==== Front Mol Biol CellMol. Biol. CellmolbiolcellmbcmbocMolecular Biology of the Cell1059-15241939-4586The American Society for Cell Biology 29953345E17-12-073810.1091/mbc.E17-12-0738MBoC Technical PerspectivesGenetically encoded lipid biosensors Wills Rachel C. Goulden Brady D. Hammond Gerald R. V. *Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 16261Kozminski Keith G. Monitoring EditorUniversity of Virginia*Address correspondence to: Gerald R. V. Hammond (ghammond@pitt.edu).01 7 2018 29 13 1526 1532 23 3 2018 19 4 2018 24 4 2018 © 2018 Wills et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.2018This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License.Lipids convey both structural and functional properties to eukaryotic membranes. Understanding the basic lipid composition and the dynamics of these important molecules, in the context of cellular membranes, can shed light on signaling, metabolism, trafficking, and even membrane identity. The development of genetically encoded lipid biosensors has allowed for the visualization of specific lipids inside individual, living cells. However, a number of caveats and considerations have emerged with the overexpression of these biosensors. In this Technical Perspective, we provide a current list of available genetically encoded lipid biosensors, together with criteria that determine their veracity. We also provide some suggestions for the optimal utilization of these biosensors when both designing experiments and interpreting results. ==== Body INTRODUCTION Lipids are central molecules in cell biology. They are the building blocks for membranes, which give structure to eukaryotic cells. Yet the diversity of lipid species is paramount, too, since different lipids convey different physical and thus functional properties on membranes. Membrane fluidity, curvature, peripheral protein recruitment, as well as membrane protein regulation are all controlled by the different lipids that form a membrane (van Meer and de Kroon, 2011). It follows that understanding the intricate molecular choreography of cells requires a detailed understanding of the dynamics and disposition of lipids in addition to the more familiar proteins. Lipids present unique challenges for developing tools to study them. When compared with proteins (the biomolecules most familiar to a typical cell biologist), there are some parallels: Like proteins, antibodies have been developed to probe lipids, forming the basis of approaches akin to Western blot and immunofluorescence. The chemistry of lipid extraction for blotting is very different, and blotting is now largely being superseded by mass spectrometry approaches (Nguyen et al., 2017). Immunofluorescence of lipids is possible, though it is even more tricky and artifact prone than it is for proteins, since lipids fix poorly and membranes must be disrupted to allow access of antibodies to internal lipids. Alternatively, akin to fluorescent protein tagging, fluorophore labeled lipids have been developed and successfully used to probe lipid distribution and traffic. However, a huge caveat to these approaches is that a significant region of the hydrophobic tail or head group is replaced with the fluor. Therefore, there is greater potential for occluding crucial interactions that drive lipid localization or function. In addition, selective delivery of the lipids to the correct cellular locale can be difficult or impossible, and the lipid may be metabolized into other species that are indistinguishable by fluorescence. Therefore, in this Perspective, we focus on the genetically encoded lipid biosensors, derived from specific lipid-binding domains. These possess the advantages of being compatible with living cells, detecting the endogenous lipids, and (when used carefully) having limited effects on lipid metabolism and distribution. For a wider primer on the full range of approaches described above, we direct the reader to an excellent recent review (Maekawa and Fairn, 2014) CURRENT LIPID BIOSENSORS The association of proteins with lipids is usually mediated by specialized lipid-binding domains (Maekawa and Fairn, 2014; Hammond and Balla, 2015). In addition, a number of bacterial effectors and toxins can target host cell membranes by binding to lipids (Maekawa and Fairn, 2014; Várnai et al., 2017). These isolated domains, effectors, and toxins form the basis of genetically encoded lipid biosensors. They can be made as recombinant proteins and used for staining or blotting approaches. More commonly, they are expressed as fusions with fluorescent proteins to allow direct visualization of the lipids in cytosolic membrane