CHAPTER FIVE
Analysis, Regulation, and Roles of Endosomal Phosphoinositides Tania Maffucci, Marco Falasca1 Inositide Signalling Group, Centre for Diabetes, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Endosomal PIs 2.1 Phosphatidylinositol 3-phosphate 2.2 Phosphatidylinositol 3,5-bisphosphate 3. Analysis of PtdIns3P Levels 3.1 Metabolic labeling and HPLC analysis 3.2 Liquid chromatography–mass spectrometry 3.3 Novel assay to measure PtdIns3P levels 4. Monitoring PtdIns3P Intracellular Localization 4.1 PtdIns3P-binding domains 4.2 Immunofluorescence microscopy analysis 4.3 Immunofluorescence analysis: Pro&Con 4.4 Immunofluorescence using anti-PtdIns3P antibody 4.5 Immunoelectron microscopy analysis 5. Endosomal PtdIns3P 5.1 Regulation of the endosomal pool of PtdIns3P 5.2 Cellular functions regulated by the endosomal pool of PtdIns3P 6. Phosphatidylinositol 3,5-Bisphosphate 6.1 Intracellular levels 6.2 Intracellular localization 6.3 Intracellular roles Acknowledgments References
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Abstract Phosphoinositides (PIs) are minor lipid components of cellular membranes that play critical roles in membrane dynamics, trafficking, and cellular signaling. Among the seven naturally occurring PIs, the monophosphate phosphatidylinositol 3-phosphate (PtdIns3P) and the bisphosphate phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] have been mainly associated with endosomes and endosomal functions. Metabolic labeling and HPLC analysis revealed that a bulk of PtdIns3P is constitutively present Methods in Enzymology, Volume 535 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-397925-4.00005-5
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2014 Elsevier Inc. All rights reserved.
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in cells, making it the only detectable product of the enzymes phosphoinositide 3-kinases in unstimulated, normal cells. The use of specific tagged-PtdIns3P-binding domains later demonstrated that this constitutive PtdIns3P accumulates in endosomes where it critically regulates trafficking and membrane dynamics.
1. INTRODUCTION Phosphoinositides (PIs) are phospholipids comprising a water-soluble head group (myo-inositol) linked by a glycerol moiety to two fatty acid chains, usually a saturated C18 residue (stearoyl) in the 1-position and a tetra-unsaturated C20 residue (arachidonoyl) in the 2-position (Michell, 2008). The founding member of the PIs family is the unphosphorylated phosphatidylinositol (PtdIns), primarily synthesized in the endoplasmic reticulum and then delivered to other membranes by vesicular transport or via cytosolic PtdIns transfer protein (Di Paolo & De Camilli, 2006). PIs derive from phosphorylation of the hydroxyls at positions 3-, 4- and 5- within the myo-inositol headgroup of PtdIns and the different possible combinations generate seven distinct derivatives, which can be interconverted into each other by the action of specific kinases or phosphatases. PIs play many intracellular roles either as component of cellular membranes or by regulating the activity of targeted proteins temporally and/or spatially. Because of their lipid tail, PIs are obligatory membrane-bound; therefore, they can mark specific membrane compartments or subdomains within a membrane. On the other hand, PIs also possess a soluble headgroup, which allows them to bind to cytosolic proteins and to mediate the association of these proteins to specific membranes (spatial regulation). In addition, since some PIs are only synthesized upon cellular stimulation in normal cells, they can also regulate activation of target proteins temporally (Maffucci, 2012).
2. ENDOSOMAL PIs Two PIs have been mainly associated with endosomes and endosomal functions: the monophosphate phosphatidylinositol 3-phosphate (PtdIns3P) and the bisphosphate phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] (Nicot & Laporte, 2008).
2.1. Phosphatidylinositol 3-phosphate PtdIns3P comprises 0.1–0.5% of all PIs (Falasca & Maffucci, 2006) and almost 0.002% of total membrane lipids (Stephens, McGregor, &
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Hawkins, 2000). PtdIns3P is the most abundant among the PIs phosphorylated at position 3 in resting mammalian cells and its intracellular levels are regulated by a coordinated action of kinases and phosphatases (Falasca & Maffucci, 2006, 2009). Synthesis of PtdIns3P is catalyzed by members of the family of enzymes phosphoinositide 3-kinase (PI3K). Three classes of PI3Ks exist (Falasca & Maffucci, 2007; Vanhaesebroeck, Guillermet-Guibert, Graupera, & Bilanges, 2010) and synthesis of PtdIns3P occurs in vivo through phosphorylation of PtdIns by class II or class III PI3K isoforms. No evidence so far has supported a role for class I PI3Ks in direct synthesis of PtdIns3P from PtdIns in vivo. Class III comprises only one PI3K isoform, vacuolar protein sorting 34 (Vps34), which is the most ancient forms of PI3Ks and the only one conserved from lower eukaryotes to plants and mammals (Backer, 2008; Engelman, Luo, & Cantley, 2006). It is still not clear whether the activity of Vps34 is modulated by cellular stimulation (Vanhaesebroeck et al., 2010) although it has been suggested that the basal activation of hVps34 may be regulated by nutrients (Byfield, Murray, & Backer, 2005). In contrast to the other PI3Ks, which can produce three 3-phosphorylated PIs at least in vitro (PtdIns3P, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate), hVps34 can only generate PtdIns3P (Backer, 2008). Class II comprises three PI3K isoforms, PI3K-C2a (Domin et al., 1997), PI3K-C2b (Brown, Ho, Weber-Hall, Shipley, & Fry, 1997), and PI3K-C2g (Misawa et al., 1998; Ono et al., 1998; Rozycka et al., 1998). Several studies have now demonstrated that at least some members of the class II PI3K subfamily catalyze synthesis of PtdIns3P in vivo (Falasca & Maffucci, 2012; Mazza & Maffucci, 2011). PI3K-C2a regulates an insulin-dependent, plasma membrane (PM)-associated pool of PtdIns3P in muscle cells (Falasca et al., 2007; Maffucci, Brancaccio, Piccolo, Stein, & Falasca, 2003) and it generates PtdIns3P in large densecore vesicles (LDCVs) of PC12 upon stimulation of exocytosis (Wen et al., 2008). Recently, studies using MEFs upon induction of PI3K-C2a knockout (Yoshioka et al., 2012) and endothelial cells (Biswas et al., 2013) have confirmed PtdIns3P as the main specific product of PI3KC2a. Similarly, PI3K-C2b regulates lysophosphatidic acid-induced and sphingosine-1-phosphate-dependent synthesis of PtdIns3P in cancer (Maffucci et al., 2005) and endothelial cells (Tibolla et al., 2013), respectively. It has also been proposed that PI3K-C2b regulates a nuclear pool of PtdIns3P during cell cycle progression (Visnjic´ et al., 2003). As far as we know, no study so far has investigated whether PI3K-C2g also catalyzes PtdIns3P synthesis in vivo.
