSI. Spinalis muscle had the highest amount of identified PI molecular species, followed by loin, transparent tissue, and intermuscular fat tissue. The diacyl-PI molecular species containing hexadecadienoic, oleic, linoleic and eicosadienoic acids at the sn-2 position were mainly abundant in the loin and spinalis muscle, whereas those containing mead, arachidonic, docosatetraenoic, and docosapentaenoic acids at the sn-2 position were mainly abundant in both muscles as well as transparent tissues. Notably, the balance of PI molecular species differed among the tissues depending on fatty acid compositions at the sn-2 position. These results suggested that MALDI-MSI is a promising tool for assessing the association between individual pork tissues and the protective effects of PI molecular species against diseases related to pregnancy and cancer. To the best of our knowledge, this is the first report showing tissue-specific distributions of PI molecular species in pork chop using MALDI-MSI.

Analysis of Phosphoinositides from Complex Plant Samples by Solid-Phase Adsorption Chromatography and Subsequent Quantification via Thin-Layer and Gas Chromatography.

The determination of phosphoinositide molecular species in plant material is challenging because of their low abundance concurrent with a very high abundance of other membrane lipids, such as plastidial glycolipids. Phosphoinositides harbor an inositol headgroup which carries one or more phosphate groups at different positions of the inositol, linked to diacylglycerol via a phosphodiester. Thus, a further analytical challenge is to distinguish the different inositol-phosphate headgroups as well as the fatty acids of the diacylglycerol backbone. The method presented in this chapter expands on previous protocols for phosphoinositide analysis by employing chromatographic enrichment of phospholipids and their separation from other, more abundant lipid classes, before analysis. Lipids extracted from plant material are first separated by solid-phase adsorption chromatography into fractions containing neutral lipids, glycolipids, or phospholipids. Lipids from the phospholipid fraction are then separated by thin-layer chromatography (TLC) according to their characteristic head groups, and the individual phosphatidylinositol-monophosphates and phosphatidylinositol-bisphosphates are isolated. Finally, the fatty acids associated with each isolated phosphatidylinositol-monophosphate or phosphatidylinositol-bisphosphate are analyzed in a quantitative fashion using gas chromatography (GC). The analysis of phosphoinositides by this combination of methods provides a cost-efficient and reliable alternative to lipidomics approaches requiring more extensive instrumentation.

Enhancing Coverage of Phosphatidylinositol Species in Canola Through Specialised Liquid Chromatography-Mass Spectrometry Buffer Conditions.

Phosphatidylinositols (PIs) constitute a minor class of phospholipid with wide-spread influence throughout various cellular functions. Monitoring the distribution of these lipids can therefore provide insight as to the state of cellular processes or reveal the development of various pathologies. The speciation of these compounds is often performed either as part of a comprehensive characterisation of lipids, or specifically targeted using the same methods, however, such methods were intended to maximise coverage of lipid classes rather than provide an in-depth analysis of any single class. In the particular case of PIs, the majority of reported molecular diversity is limited to a small proportion of the already minor class, as such the cursory glance enabled by such methods is insufficient. Therefore, this work compared the suitability of both established and novel LC-MS buffers with the aim of maximising the ionisation efficiency of PIs, in an attempt to enhance coverage of the class. Through experimentation, it was determined that a 0.25 mM ammonium fluoride buffer provided up to a 6-fold increase in signal intensity, and on average a 38-fold increase in the signal-to-noise ratio. Using these new conditions, 14 PI species, and 12 PI candidates were identified within a dilute lipid extract sourced from canola seed, compared to 0 species identified using the generalised method. As a result, it is suggested that this procedure has yielded the highest number of PI species identifications for a sample of this concentration. Methods which therefore intend to characterise PI species in dilute quantities, such as those extracted from mammalian cells, are henceforth provided with the means to conduct more comprehensive characterisations.

Simultaneous Detection of Phosphoinositide Lipids by Radioactive Metabolic Labeling.

Phosphoinositide (PPI) lipids are a crucial class of low-abundance signaling molecules that regulate many processes within cells. Methods that enable simultaneous detection of all PPI lipid species provide a wholistic snapshot of the PPI profile of cells, which is critical for probing PPI biology. Here we describe a method for the simultaneous measurement of cellular PPI levels by metabolically labeling yeast or mammalian cells with myo-3H-inositol, extracting radiolabeled glycerophosphoinositides, and separating lipid species on an anion exchange column via HPLC.

Label-Free Quantification of Phosphoinositides in Drosophila by Mass Spectrometry.

Phosphoinositides (PIs), the seven phosphorylated derivatives of phosphatidylinositol, are recognized as key molecules in the control of multiple molecular events in eukaryotic cells. Within cells, PIs are low-abundance lipids making their detection and quantification challenging. While many methods that allow radiolabeling and quantification of PIs in the context of cultured cells are available, these are not useful in the context of in vivo animal models where cell and developmental processes are best studied. In this chapter, we describe radionuclide-free, mass spectrometry-based methods for the detection and quantification of PIs from Drosophila tissues in vivo. The use of these methods should facilitate the discovery of novel modes by which PIs regulate cellular and developmental processes in complex metazoans.

Profiling of Phosphoinositide Molecular Species in Resting or Activated Human or Mouse Platelets by a LC-MS Method.