leaflets of living cells. Table 1 lists lipid-binding domains that can be used for this purpose. TABLE 1: Commonly used genetically encoded biosensors and the criteria used to assess their interpretation; see the text for a discussion. Continued aThe accuracy of this probe is disputed. Each biosensor has a number of potential caveats and pitfalls that the user should be aware of. We think of three crucial criteria that should be considered when determining the validity of a probe: Is the probe specific for the target lipid? Is the biosensor's localization dependent on that lipid? And, if it is dependent, is the lipid alone sufficient to localize the biosensor? A previous review (Hammond and Balla, 2015) has explained each of these criteria in detail. Table 1 describes whether these criteria are met, and it is intended as a guide in selecting the most appropriate biosensors. Many are available through the plasmid sharing resource Addgene (www.addgene.org). THINGS TO THINK ABOUT WHEN USING LIPID BIOSENSORS Selectivity of the biosensor A quick perusal of Table 1 reveals that relatively few probes meet all of our prescribed criteria for truly selective, unbiased lipid biosensors. However, this does not mean that they cannot be used to gather useful information about lipid localization and dynamics. However, care must be taken with the interpretation of results. This is illustrated well by the number of probes that recognize PI4P; most have additional binding sites for other ligands that bias lipid detection in compartments that also contain this ligand. They can be useful in reporting changes in PI4P levels in these individual compartments but are often blind to changes in other regions of the cell. Sometimes it can be assumed that alternative interactions with other lipids will have minimal effects; for example, as Table 1 shows, the Tubby c-terminal domain binds to PI(3,4,5)P3 and PI(3,4)P2 in addition to PI(4,5)P2. Nevertheless, even after stimulation, these other lipids are present at <5% of PI(4,5)P2 (Stephens et al., 1993) and are therefore assumed not to alter localization of the biosensor. A long-time controversy has been whether the soluble headgroup, which are usually present in the cytosol, competes with binding to the lipid. The biggest example has been competition of PH-PLCδ1 from membrane PI(4,5)P2 by the lipid's hydrolysis product, IP3. The short answer is that both reductions in the lipid as well as increases in IP3 that occur after phospholipase C activation can lead to dissociation of the biosensor from the membrane, and it is not possible to prescribe a priori the contribution of each (Xu et al., 2003). Therefore, we urge caution when interpreting loss of localization of this biosensor after phospholipase C activation. This is a perfect example of when the Tubby c-terminal domain and its variants, which do not bind IP3, are a better probe (Quinn et al., 2008). Inhibitory effects of the biosensor Perhaps the greatest fear associated with the use of lipid biosensors is that they can sequester their target lipid and hence disrupt physiologic interactions with effector proteins. For this reason, articles such as this usually advise the reader to use the lowest possible expression levels of biosensor possible (e.g., Várnai et al., 2017). This is good advice, but it reminds us of Rick Sanchez, the fictitious mad scientist from the sci-fi cartoon “Rick and Morty,” who once quipped: “Ok… well, sometimes science is more art than science… A lot of people don't get that.” Rather than leave the reader to the art of determining appropriate expression levels, we will sketch out some slightly more quantitative considerations. Most biosensors in Table 1 bind with a 1:1 stoichiometry and with high affinity. In this case, it is permissible to estimate the concentration of lipids as if they were dissolved in the three-dimensional volume of the cell (though this does not apply to tandem arrays of lipid binding domains with more than one binding site, which are much more sensitive to the high local concentration of lipid on the two-­dimensional membrane surface). Estimates of this three-dimensional-equivalent concentration in a typical mammalian cell vary from ∼1 mM for bulk lipids like phosphatidylserine (PS) or phosphatidylcholine (PC), down to ∼10 µM for PI(4,5)P2 or 100 nM for PI(3,4,5)P3 (Stephens et al., 1993; McLaughlin et al., 2002). The fraction of total expressed biosensor that binds to lipid (fbiosnesor) is related to the binding site's dissociation constant (KD) by the following relationship: From this, we can see that a biosensor must have a KD similar to or substantially lower