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Levels of PtdIns3P are further regulated through dephosphorylation at position 3 by several phosphatases, mainly belonging to the family of myotubularins (MTMs; Robinson & Dixon, 2006). Fourteen MTMs have been detected in humans, nearly half of which are predicted to be catalytically inactive. Recombinant myotubularin MTM1 and myotubularinrelated (MTMR) 1, 2, 3, 4, 6, and 7 were initially reported to selectively dephosphorylate PtdIns3P although it was later shown that some of these (such as MTM1 and MTMR2, 3, and 6) can also dephosphorylate PtdIns(3,5)P2 (Maffucci, 2012).
2.2. Phosphatidylinositol 3,5-bisphosphate PtdIns(3,5)P2 represents 0.0001% of total membrane lipids (Stephens et al., 2000) and it is usually only 0.1% or less of total cellular PIs in unstressed mammalian cells (Michell, Heath, Lemmon, & Dove, 2006). It is generally accepted that PtdIns(3,5)P2 derives from phosphorylation of PtdIns3P by a member of the family of type III PIPKs (Dove, Dong, Kobayashi, Williams, & Michell, 2009), mainly PI kinase for five positions containing a FYVE finger (PIKfyve) whose activation involves formation of a complex with the activator ArPIKfyve (Shisheva, 2008). PtdIns(3,5)P2 dephosphorylation can be mediated by different phosphatases including the 5-phosphatase FIG4/Sac3 and the scaffolding protein VAC14 although Sac3 can also associate with the complex PIKfyve/ArPIKfyve and promote maximal PtdIns(3,5)P2 synthesis. It has also been suggested that inactive MTMs can dimerize with active MTMs and change their specificity towards this PI rather than PtdIns3P (Lecompte, Poch, & Laporte, 2008). Other 5-phosphatases have been proposed to be able to dephosphorylate PtdIns(3,5)P2 including 72 kDa 5-phosphatase/INPP5E or SHIP2 (Ooms et al., 2009), but it is not known whether the endogenous enzymes have a role in modulating the levels of PtdIns(3,5)P2 in vivo.
3. ANALYSIS OF PtdIns3P LEVELS 3.1. Metabolic labeling and HPLC analysis The classical experimental procedure to quantitatively measure the intracellular levels of PtdIns3P involves labeling of cells with myo-[3H] inositol, lipid extraction, and HPLC analysis. Alternatively, cells can be labeled with [32P] orthophosphate.
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3.1.1 Cellular labeling Incorporation of myo-[3H] inositol is performed in inositol-free media for the time required to reach isotopic equilibrium, which can vary between cellular types and therefore needs to be optimized for the specific cell system to be used. Labeling can be performed in the presence or absence of serum depending on the cell types and the efficiency of incorporation. Similarly, optimization is required to determine the amount of myo-[3H] inositol, which allows detection of the PIs of interest. In our studies, we typically used 5 mCi myo-[3H] inositol/ml (Falasca et al., 2007; Maffucci et al., 2005). The following protocol refers to cells plated in a 6-well plate.
3.1.2 Lipid extraction Following incorporation, cells are lysed and lipids are extracted by phase separation using a mix of water/hydrochloric acid/methanol/chloroform. [3H]PIs containing two acyl chains accumulate in the organic phase, whereas the water-soluble [3H]inositol phosphates accumulate in the aqueous phase. 1. Lyse cells with 500 ml ice-cold HCl 1 M þ 1 mM tetrabutylammonium hydrogen sulfate/well, scrape, and collect the lysates in glass Chromacol vials. 2. Wash each well with 670 ml ice-cold CH3OH, collect, and transfer to the corresponding vial. 3. Prepare a mix of CHCl3:PIs from bovine brain (1.33 ml CHCl3 þ 2 mg PIs/sample). 4. Add 1.332 ml of mix/sample. 5. Vortex. 6. Centrifuge (1500 rpm, 5 min, room temperature). 7. Separate aqueous (upper) from organic (lower) phase. 8. Prepare washing mix (3.545 ml HCl 1 M þ 18.75 ml tetrabutylammonium hydrogen sulfate 1 M þ 187.5 ml EDTA 0.5 M, 5 ml CH3OH, and 10 ml CHCl3). 9. Add 1 ml of aqueous washing phase (upper) to the sample organic phase. 10. Add 1.5 ml of organic (lower) washing phase to the sample aqueous phase. 11. Vortex. 12. Centrifuge (1500 rpm, 5 min, room temperature). 13. Separate aqueous (upper) from organic (lower) phase.