Our knowledge of the role and biology of the different phosphoinositides has greatly expanded over recent years. Reversible phosphorylation by specific kinases and phosphatases of positions 3, 4, and 5 on the inositol ring is a highly dynamic process playing a critical role in the regulation of the spatiotemporal recruitment and binding of effector proteins. The specific phosphoinositide kinases and phosphatases are key players in the control of many cellular functions, including proliferation, survival, intracellular trafficking, or cytoskeleton reorganization. Several of these enzymes are mutated in human diseases. The impact of the fatty acid composition of phosphoinositides in their function is much less understood. There is an important molecular diversity in the fatty acid side chains of PI. While stearic and arachidonic fatty acids are the major acyl species in PIP, PIP2, and PIP3, other fatty acid combinations are also found. The role of these different molecular species is still unknown, but it is important to quantify these different molecules and their potential changes during cell stimulation to better characterize this emerging field. Here, we describe a sensitive high-performance liquid chromatography-mass spectrometry method that we used for the first time to profile the changes in phosphoinositide molecular species (summed fatty acyl chain profiles) in human and mouse platelets under resting conditions and following stimulation. This method can be applied to other hematopoietic primary cells isolated from human or experimental animal models.

Detection of Plasma Membrane Phosphoinositide Dynamics Using Genetically Encoded Fluorescent Protein Probes.

The dynamic phosphorylation of phosphatidylinositol produces seven distinct but interconvertible phosphatidylinositol phosphates (PIPs). Each PIP exhibits specific enrichment in a subset of membrane compartments as a result of dynamic phosphorylation and dephosphorylation by lipid kinases and phosphatases, and/or by vesicle-mediated transport. Several PIPs are found within the plasma membrane, such as phosphatidylinositol-4-phosphate [PI(4)P], phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2], and phosphatidylinositol-3,4,5-trisphosphate (PIP3), and these control many aspects of cell physiology, including receptor signaling and membrane traffic. As a result, measurement of the cell surface abundance of these PIPs is a valuable resource to allow understanding of the regulation and function of these cell surface lipids. Here, we describe methods based on quantification of the localization of genetically encoded fluorescent PIP probes to the cell surface by either spinning disc confocal microscopy or total internal reflection fluorescence microscopy that allow detection of changes in cell surface levels of PI(4,5)P2, PI(3,4)P2, and PIP3. These methods can also be applied to the measurement of other PIPs or lipid species at the cell surface, and thus represent a useful resource for the study of the cell biology of PIPs.

Quantification of Genetically Encoded Lipid Biosensors.

Lipids, like phosphoinositides, can be visualized in living cells in real time using genetically encoded biosensors and fluorescence microscopy. Sensor localization can be quantified by determining the fluorescence intensity of each fluorophore. Enrichment of lipids at membranes can be determined by generating and applying an organelle-specific binary mask. In this chapter, we provide a detailed list of reagents and methods to visualize and quantify relative lipid levels. Applying this approach, changes in lipid levels can be assessed in cases when lipid metabolizing enzymes are mutated or otherwise altered.

Imaging the Nanoscale Distribution of Phosphoinositides in the Cell Plasma Membrane with Single-Molecule Localization Super-Resolution Microscopy.

Phosphoinositides make up only a small fraction of cellular phospholipids yet control cell function in a fundamental manner. Through protein interactions, phosphoinositides define cellular organelle identity and regulate protein function and organization and recruitment at the cytosol-membrane interface. As a result, perturbations on phosphoinositide metabolism alter cell physiology and lead to a wide range of human diseases, including cancer and diabetes. Among seven phosphoinositide members, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2, also known as PI(4,5)P2 or PIP2) is abundant in the plasma membrane. Besides its role in the second messenger pathway of phospholipase C that cleaves PtdIns(4,5)P2 to form diacylglycerol and inositol-1,4,5-trisphosphate (IP3), PtdIns(4,5)P2 regulates membrane trafficking and the function of the cytoskeleton, ion channels, and transporters. The nanoscale organization of PtdIns(4,5)P2 in the plasma membrane becomes essential to understand cellular signaling specificity in time and space. Here, we describe a single-molecule method to visualize the nanoscale distribution of PtdIns(4,5)P2 in the plasma membrane by using super-resolution microscopy and the dual-color fluorescent probes based on the PLCδ1 pleckstrin homology (PH) domain. This approach can be extended to image other phosphoinositides by changing the specific probes.

A Real-Time Phosphatidylinositol 4-Phosphate 5-Kinase Assay Using Fluorescence Spectroscopy.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is an enzyme that converts phosphatidylinositol 4-phosphate [PI4P] to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. PIP5K plays a key role in the regulation of vesicular transport, cytoskeleton reorganization, and cell division. In general, to investigate an enzymatic activity of PIP5K, the amount of incorporated [P32] ATP into PI(4,5)P2 fraction is measured in in vitro reconstitution experiments. However, tools to monitor dynamic changes in its activity in real time have been lacking. Recently, we have developed a novel PIP5K assay using fluorescence spectroscopy. Compared to conventional methods in which lipids extraction steps are needed, our method is easy and quick to perform and enables a real-time analysis. This chapter provides a protocol to set up and perform the novel PIP5K assay we have recently established.

Exploring Phosphoinositide Binding Using Native Mass Spectrometry.

Phosphoinositides interact with proteins to fulfill various functions in the cell. In many cases, they specifically recruit peripheral membrane proteins to biological membranes. The analysis of their interactions with proteins is therefore essential for understanding the underlying processes. Native mass spectrometry (MS) preserves noncovalent interactions in the gas phase of a mass spectrometer and is therefore well-suited to study protein-phosphoinositide interactions. In this protocol, we describe the application of native MS to integral and peripheral membrane proteins and their interactions with lipids. We discuss sample and instrumental requirements, the realization of experiments, and the data analysis workflow. We further describe a biochemical assay to proof interactions of peripheral membrane proteins with lipids.