than that of the effective lipid concentration for appreciable localization when expressed in cells. This is either known to be the case or inferred from the localization of the biosensors listed in Table 1. Given these dissociation constants, it follows that if the concentration of biosensor in cells approaches or exceeds the concentration of lipid, a significant fraction of the lipid will be sequestered. This may begin to displace endogenous proteins and interactions, causing inhibitory effects. How likely is this? Typical yields for protein expression in mammalian systems reveal an upper limit of ∼100 µM for overexpressed protein in the cytosol (estimated as 1 mg/ml culture volume, 50-kDa protein, 2.5 × 106 cells/ml, 15-pl cell volume), whereas expression of lipid biosensors in cells has been found in the range of 1–10 µM (Xu et al., 2003). So, sequestering of significant fractions is unlikely for bulk lipids like PS or cholesterol, whereas it is a real concern with less abundant lipids, that is, the phosphoinositides. Indeed, dominant negative effects have been observed with the PI(4,5)P2 biosensor PH-PLCδ1 (Várnai and Balla, 1998; Holz et al., 2000). However, such dominant negative effects have been rare, and it appears that cells compensate by synthesizing more PI(4,5)P2 in response to biosensor expression (Traynor-Kaplan et al., 2017), likely keeping the free lipid concentration constant. This may explain how it is possible to use the PI(3,4,5)P3 biosensors listed in Table 1, since these have KD ≈ [PI(3,4,5)P3] ≈ 100 nM, and likely sequester the majority of synthesized lipid. By contrast, we estimate that HeLa cells contain ∼500 nM of endogenous effector protein from published proteomic data (Hein et al., 2015). Simple sequestering of the lipid is not the only potential concern when using biosensors. Several of the lipid binding domains listed in Table 1 also have protein ligands, which may be present at much lower effective cytosolic concentrations (Hein et al., 2015), and therefore be much more susceptible to sequestration. Furthermore, the accumulation of high densities of lipid binding proteins on membranes can have other deleterious effects due to protein crowding, such as membrane deformation (Stachowiak et al., 2012). For this reason, it is still always advisable to keep biosensor expression levels as low as practicable for imaging purposes. However, with modern instruments, ∼1 µM is adequate for high-quality images and should not cause deleterious effects for most lipids. The treachery of images Belgian surrealist painter René Magritte famously produced a painting of a pipe with an accompanying legend, “Ceci n'est pas une pipe” (“This is not a pipe”); this illustrated the fact that the painting is merely a representation of an object, distinct from the real thing. Similarly, it is important to bear in mind that the biosensor is not the same as the lipid it is being used to detect. First, biosensors are in equilibrium with the free pool of lipid and will not interact with lipids bound to effector proteins; similarly, effector proteins cannot bind to biosensor-bound lipids. Given that lipid–biosensor complexes can diffuse several microns in the plane of the membrane (Hammond et al., 2009), biosensors often cannot detect local enrichment of lipids associated with specific protein complexes, unless the biosensor has a low affinity (and hence rapid dissociation rate) or is specifically targeted to that protein complex via a secondary, low-affinity interaction (Trexler et al., 2016; He et al., 2017). That said, most complexes of biosensor or effector protein last only a few seconds—so effector-bound, free, and biosensor-bound lipids rapidly equilibrate with each other. A second key point is that the biosensor is expressed in the cell independently of the presence of its lipid target; the lipid only influences the localization of the biosensor. Therefore, changes in local concentrations of the lipid cause changes in biosensor localization and not overall changes in its expression level or total fluorescence—this key point is occasionally lost in the literature. For the purposes of quantification, it is necessary to keep this concept of localization and measure fluorescence intensity changes relative to the membrane compartment of interest. This can be accomplished in a number of ways; for example, by generating a mask corresponding to specific organelles via expression of secondary markers and measuring intensity in the masked region relative to the rest of the cell, by selectively imaging the plasma membrane via total internal reflection fluorescence microscopy (TIRFM), or