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3.1.3 PIs deacylation Extracted PIs are then deacylated using a mix of monomethylamine/methanol/water/butanol to avoid that acyl chains affect the binding to the HPLC column. 1. Dry samples (speed vac). 2. Add 500 ml of monomethylamine solution (CH3NH2:CH3OH:H2O: C4H9OH 5:4:3:1). 3. Vortex. 4. Incubate at 53 C, 45 min. 5. Cool down. 6. Centrifuge samples (2500 rpm, 1 min, room temperature). 7. Dry samples (speed vac) for at least 1 h. 3.1.4 Glycerophosphoinositide extraction A mix butanol/petroleum ether/ethyl acetate is used to separate the organic phase containing the acyl chains from the aqueous phase containing the [3H] glycerophosphoinositides (gPIs). 1. Prepare a mix C4H9OH:C6H14:C4H8O2 (20:4:1). 2. Add 600 ml mix/sample. 3. Add 500 ml of distilled water/sample. 4. Vortex. 5. Centrifuge (2500 rpm, 1 min, room temperature). 6. Discard upper phase. 7. Add 600 ml mix/sample. 8. Vortex. 9. Centrifuge (2500 rpm, 1 min, room temperature). 10. Discard upper phase. 11. Dry samples (speed vac). 12. Resuspend pellet of gPIs in 600 ml H2O. 3.1.5 HPLC analysis gPIs are separated by HPLC using PartiSphere™ 5 mm strong anion exchange column (25 4.6 mm). We optimized a protocol to better separate PtdIns3P using a nonlinear gradient of 1 mM EDTA (buffer A) and 1.3 M (NH4)2HPO4 þ 1 mM EDTA (pH 3.8) (buffer B): 0–1 min, 0% buffer B; 1–40 min, 0–5% buffer B; 40–41 min, 5–15% buffer B; 41–75 min, 15–24% buffer B; 75–76 min, 24–33% buffer B; 76–95 min, 33–60% buffer B; 95–96 min, 60–100% buffer B; 96–100 min, 100% buffer B;
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100–101 min, 100% to 0% buffer B; and 101–121 min, 0% buffer B wash. Fractions are collected (1 ml/min) and radioactivity determined using appropriate scintillation liquids. 3.1.6 Pro&Con This method allows an accurate and quantitative analysis of PtdIns3P levels in cell lines and in some primary cells. One of the main limitations of this approach is the fact that many primary cells cannot be maintained in culture for a sufficient length of time to allow equilibrium radiolabeling. In addition, this technique cannot be used to directly quantify PtdIns3P levels in samples such as biopsies. Because of the many steps of the experimental procedures, it can also be less sensitive to detect trace amounts of PIs or to monitor subtle changes in specific intracellular compartments.
3.2. Liquid chromatography–mass spectrometry An experimental protocol to analyze all PIs in total lipid extract was developed a few years ago based on liquid chromatography coupled directly to a mass spectrometry detector (Pettitt, Dove, Lubben, Calaminus, & Wakelam, 2006). This approach provides a very sensitive analysis of all PIs without prior fractionation and modification. However, the experimental procedure and protocol requires specific instrument and expertise.
3.3. Novel assay to measure PtdIns3P levels Recently, a novel mass assay has been described to quantify PtdIns3P, which can potentially overcome the difficulties of primary cells radiolabeling and also allow the quantification of PtdIns3P in various biological samples such as biopsies. The method is based on the use of recombinant PYKfyve and [g-32P]ATP and the quantification of radiolabeled PtdIns(3,5)P2 from PtdIns3P (Chicanne et al., 2012). In this protocol, lipids are extracted from the sample of interest and resolved by Thin Layer Chromatography (TLC). PIs are then scraped off, extracted from silica, and then combined with phosphatidylethanolamine to allow vesicle formation. Lipid kinase assay is then performed using recombinant GST-PIKfyve and [g-32P]ATP and phosphorylated lipids are extracted and separated by TLC. The radioactive bisphosphates are then scraped off, deacylated, and analyzed by HPLC.