by using resonant energy transfer approaches to detect biosensor accumulated at a specific membrane. Several recent articles have described these approaches in detail (Hammond and Balla, 2015; Várnai et al., 2017) We illustrate the need to be mindful when interpreting localization in Figure 1. The figure shows cells expressing a high-affinity PI4P probe, GFP-P4C, before and after a treatment. We visualize an increase in fluorescence on endosomes posttreatment. This observation could be the result of an increase in PI4P on endosomes or a loss of lipid where the probe was initially bound. The change in Figure 1 is actually the result of a PI4P phosphatase being recruited to the plasma membrane, leading to degradation of this lipid and loss of P4C localization there. The P4C relocalized to organelles where PI4P was still present, which is observed as an increase in fluorescence at these membranes. FIGURE 1: Ceci n'est pas une PIP (“This is not a PIP”). This COS-7 cell is expressing GFP-P4C, a PI4P biosensor. The images show the cell before and after a treatment that induces relocalization of the probe. What do you suppose this treatment was? Read the text for the answer. Go low or go high? The most striking fluorescence images of cell structure are produced when there is the highest contrast between the cellular structure and the rest of the cell. For lipid biosensors, this will correspond to a high fraction of biosensor bound to membrane, that is, a high-affinity (or low KD). High-affinity probes can be made by tandem dimers or trimers of lower-affinity domains, with a resulting multiplicative increase in affinity—though this approach has pros and cons. For example, a tandem dimer of the P4M PI4P probe allows enhanced detection of PM and endosomal pools of PI4P; conversely, the increased affinity can lead to greater sequestration of lipid and even distort organelles at higher expression levels (Hammond et al., 2014). High-affinity probes come with another setback: if the majority of biosensor is already bound to lipid, then it will not detect increases in that lipid. Figure 2 shows a single (P4Mx1) or tandem (P4Mx2) PI4P probe after the recruitment of a PI(4,5)P2 phosphatase, which elevates PI4P in the plasma membrane. We can observe an increase of the P4Mx1 probe at the membrane after the recruitment of the phosphatase, while little translocation of P4Mx2 is observed—which could be misinterpreted as a lack of lipid production. Indeed, a little trumpeted fact about the popular PI(4,5)P2 probe, PH-PLCδ1, is that it is poorly able to detect increases in plasma membrane PI(4,5)P2 (Suh et al., 2006) FIGURE 2: Go high or go low (affinity). These HeLa cells are expressing either a monomeric (top) or a dimeric (bottom) version of GFP-P4M, a PI4P biosensor. The images show the cells before and after activation of a 5-phosphatase that increases PI4P in the plasma membrane. The lower affinity biosensor is able to detect small changes in PI4P synthesis and relocalize to the PM (note how the cytoplasm looks dimmer in the “after” image), whereas the higher-affinity probe is already saturated at the plasma membrane. Lower-affinity probes produce much less striking images—but can be much more informative regarding changes in lipid concentrations. They are also much less prone to the inhibitory effects discussed above, and their shorter lifetime in complex with lipids can allow them to report on local lipid accumulations missed by higher-affinity domains (Kabachinski et al., 2014; Trexler et al., 2016). However, the low affinity can also make it hard to quantify decreases in lipid concentration in a particular membrane, for example, the drop in PI(4,5)P2 levels after PLC activation. For these reasons, in our lab we often employ both low- and high-affinity biosensors, when they are available, for a given lipid species. CONCLUSION The aim of this Perspective was to provide a current (perhaps fleetingly so) overview of the available genetically encoded lipid biosensors. As we have discussed, these tools may have some limitations, but when controlled, quantified, and interpreted properly, these probes provide invaluable information regarding lipid dynamics. There are a large number of probes available for a wide selection of lipids. However, some lipids still lack high-quality biosensors or any biosensor at all (e.g., PI). The area of biosensor development is a quickly moving field with continuous advancements; new lipid-binding domains are continually being recognized or modified to improve their efficacy. Therefore, we expect the current list (Table 1) to become rapidly outdated. However, the principles