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4. MONITORING PtdIns3P INTRACELLULAR LOCALIZATION 4.1. PtdIns3P-binding domains The identification of protein domains specifically able to bind PtdIns3P has allowed the development of protocols to visualize the specific intracellular compartment where this PI is synthesized. Fluorescent-tagged PtdIns3Pbinding domains followed by confocal microscopy analysis and GST-tagged domains and electron microscopy analysis have been extensively used to assess PtdIns3P cellular localization. 4.1.1 Fab1/YOTB/Vac1/EEA1 domain The best characterized and most used PtdIns3P-binding domain is the Fab1/YOTB/Vac1/EEA1 (FYVE) domain, a zinc fingers module of about 60–70 amino acids (Stenmark & Aasland, 1999) consisting of two doublestranded antiparallel b-sheets and a small C-terminal a-helix. The structure is held together by two tetrahedrally coordinated Zn2þ ions (Lemmon, 2008), which are critical for preservation of the structure and function of the domain (Kutateladze, 2006). Specificity of FYVE domain for PtdIns3P seems to derive from hydrogen bonds with the 4-, 5- and 6-hydroxyl groups and with the 1- and 3-phosphates (Lemmon, 2008). Nonspecific electrostatic interaction between basic residues within the FYVE domain and negatively charged phospholipids within the membrane, such as phosphatidylserine and phosphatidic acid, in early endosomes can facilitate membrane recruitment of FYVE domain-containing proteins (Kutateladze, 2006). Dimerization is often required for proper endosomal targeting. Green Fluorescent Protein (GFP)-tagged tandem FYVE domains (GFP–2XFYVE) of specific proteins, such as early endosome antigen 1 (EEA1) and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), have been used to study PtdIns3P intracellular localization. For instance, this tool allowed the visualization of a pool of PtdIns3P in endosomes (Gillooly et al., 2000) and it also allowed to identify the de novo synthesis of PtdIns3P at the PM (Maffucci, Brancaccio, et al., 2003; Maffucci et al., 2005) or in LDCV (Wen et al., 2008). More recently, a distinct approach has been used to control dimerization of the FYVE domain probe intracellularly (Hayakawa et al., 2004; Stuffers et al., 2010). Specifically, the monomeric FYVE domain of Hrs was fused to the rapamycin-binding protein FKBP (Fv). The resulting GFP-Fv-FYVE can homodimerize in the presence of a rapamycin derivative, allowing
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controlled dimerization and endosomal targeting of the probe and more specific analysis of PtdIns3P endosomal localization (Stuffers et al., 2010). 4.1.2 Phox homology domain Phox homology (PX) domains are regions of 130 amino acids named after the two phagocyte NADPH oxidase subunits p40phox and p47phox (Kutateladze, 2007). PX domains show a highly conserved threedimensional structure consisting of three-stranded b-sheet and a subdomain of three to four a-helices. All PX domains found in S. cerevisiae bind PtdIns3P (Lemmon, 2008) although PX domains able to bind PtdIns(3,5) P2 and PtdIns(3,4)P2 have also been found in mammals. Nevertheless, it is still generally accepted that PX domains preferentially bind PtdIns3P (Lemmon, 2008). There are only few examples of PX domains able to bind PtdIns3P with high affinity and whose interaction with this PI is sufficient to target them to membranes. GFP-PX domains were used together with GFP-FYVE to visualize changes in the levels and subcellular localization of PtdIns3P during phagosome formation (Ellson et al., 2001). 4.1.3 Pleckstrin homology domain Pleckstrin homology (PH) domains are modules of about 100 amino acids (Maffucci & Falasca, 2001). Although very different in their primary structure, all PH domains possess a similar tertiary structure, consisting of a 7-stranded b-sandwich structure formed by two near-orthogonal b-sheets (Lemmon, 2008). Most PH domains show either low specificity or low affinity (or both) for PIs and require additional mechanisms to guarantee the specific targeting of the host protein (Maffucci & Falasca, 2001). Examples of PtdIns3P-binding PH domains include the amino terminal PH domain of phospholipase C (PLC)b1 (Razzini, Brancaccio, Lemmon, Guarnieri, & Falasca, 2000) and PH domain of insulin receptor substrate (IRS) 3 (Maffucci, Razzini, et al., 2003). In its tagged form, the PLCb1 PH domain first allowed us to visualize a pool of PtdIns3P specifically localized at the PM, which is important for PLCb1 activation (Razzini et al., 2000). Similarly, comparison of the intracellular localization of tagged full-length IRS3, the isolated PH domain, and mutants unable to bind PtdIns3P led us to the identification of a nuclear pool of PtdIns3P (Maffucci, Razzini, et al., 2003), later also observed using the PtdIns3Pbinding PH domain of casein kinase 2-interacting protein-1 (Safi et al., 2004).
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4.2. Immunofluorescence microscopy analysis PtdIns3P-binding PH domain has been used both in their GFP-tagged and GST-tagged forms. GFP-tagged domains allow not only direct immunofluorescence analysis on fixed cells but also in vivo imaging of PtdIns3P dynamics. 4.2.1 Sample preparation 1. Plate cells on glass coverslips at least one day before starting the experiment. If cells are stably transfected with the specific GFP-tagged or GST-tagged PtdIns3P-binding domain, go to Step 3. 2. For transient experiment, transfect cells with cDNA expressing the tagged PtdIns3P-binding domain of choice. Several transfection reagents can be used according to the specific cell lines and this needs to be optimized in order to achieve a high efficiency of transfection. 3. Overexpressed proteins are usually easily detectable 24 h posttransfection. If no cellular stimulation is required, coverslips can be fixed at this stage. Alternatively, cells can be serum-starved for an appropriate length of time (depending on the cell type, usually from 6 to 24 h), stimulated as required, and then fixed. 4. Wash coverslips three times with PBS. 5. Fix coverslips with paraformaldehyde 4% in PBS for 15–30 min at room temperature. 6. For GST domains expressing cells and to perform colocalization analysis with specific intracellular compartments markers (for instance, EEA1 for early endosomes), permeabilize cells with 0.1–0.25% Triton X-100 for 5 min at room temperature. 7. Block coverslips with 1% bovine serum albumin (BSA) in PBS for 30 min at room temperature. 8. Incubate coverslips with the required primary antibodies diluted in PBS/BSA for at least 1 h. Dilution depends on the specific primary antibody and needs to be optimized. 9. Wash coverslips three times with PBS. 10. Incubate with the required specific fluorescent secondary antibodies (for instance, Alexa 488- or Alexa 594-conjugated antibodies) for 1 h. 11. Wash coverslips two times with PBS. 12. If required, incubate with 40 ,6-diamidino-2-phenylindole to stain nuclei in PBS for 5 min. 13. Wash coverslips three times with PBS and once with H2O. 14. Mount coverslips using an antiphotobleaching agent.
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4.3. Immunofluorescence analysis: Pro&Con Coverslips can then be analyzed using an inverted fluorescence microscope. For a more precise, accurate analysis, the use of confocal microscope is suggested. Quantification of the degree of colocalization of PtdIns3P and the distinct intracellular compartments markers can be performed (Wen et al., 2008). Quantification of the number of 2XFYVE-positive vesicles/cell has also been performed in some studies (Biswas et al., 2013; Yoshioka et al., 2012). Similarly, changes in fluorescence intensity of 2XFYVE-positive vesicles have been quantified (Wen et al., 2008). Despite this, it must be noted that the immunofluorescence staining remains mainly a qualitative assay. It is not clear whether these domains are indeed able to bind all PtdIns3P intracellular pools: for instance, no nuclear localization has been detected using tagged-2XFYVE domains, indicating that these probes may selectively recognize distinct PtdIns3P pools. Another complication in data interpretation derives from the fact that overexpression of these domains can affect and impair the normal intracellular functions regulated by PtdIns3P.
4.4. Immunofluorescence using anti-PtdIns3P antibody Specific anti-PI antibodies have been developed and used in some studies to visualize the PIs. For instance, a couple of studies suggested that amino acids are able to stimulate PtdIns3P synthesis in vivo based on the use of an antiPtdIns3P antibody and indirect analysis of fluorescence (Gulati et al., 2008; Nobukuni et al., 2005). This approach was also used to detect a perinuclear PtdIns3P in zebrafish myofibers (Dowling et al., 2009), but it is still not widely accepted.
4.5. Immunoelectron microscopy analysis An alternative method to detect the specific intracellular compartment and structure where PtdIns3P accumulates is based on transfection of cells with GST-tagged FYVE domains and immunoelectron microscopy analysis using anti-GST antibody and protein A-gold (Gillooly et al., 2000; Wen et al., 2008).
5. ENDOSOMAL PtdIns3P 5.1. Regulation of the endosomal pool of PtdIns3P Different pools of PtdIns3P have been detected intracellularly, a “constitutive” pool, already detectable in resting cells, and regulated pools, generated
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upon cellular stimulation (Falasca & Maffucci, 2009). Originally, studies showed that the “constitutive” PtdIns3P is specifically localized in endosomes, mostly on early endosomal membranes and on the internal membranes of multivesicular endosomes (Gillooly et al., 2000). Spatiotemporal analysis of PtdIns3P endosomal distribution, obtained using the “controlled dimerization” of FYVE domain, has revealed that the GFP-Fv-FYVE domain associates very rapidly with EEA1-positive, early endosomes compartment and only later with CD63-containing late endosomes (Stuffers et al., 2010). It is generally accepted that hVps34 is responsible for regulation of this PtdIns3P pool. However, the observation that downregulation of hVps34 affects late endosomal structure but not early endosome morphology and trafficking pathways ( Johnson, Overmeyer, Gunning, & Maltese, 2006) first suggested that hVps34 might not be the only enzyme responsible for the synthesis of the endosomal PtdIns3P. Indeed, it was later shown that PtdIns3P can be generated in early endosomes through an enzymatic cascade involving class I PI3K and the sequential action of PI 5- and PI 4-phosphatases (Shin et al., 2005). More recently, it has been reported that downregulation of PI3K-C2a in endothelial cells decreases the number of PtdIns3P-enriched endosomes (Biswas et al., 2013; Yoshioka et al., 2012), suggesting that class II PI3K(s) can also be involved in regulation of endosomal PtdIns3P. Finally, the endosomal pool of PtdIns3P is also regulated by phosphatases, specifically MTM1, which localizes to Rab5-positive early endosomes (Cao, Laporte, Backer, Wandinger-Ness, & Stein, 2007) where it regulates PtdIns3P levels (Cao, Backer, Laporte, Bedrick, & Wandinger-Ness, 2008), and MTMR2, which localizes to Rab7-positive late endosomes (Cao et al., 2007) where it can regulate late endosomal PtdIns3P levels (Cao et al., 2008).
5.2. Cellular functions regulated by the endosomal pool of PtdIns3P The endosomal pool of PtdIns3P controls membrane transport and membrane dynamics mostly by recruiting key proteins containing FYVE, PX, and PH domains (Lindmo & Stenmark, 2006) including EEA1, which has a critical role in endosome fusion, and Hrs, which regulates the first steps of receptor sorting and internalization within the multivesicular bodies (MVBs). Endosomal pool of PtdIns3P can also contribute to growth factor signaling through regulation of maturation of an early endocytic intermediate (Zoncu et al., 2009).
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It is now becoming increasingly clear that the endosomal PtdIns3P has also a role in the control of autophagy. PtdIns3P has a well-established and key role in various steps of the autophagy process, including autophagosome biogenesis, maturation, and intracellular transport (Dall’Armi, Devereaux, & Di Paolo, 2013). Specifically related to the endosomal PtdIns3P pool is the control of the maturation steps, which involve fusion of the autophagosome with the endosomes, generating the amphisomes that then fuse with the lysosomes (Dall’Armi et al., 2013).
6. PHOSPHATIDYLINOSITOL 3,5-BISPHOSPHATE 6.1. Intracellular levels PtdIns(3,5)P2 was first identified in a study using [3H]inositol-labeled S. cerevisiae grown almost to isotopic equilibrium in an inositol-free and/or low-phosphate minimal medium and HPLC analysis (Dove et al., 1997). Subsequent studies using this technique were performed in yeasts (Dove et al., 2002; Phelan, Millson, Parker, Piper, & Cooke, 2006) and mammalian cell lines ( Jefferies et al., 2008; Sbrissa & Shisheva, 2005).