and pitfalls we have outlined will be as applicable for future generations of lipid biosensors as they are for the current suite. DOI:10.1091/mbc.E17-12-0738 We apologize to the large number of colleagues whose fine work we have had to exclude due to space limitations. We are grateful to James P. Zewe for helpful comments on the manuscript. This work was supported by National Institutes of Health grant 1R35GM119412-01. Abbreviations used: Cholcholesterol DAGdiacylglycerol EEearly endosome ERendoplasmic reticulum IP 3inositol 1,4,5-trisphosphate LElate endosome NEnuclear envelope PAphosphatidic acid PI3Pphosphatidylinositol 3-phosphate PI4Pphosphatidylinositol 4-phosphate PI5Pphosphatidylinositol 5-phosphate PI(3,4)P 2phosphatidylinositol 3,4-bisphosphate PI(3,5)P 2phosphatidylinositol 3,5-bisphosphate PI(4,5)P 2phosphatidylinositol 4,5-bisphosphate PI(3,4,5)P 3phosphatidylinositol 3,4,5-­trisphosphate PMplasma membrane PSphosphatidylserine SMsphingomyelin. ==== Refs REFERENCES Abe M Makino A Hullin-Matsuda F Kamijo K Ohno-Iwashita Y Hanada K Mizuno H Miyawaki A Kobayashi T (2012 ). A role for sphingomyelin-rich lipid domains in the accumulation of phosphatidylinositol-4,5-bisphosphate to the cleavage furrow during cytokinesis . Mol Cell Biol , 1396 –1407 .22331463 Bohdanowicz M Schlam D Hermansson M Rizzuti D Fairn G Ueyama T Somerharju P Du G Grinstein S (2013 ). Phosphatidic acid is required for the constitutive ruffling and macropinocytosis of phagocytes . Mol Biol Cell , 1700 –1712 .23576545 Bravo J Karathanassis D Pacold CM Pacold ME Ellson CD Anderson KE Butler PJ Lavenir I Perisic O Hawkins PT , et al (2001 ). The crystal structure of the PX domain from p40phox bound to phosphatidylinositol 3-phosphate . Mol Cell , 829 –839 .11684018 Brombacher E Urwyler S Ragaz C Weber S Kami K Overduin M Hilbi H (2009 ). Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of Legionella pneumophila . J Biol Chem , 4846 –4856 .19095644 Burd C Emr S (1998 ). Phosphatidylinositol(3)-phosphate signaling mediated by specific binding to RING FYVE domains . Mol Cell , 157 –162 .9702203 Chen J Deng F Li J Wang J (2008 ). Selective binding of phorbol esters and diacylglycerol by individual C1 domains of the PKD family . Biochem J , 333 –342 . Chung J Torta F Masai K Lucast L Czapla H Tanner L Narayanaswamy P Wenk M Nakatsu F Camilli P (2015 ). PI4P/phosphatidylserine countertransport at ORP5- and ORP8-mediated ER–plasma membrane contacts . Science , 428 –432 . Cohen L Honda A Varnai P Brown F Balla T Donaldson J (2007 ). Active Arf6 recruits ARNO/cytohesin GEFs to the PM by binding their PH domains . Mol Biol Cell , 2244 –2253 .17409355 Dolinsky S Haneburger I Cichy A Hannemann M Itzen A Hilbi H (2014 ). The Legionella longbeachae Icm/Dot substrate SidC selectively binds phosphatidylinositol 4-phosphate with nanomolar affinity and promotes pathogen vacuole-endoplasmic reticulum interactions . Infect Immun , 4021 –4033 .25024371 Domart M-C Hobday TM Peddie CJ Chung GH Wang A Yeh K Jethwa N Zhang Q Wakelam MJ Woscholski R , et al (2012 ). Acute manipulation of diacylglycerol reveals roles in nuclear envelope assembly & endoplasmic reticulum morphology . PLoS One , e51150 .23227247 Dowler S Currie R Campbell D Deak M Kular G Downes C Alessi D (2000 ). Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities . Biochem J , 19 –31 . Ellson CD Gobert-Gosse S Anderson KE Davidson K Erdjument-­Bromage H Tempst P Thuring JW Cooper MA Lim Z-Y Holmes AB , et al (2001 ). PtdIns(3)P regulates the neutrophil oxidase complex by binding to the PX domain of p40phox . Nat Cell Biol , 679 –682 .11433301 Ford M Pearse B Higgins M Vallis Y Owen D Gibson A Hopkins C Evans P McMahon H (2001 ). Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes . Science , 1051 –1055 . Frech M Andjelkovic M Ingley E Reddy K Falck J Hemmings B (1997 ). High affinity binding of inositol phosphates and phosphoinositides to the pleckstrin homology domain of RAC/protein kinase B and their influence on kinase activity . J Biol Chem , 8474 –8481 .9079675 Fukuda M Kojima T Kabayama H Mikoshiba K (1996 ). Mutation of the pleckstrin homology domain of Bruton's tyrosine kinase in immunodeficiency impaired inositol 1,3,4,5-tetrakisphosphate binding capacity . J Biol Chem , 30303 –30306 .8939985 Garcia P Gupta R Shah S Morris AJ Rudge SA Scarlata S Petrova V McLaughlin S Rebecchi MJ (1995 ). The pleckstrin homology domain of phospholipase C-.delta.1 binds with high affinity to phosphatidylinositol 4,5-bisphosphate in bilayer membranes . Biochemistry , 16228 –16234 .8519781 Gaullier J-M Rønning E Gillooly D Stenmark H (2000 ). Interaction of the EEA1 FYVE finger with phosphatidylinositol 3-phosphate and early endosomes. Role of conserved residues . J Biol Chem , 24595 –24600 .10807926 Gaullier JM Simonsen A D'Arrigo A Bremnes B Stenmark H Aasland R (1998 ). FYVE fingers bind PtdIns(3)P . Nature , 432 –433 .9697764 Ghai R Du X Wang H Dong J Ferguson C Brown A Parton R Wu J-W Yang H (2017 ). ORP5 and ORP8 bind phosphatidylinositol-4, 5-biphosphate (PtdIns(4,5) P 2) and regulate its level at the plasma membrane . Nat Commun , 757 .28970484 Gillooly D Morrow I Lindsay M Gould R Bryant N Gaullier J Parton R Stenmark H (2000 ). Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells . Embo J , 4577 –4588 .10970851 Gozani O Karuman P Jones DR Ivanov D Cha J Lugovskoy AA Baird CL Zhu H Field SJ Lessnick SL , et al (2003 ). The PHD finger of the chromatin-associated protein ING2 functions as a nuclear phosphoinositide receptor . Cell , 99 –111 . Gray A Kaay J Downes C (1999 ). The pleckstrin homology domains of protein kinase B and GRP1 (general receptor for phosphoinositides-1) are sensitive and selective probes for the cellular detection of phosphatidylinositol 3,4-bisphosphate and/or phosphatidylinositol 3,4,5-trisphosphate in vivo . Biochem J , 929 –936 .10585883 Halaszovich CR Schreiber DN Oliver D (2009 ). Ci-VSP is a depolarization-activated phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate 5’-phosphatase . J Biol Chem , 2106 –2113 .19047057 Hammond G Machner M Balla T (2014 ). A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi . J Cell Biol , 113 –126 . Hammond G Sim Y Lagnado L Irvine R (2009 ). Reversible binding and rapid diffusion of proteins in complex with inositol lipids serves to coordinate free movement with spatial information . J Cell Biol , 297 –308 .19153221 Hammond G Takasuga S Sasaki T Balla T (2015 ). The ML1Nx2 phosphatidylinositol 3,5-bisphosphate probe shows poor selectivity in cells . PLoS One , e0139957 .26460749 Hammond GR Balla T (2015 ). Polyphosphoinositide binding domains: key to inositol lipid biology . Biochim Biophys Acta , 746 –758 . He K Marsland R Upadhyayula S Song E Dang S Capraro BR Wang W Skillern W Gaudin R Ma M (2017 ). Dynamics of phosphoinositide conversion in clathrin-mediated endocytic traffic . Nature , 410 . Hein M Hubner NC Poser I Cox J Nagaraj N Toyoda Y Gak IA Weisswange I Mansfeld J Buchholz F , et al (2015 ). A human interactome in three quantitative dimensions organized by stoichiometries and abundances . Cell , 712 –723 . Hirose K Kadowaki S Tanabe M Takeshima H Iino M (1999 ). Spatiotemporal dynamics of inositol 1,4,5-trisphosphate that underlies complex Ca2+ mobilization patterns . Science , 1527 –1530 .10348740 Hofmann I Thompson A Sanderson C Munro S (2007 ). The Arl4 family of small G proteins can recruit the cytohesin Arf6 exchange factors to the plasma membrane . Curr Biol , 711 –716 .17398095 Holz RW Hlubek MD Sorensen SD Fisher SK Balla T Ozaki S Prestwich GD Stuenkel EL Bittner MA (2000 ). A pleckstrin homology domain specific for Ptdins-4-4-P2 and fused to green fluorescent protein identifies plasma membrane Ptdins-4-5-P2 as being important in exocytosis . J Biol Chem , 17878 –17885 .10747966 Itoh T Koshiba S Kigawa T Kikuchi A Yokoyama S Takenawa T (2001 ). Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis . Science , 1047 –1051 . Kabachinski G Yamaga M Kielar-Grevstad MD Bruinsma S Martin TF (2014 ). CAPS and Munc13 utilize distinct PIP2-linked mechanisms to promote vesicle exocytosis . Mol Biol Cell , 508 –521 .24356451 Kanai F Liu H Field S Akbary H Matsuo T Brown G Cantley L Yaffe M (2001 ). The PX domains of p47phox and p40phox bind to lipid products of PI(3)K . Nat Cell Biol , 675 –678 .11433300 Kim YJ Guzman-Hernandez ML Balla T (2011 ). A highly dynamic ER-derived phosphatidylinositol-synthesizing organelle supplies phosphoinositides to cellular membranes . Dev Cell , 813 –824 .22075145 Kimber WA Trinkle-Mulcahy L Cheung PC Deak M Marsden LJ Kieloch A Watt S Javier RT Gray A Downes C , et al (2002 ). Evidence that the tandem-pleckstrin-homology-domain-containing protein TAPP1 interacts with Ptd(3,4)P2 and the multi-PDZ-domain-containing protein MUPP1 in vivo . Biochem J , 525 –536 . Kiyokawa E Baba T Otsuka N Makino A Ohno S Kobayashi T (2005). Spatial and functional heterogeneity of sphingolipid-rich membrane domains . J Biol Chem , 24072 –24084 .15840575 Klarlund J Guilherme A Holik J Virbasius J Chawla A Czech M (1997 ). Signaling by phosphoinositide-3,4,5-trisphosphate through proteins containing pleckstrin and Sec7 homology domains . Science , 1927 –1930 . Kontos CD Stauffer TP Yang WP York JD Huang L Blanar MA Meyer T Peters KG (1998 ). Tyrosine 1101 of Tie2 is the major site of association of p85 and is required for activation of phosphatidylinositol 3-kinase and Akt . Mol Cell Biol , 4131 –4140 .9632797 Lee S Várnai P Balla A Jalink K Rhee S-G Balla T (2004 ). The pleckstrin homology domain of phosphoinositide-specific phospholipase Cδ4 is not a critical determinant of the membrane localization of the enzyme . J Biol Chem , 24362 –24371 .15037625 Lemmon M Ferguson K O'Brien R Sigler P Schlessinger J (1995 ). Specific and high-affinity binding of inositol phosphates to an isolated pleckstrin homology domain . Proc Natl Acad Sci USA , 10472 –10476 .7479822 Lenoir M Grzybek M Majkowski M Rajesh S Kaur J Whittaker S Coskun Ü Overduin M (2015 ). Structural basis of dynamic membrane recognition by trans-Golgi network specific FAPP proteins . J Mol Biol , 966 –981 .25579996 Levin R Hammond G Balla T Camilli P Fairn G Grinstein S (2017 ). Multiphasic dynamics of phosphatidylinositol 4-phosphate during phagocytosis . Mol Biol Cell , 128 –140 .28035045 Levine TP , Munro S (2002). Targeting of Golgi-specific pleckstrin homology domains involves both PtdIns 4-kinase-dependent and -independent components . Curr Biol , 695 –704 .12007412 Li C-C Chiang T-C Wu T-S Pacheco-Rodriguez G Moss J Lee F-J (2007 ). ARL4D recruits cytohesin-2/ARNO to modulate actin remodeling . Mol Biol Cell , 4420 –4437 .17804820 Li X Wang X Zhang X Zhao M Tsang W Zhang Y Yau R Weisman L Xu H (2013 ). Genetically encoded fluorescent probe to visualize intracellular phosphatidylinositol 3,5-bisphosphate localization and dynamics . Proc Nat Acad Sci USA , 21165 –21170 .24324172 Liu S-L Sheng R Jung J Wang L Stec E O’Connor MJ Song S Bikkavilli R Winn RA Lee D , et al (2016 ). Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol . Nat Chem Biol , 268 –274 . Maeda K Anand K Chiapparino A Kumar A Poletto M Kaksonen M Gavin A-C (2013 ). Interactome map uncovers phosphatidylserine transport by oxysterol-binding proteins . Nature , 257 -261 . Maekawa M Fairn G (2014 ). Molecular probes to visualize the location, organization and dynamics of lipids . J Cell Sci , 4801 –4812 .25179600 Maekawa M Fairn G (2015 ). Complementary probes reveal that phosphatidylserine is required for the proper transbilayer distribution of cholesterol . J Cell Sci , 1422 –1433 .25663704 Manna D Albanese A Park W Cho W (2007 ). Mechanistic basis of differential cellular responses of phosphatidylinositol 3,4-bisphosphate- and phosphatidylinositol 3,4,5-trisphosphate-binding pleckstrin homology domains . J Biol Chem , 32093 –32105 .17823121 Marshall AJ Krahn AK Ma K Duronio V Hou S (2002 ). TAPP1 and TAPP2 are targets of phosphatidylinositol 3-kinase signaling in B cells: sustained plasma membrane recruitment triggered by the B-cell antigen receptor . Mol Cell Biol , 5479 –5491 .12101241 McLaughlin S Wang J Gambhir A Murray D (2002 ). PIP2 and proteins: interactions, organization, and information flow . Biophysics Biomol Struct , 151 –175 . Nguyen A Rudge S Zhang Q Wakelam M (2017 ). Using lipidomics analysis to determine signalling and metabolic changes in cells . Curr Opin Biotech , 96 –103 .27816901 Pendaries C Tronchère H Arbibe L Mounier J Gozani O Cantley L Fry M Gaits-Iacovoni F Sansonetti P Payrastre B (2006 ). PtdIns(5)P activates the host cell PI3-kinase/Akt pathway during Shigella flexneri infection . Embo J , 1024 –1034 .16482216 Quinn K Behe P Tinker A (2008 ). Monitoring changes in membrane phosphatidylinositol 4,5-bisphosphate in living cells using a domain from the transcription factor tubby . J Physiology , 2855 –2871 . Rameh LE Arvidsson A Carraway KL Couvillon AD Rathbun G Crompton A VanRenterghem B Czech MP Ravichandran KS Burakoff SJ , et al (1997 ). A comparative analysis of the phosphoinositide binding specificity of pleckstrin homology domains . J Biol Chem , 22059 –22066 .9268346 Salim K Bottomley M Querfurth E Zvelebil M Gout I Scaife R Margolis R Gigg R Smith C Driscoll P , et al (1996 ). Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton's tyrosine kinase . EMBO J , 6241 –6250 .8947047 Sankaran V Klein D Sachdeva M Lemmon M (2001 ). High-affinity binding of a FYVE domain to phosphatidylinositol 3-phosphate requires intact phospholipid but not FYVE domain oligomerization . Biochemistry , 8581 –8587 .11456498 Schoebel S Blankenfeldt W Goody R Itzen A (2010 ). High-affinity binding of phosphatidylinositol 4-phosphate by Legionella pneumophila DrrA . Embo Rep , 598 –604 .20616805 Shimada Y Maruya M Iwashita S Ohno-Iwashita Y (2002). The C-terminal domain of perfringolysin O is an essential cholesterol-binding unit targeting to cholesterol-rich microdomains . Eur J Biochem , 6195 –6203 .12473115 Sohn M Korzeniowski M Zewe JP Wills RC Hammond G Humpolickova J Vrzal L Chalupska D Veverka V Fairn GD , et al (2018 ). PI(4,5)P2 controls plasma membrane PI4P and PS levels via ORP5/8 recruitment to ER–PM contact sites . J Cell Biol , 1797 –1813 .29472386 Stachowiak J Schmid E Ryan C Ann H Sasaki D Sherman M Geissler P Fletcher D Hayden C (2012 ). Membrane bending by protein–protein crowding . Nat Cell Biol , 944 –949 .22902598 Stahelin RV Digman MA Medkova M Ananthanarayanan B Melowic HR Rafter JD Cho W (2005). Diacylglycerol-induced membrane targeting and activation of protein kinase Cϵ: Mechanistic differences between protein kinases Cδ and Cϵ . J Biol Chem , 19784 –19793 .15769752 Stauffer T Ahn S Meyer T (1998 ). Receptor-induced transient reduction in plasma membrane PtdIns(4,5)P2 concentration monitored in living cells . Curr Biol , 343 –346 .9512420 Stephens LR Jackson TR Hawkins PT (1993 ). Agonist-stimulated synthesis of phosphatidylinositol(3,4,5)-trisphosphate: a new intracellular signalling system ? Biochim Biophys Acta , 27 –75 . Suh B-C Inoue T Meyer T Hille B (2006 ). Rapid chemically induced changes of PtdIns(4,5)P2 gate KCNQ ion channels . Science , 1454 –1457 . Szentpetery Z Balla A Kim Y Lemmon M Balla T (2009 ). Live cell imaging with protein domains capable of recognizing phosphatidylinositol 4,5-bisphosphate; a comparative study . Bmc Cell Biol , 1 –20 .19134226 Szentpetery Z Várnai P Balla T (2010 ). Acute manipulation of Golgi phosphoinositides to assess their importance in cellular trafficking and signaling . Proc Natl Acad Sci USA , 8225 –8230 .20404150 Thomas C Dowler S Deak M Alessi D And Aalten D (2001 ). Crystal structure of the phosphatidylinositol 3,4-bisphosphate-binding pleckstrin homology (PH) domain of tandem PH-domain-containing protein 1 (TAPP1): molecular basis of lipid specificity . Biochem J , 287 –294 . Traynor-Kaplan A Kruse M Dickson E Dai G Vivas O Yu H Whittington D Hille B (2017 ). Fatty-acyl chain profiles of cellular phosphoinositides . Biochim Biophys Acta , 513 –522 . Trexler A Sochacki K Taraska J (2016 ). Imaging the recruitment and loss of proteins and lipids at single sites of calcium-triggered exocytosis . Mol Biol Cell , 2423 –2434 .27307587 van Meer G de Kroon A (2011 ). Lipid map of the mammalian cell . J Cell Sci , 5 –8 .21172818 Várnai P Balla T (1998 ). Visualization of phosphoinositides that bind pleckstrin homology domains: calcium- and agonist-induced dynamic changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools . J Cell Biol , 501 –510 .9786958 Várnai P Gulyás G Tóth DJ Sohn M Sengupta N Balla T (2017 ). Quantifying lipid changes in various membrane compartments using lipid binding protein domains . Cell Calcium , 72 –82 .28088320 Vecchio K Stahelin R (2018 ). Investigation of the phosphatidylserine binding properties of the lipid biosensor, Lactadherin C2 (LactC2), in different membrane environments . J Bioenerg Biomembr , 1 –10 .29426977 Venkateswarlu K Oatey P Tavaré J Cullen P (1998 ). Insulin-dependent translocation of ARNO to the plasma membrane of adipocytes requires phosphatidylinositol 3-kinase . Curr Biol , 463 –466 .9550703 Watton S Downward J (1999 ). Akt/PKB localisation and 3′ phosphoinositide generation at sites of epithelial cell–matrix and cell–cell interaction . Curr Biol , 433 –436 .10226029 Weber S Wagner M Hilbi H (2014 ). Live-cell imaging of phosphoinositide dynamics and membrane architecture during Legionella infection . Mbio , e00839 –13 .24473127 Xu C Watras J Loew L (2003 ). Kinetic analysis of receptor-activated phosphoinositide turnover . J Cell Biol , 779 –791 .12771127 Yamaji A Sekizawa Y Emoto K Sakuraba H Inoue K Kobayashi H Umeda M (1998 ). Lysenin, a novel sphingomyelin-specific binding protein . J Biol Chem , 5300 –5306 .9478988 Yeung T Gilbert GE Shi J Silvius J Kapus A Grinstein S (2008 ). Membrane phosphatidylserine regulates surface charge and protein localization . Science , 210 –213 . Yoon Y Lee P Kurilova S Cho W (2011 ). In situ quantitative imaging of cellular lipids using molecular sensors . Nat Chem , 868 –874 .22024883 Zewe J Wills R Sangappa S Goulden B Hammond G (2018 ). SAC1 degrades its lipid substrate PtdIns4P in the endoplasmic reticulum to maintain a steep chemical gradient with donor membranes . Elife , e35588 .29461204 Zhang F Wang Z Lu M Yonekubo Y Liang X Zhang Y Wu P Zhou Y Grinstein S Hancock JF , et al (2014 ). Temporal production of the signaling lipid phosphatidic acid by phospholipase D2 determines the output of extracellular signal-regulated kinase signaling in cancer cells . Mol Cell Biol , 84 –95 .24164897