6.2. Intracellular localization Because specific PtdIns(3,5)P2-binding domains have not been described, information on the intracellular localization of this PI has mostly been derived from the localization of PIKfyve. The endogenous enzyme localizes to discrete peripheral punctae resembling endosomes in 3T3L1 adipocytes (Shisheva, 2008). Endosomal localization (both on early and late endosomes) and localization in MVBs and at the trans-Golgi network was reported for the overexpressed wild-type PIKfyve. However, it has also been suggested that the localization is strongly dependent on the cell type, the level of protein expressed, and on the ratio between PtdIns(3,5)P2 and PtdIns3P (Shisheva, 2008).
6.3. Intracellular roles PtdIns(3,5)P2 has a well-established role in membrane trafficking and it is currently believed that specific cargo molecules can stimulate synthesis of this PI to regulate their correct endosome–lysosome trafficking (Dove et al., 2009). Accumulation of swollen intracellular vacuoles, enlarged late endosomes, and abnormal MVB has been observed in cultured cells, in animal models, and in human tissues as a result of defects in PtdIns(3,5)P2
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synthesis. Recent data have also revealed a critical role for PtdIns(3,5)P2 in autophagy in the central nervous system (Ferguson, Lenk, & Meisler, 2009, 2010).
ACKNOWLEDGMENTS Work in our laboratory is supported by Pancreatic Cancer Research Fund (M. F.), Bowel and Cancer Research (M. F.), Prostate Cancer UK (M. F.), Diabetes UK (T. M.) and Barts and The London Charity-Cancer Fund (T. M.).
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Maffucci, T., Brancaccio, A., Piccolo, E., Stein, R. C., & Falasca, M. (2003). Insulin induces phosphatidylinositol-3-phosphate formation through TC10 activation. The EMBO Journal, 22, 4178–4189. Maffucci, T., Cooke, F. T., Foster, F. M., Traer, C. J., Fry, M. J., & Falasca, M. (2005). Class II phosphoinositide 3-kinase defines a novel signaling pathway in cell migration. The Journal of Cell Biology, 169, 789–799. Maffucci, T., & Falasca, M. (2001). Specificity in pleckstrin homology (PH) domain membrane targeting: A role for a phosphoinositide-protein co-operative mechanism. FEBS Letters, 506, 173–179. Maffucci, T., Razzini, G., Ingrosso, A., Chen, H., Iacobelli, S., Sciacchitano, S., et al. (2003). Role of pleckstrin homology domain in regulating membrane targeting and metabolic function of insulin receptor substrate 3. Molecular Endocrinology, 17, 1568–1579. Mazza, S., & Maffucci, T. (2011). Class II phosphoinositide 3-kinase C2alpha: What we learned so far. International Journal of Biochemistry and Molecular Biology, 2, 168–182. Michell, R. H. (2008). Inositol derivatives: Evolution and functions. Nature Reviews Molecular Cell Biology, 9, 151–161. Michell, R. H., Heath, V. L., Lemmon, M. A., & Dove, S. K. (2006). Phosphatidylinositol 3,5-bisphosphate: Metabolism and cellular functions. Trends in Biochemical Sciences, 31, 52–63. Misawa, H., Ohtsubo, M., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., & Yoshimura, A. (1998). Cloning and characterization of a novel class II phosphoinositide 3-kinase containing C2 domain. Biochemical and Biophysical Research Communications, 244, 531–539. Nicot, A. S., & Laporte, J. (2008). Endosomal phosphoinositides and human diseases. Traffic, 9, 1240–1249. Nobukuni, T., Joaquin, M., Roccio, M., Dann, S. G., Kim, S. Y., Gulati, P., et al. (2005). Amino acids mediate mTOR/raptor signaling through activation of class 3 phosphatidylinositol 3OH-kinase. Proceedings of the National Academy of Sciences of the United States of America, 102, 14238–14243. Ono, F., Nakagawa, T., Saito, S., Owada, Y., Sakagami, H., Goto, K., et al. (1998). A novel class II phosphoinositide 3-kinase predominantly expressed in the liver and its enhanced expression during liver regeneration. The Journal of Biological Chemistry, 273, 7731–7736. Ooms, L. M., Horan, K. A., Rahman, P., Seaton, G., Gurung, R., Kethesparan, D., et al. (2009). The role of the inositol polyphosphate 5-phosphatases in cellular function and human disease. The Biochemical Journal, 419, 29–49. Pettitt, T. R., Dove, S. K., Lubben, A., Calaminus, S. D., & Wakelam, M. J. (2006). Analysis of intact phosphoinositides in biological samples. Journal of Lipid Research, 47, 1588–1596. Phelan, J. P., Millson, S. H., Parker, P. J., Piper, P. W., & Cooke, F. T. (2006). Fab1p and AP-1 are required for trafficking of endogenously ubiquitylated cargoes to the vacuole lumen in S. cerevisiae. Journal of Cell Science, 119, 4225–4234. Razzini, G., Brancaccio, A., Lemmon, M. A., Guarnieri, S., & Falasca, M. (2000). The role of the pleckstrin homology domain in membrane targeting and activation of phospholipase Cbeta(1). The Journal of Biological Chemistry, 275, 14873–14881. Robinson, F. L., & Dixon, J. E. (2006). Myotubularin phosphatases: Policing 3-phosphoinositides. Trends in Cell Biology, 16, 403–412. Rozycka, M., Lu, Y. J., Brown, R. A., Lau, M. R., Shipley, J. M., & Fry, M. J. (1998). cDNA cloning of a third human C2-domain-containing class II phosphoinositide 3-kinase, PI3K-C2gamma, and chromosomal assignment of this gene (PIK3C2G) to 12p12. Genomics, 54, 569–574. Safi, S., Vandromme, M., Caussanel, S., Valdacci, L., Baas, D., Vidal, M., et al. (2004). Role for the pleckstrin homology domain-containing protein CKIP-1 in phosphatidylinositol 3-kinase-regulated muscle differentiation. Molecular and Cellular Biology, 24, 1245–1255.
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Sbrissa, D., & Shisheva, A. (2005). Acquisition of unprecedented phosphatidylinositol 3,5bisphosphate rise in hyperosmotically stressed 3T3-L1 adipocytes, mediated by ArPIKfyve-PIKfyve pathway. The Journal of Biological Chemistry, 280, 7883–7889. Shin, H. W., Hayashi, M., Christoforidis, S., Lacas-Gervais, S., Hoepfner, S., Wenk, M. R., et al. (2005). An enzymatic cascade of Rab5 effectors regulates phosphoinositide turnover in the endocytic pathway. The Journal of Cell Biology, 170, 607–618. Shisheva, A. (2008). PIKfyve: Partners, significance, debates and paradoxes. Cell Biology International, 32, 591–604. Stenmark, H., & Aasland, R. (1999). FYVE-finger proteins—Effectors of inositol lipid. Journal of Cell Science, 112, 4175–4183. Stephens, L., McGregor, A., & Hawkins, P. (2000). Phosphoinositide 3-kinases: Regulation by cell-surface receptors and functions of 3-phosphorylated lipids. In S. Cockcroft (Ed.), Biology of phosphoinositides. Oxford, UK: Oxford Univ. Press. Stuffers, S., Malerød, L., Schink, K. O., Corvera, S., Stenmark, H., & Brech, A. (2010). Time-resolved ultrastructural detection of phosphatidylinositol 3-phosphate. The Journal of Histochemistry and Cytochemistry, 58, 1025–1032. Tibolla, G., Pin˜eiro, R., Chiozzotto, D., Mavrommati, I., Wheeler, A. P., Norata, G. D., et al. (2013). Class II phosphoinositide 3-kinases contribute to endothelial cells morphogenesis. PLoS One, 8, e53808. Vanhaesebroeck, B., Guillermet-Guibert, J., Graupera, M., & Bilanges, B. (2010). The emerging mechanisms of isoform-specific PI3K signalling. Nature Reviews Molecular Cell Biology, 11, 329–341. Visnjic´, D., Curic´, J., Crljen, V., Batinic´, D., Volinia, S., & Banfic´, H. (2003). Nuclear phosphoinositide 3-kinase C2beta activation during G2/M phase of the cell cycle in HL-60 cells. Biochimica et Biophysica Acta, 631, 61–71. Wen, P. J., Osborne, S. L., Morrow, I. C., Parton, R. G., Domin, J., & Meunier, F. A. (2008). Ca2þ -regulated pool of phosphatidylinositol-3-phosphate produced by phosphatidylinositol 3-kinase C2alpha on neurosecretory vesicles. Molecular Biology of the Cell, 12, 5593–5603. Yoshioka, K., Yoshida, K., Cui, H., Wakayama, T., Takuwa, N., Okamoto, Y., et al. (2012). Endothelial PI3K-C2a, a class II PI3K, has an essential role in angiogenesis and vascular barrier function. Nature Medicine, 18, 1560–1569. Zoncu, R., Perera, R. M., Balkin, D. M., Pirruccello, M., Toomre, D., & De Camilli, P. (2009). A phosphoinositide switch controls the maturation and signaling properties of APPL endosomes. Cell, 136, 1110–1121.
Analysis, Regulation, and Roles of Endosomal Phosphoinositides Tania Maffucci, Marco Falasca1 Inositide Signalling Group, Centre for Diabetes, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Endosomal PIs 2.1 Phosphatidylinositol 3-phosphate 2.2 Phosphatidylinositol 3,5-bisphosphate 3. Analysis of PtdIns3P Levels 3.1 Metabolic labeling and HPLC analysis 3.2 Liquid chromatography–mass spectrometry 3.3 Novel assay to measure PtdIns3P levels 4. Monitoring PtdIns3P Intracellular Localization 4.1 PtdIns3P-binding domains 4.2 Immunofluorescence microscopy analysis 4.3 Immunofluorescence analysis: Pro&Con 4.4 Immunofluorescence using anti-PtdIns3P antibody 4.5 Immunoelectron microscopy analysis 5. Endosomal PtdIns3P 5.1 Regulation of the endosomal pool of PtdIns3P 5.2 Cellular functions regulated by the endosomal pool of PtdIns3P 6. Phosphatidylinositol 3,5-Bisphosphate 6.1 Intracellular levels 6.2 Intracellular localization 6.3 Intracellular roles Acknowledgments References
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Abstract Phosphoinositides (PIs) are minor lipid components of cellular membranes that play critical roles in membrane dynamics, trafficking, and cellular signaling. Among the seven naturally occurring PIs, the monophosphate phosphatidylinositol 3-phosphate (PtdIns3P) and the bisphosphate phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] have been mainly associated with endosomes and endosomal functions. Metabolic labeling and HPLC analysis revealed that a bulk of PtdIns3P is constitutively present Methods in Enzymology, Volume 535 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-397925-4.00005-5
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in cells, making it the only detectable product of the enzymes phosphoinositide 3-kinases in unstimulated, normal cells. The use of specific tagged-PtdIns3P-binding domains later demonstrated that this constitutive PtdIns3P accumulates in endosomes where it critically regulates trafficking and membrane dynamics.
1. INTRODUCTION Phosphoinositides (PIs) are phospholipids comprising a water-soluble head group (myo-inositol) linked by a glycerol moiety to two fatty acid chains, usually a saturated C18 residue (stearoyl) in the 1-position and a tetra-unsaturated C20 residue (arachidonoyl) in the 2-position (Michell, 2008). The founding member of the PIs family is the unphosphorylated phosphatidylinositol (PtdIns), primarily synthesized in the endoplasmic reticulum and then delivered to other membranes by vesicular transport or via cytosolic PtdIns transfer protein (Di Paolo & De Camilli, 2006). PIs derive from phosphorylation of the hydroxyls at positions 3-, 4- and 5- within the myo-inositol headgroup of PtdIns and the different possible combinations generate seven distinct derivatives, which can be interconverted into each other by the action of specific kinases or phosphatases. PIs play many intracellular roles either as component of cellular membranes or by regulating the activity of targeted proteins temporally and/or spatially. Because of their lipid tail, PIs are obligatory membrane-bound; therefore, they can mark specific membrane compartments or subdomains within a membrane. On the other hand, PIs also possess a soluble headgroup, which allows them to bind to cytosolic proteins and to mediate the association of these proteins to specific membranes (spatial regulation). In addition, since some PIs are only synthesized upon cellular stimulation in normal cells, they can also regulate activation of target proteins temporally (Maffucci, 2012).
2. ENDOSOMAL PIs Two PIs have been mainly associated with endosomes and endosomal functions: the monophosphate phosphatidylinositol 3-phosphate (PtdIns3P) and the bisphosphate phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] (Nicot & Laporte, 2008).
2.1. Phosphatidylinositol 3-phosphate PtdIns3P comprises 0.1–0.5% of all PIs (Falasca & Maffucci, 2006) and almost 0.002% of total membrane lipids (Stephens, McGregor, &
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Hawkins, 2000). PtdIns3P is the most abundant among the PIs phosphorylated at position 3 in resting mammalian cells and its intracellular levels are regulated by a coordinated action of kinases and phosphatases (Falasca & Maffucci, 2006, 2009). Synthesis of PtdIns3P is catalyzed by members of the family of enzymes phosphoinositide 3-kinase (PI3K). Three classes of PI3Ks exist (Falasca & Maffucci, 2007; Vanhaesebroeck, Guillermet-Guibert, Graupera, & Bilanges, 2010) and synthesis of PtdIns3P occurs in vivo through phosphorylation of PtdIns by class II or class III PI3K isoforms. No evidence so far has supported a role for class I PI3Ks in direct synthesis of PtdIns3P from PtdIns in vivo. Class III comprises only one PI3K isoform, vacuolar protein sorting 34 (Vps34), which is the most ancient forms of PI3Ks and the only one conserved from lower eukaryotes to plants and mammals (Backer, 2008; Engelman, Luo, & Cantley, 2006). It is still not clear whether the activity of Vps34 is modulated by cellular stimulation (Vanhaesebroeck et al., 2010) although it has been suggested that the basal activation of hVps34 may be regulated by nutrients (Byfield, Murray, & Backer, 2005). In contrast to the other PI3Ks, which can produce three 3-phosphorylated PIs at least in vitro (PtdIns3P, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate), hVps34 can only generate PtdIns3P (Backer, 2008). Class II comprises three PI3K isoforms, PI3K-C2a (Domin et al., 1997), PI3K-C2b (Brown, Ho, Weber-Hall, Shipley, & Fry, 1997), and PI3K-C2g (Misawa et al., 1998; Ono et al., 1998; Rozycka et al., 1998). Several studies have now demonstrated that at least some members of the class II PI3K subfamily catalyze synthesis of PtdIns3P in vivo (Falasca & Maffucci, 2012; Mazza & Maffucci, 2011). PI3K-C2a regulates an insulin-dependent, plasma membrane (PM)-associated pool of PtdIns3P in muscle cells (Falasca et al., 2007; Maffucci, Brancaccio, Piccolo, Stein, & Falasca, 2003) and it generates PtdIns3P in large densecore vesicles (LDCVs) of PC12 upon stimulation of exocytosis (Wen et al., 2008). Recently, studies using MEFs upon induction of PI3K-C2a knockout (Yoshioka et al., 2012) and endothelial cells (Biswas et al., 2013) have confirmed PtdIns3P as the main specific product of PI3KC2a. Similarly, PI3K-C2b regulates lysophosphatidic acid-induced and sphingosine-1-phosphate-dependent synthesis of PtdIns3P in cancer (Maffucci et al., 2005) and endothelial cells (Tibolla et al., 2013), respectively. It has also been proposed that PI3K-C2b regulates a nuclear pool of PtdIns3P during cell cycle progression (Visnjic´ et al., 2003). As far as we know, no study so far has investigated whether PI3K-C2g also catalyzes PtdIns3P synthesis in vivo.
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Levels of PtdIns3P are further regulated through dephosphorylation at position 3 by several phosphatases, mainly belonging to the family of myotubularins (MTMs; Robinson & Dixon, 2006). Fourteen MTMs have been detected in humans, nearly half of which are predicted to be catalytically inactive. Recombinant myotubularin MTM1 and myotubularinrelated (MTMR) 1, 2, 3, 4, 6, and 7 were initially reported to selectively dephosphorylate PtdIns3P although it was later shown that some of these (such as MTM1 and MTMR2, 3, and 6) can also dephosphorylate PtdIns(3,5)P2 (Maffucci, 2012).
2.2. Phosphatidylinositol 3,5-bisphosphate PtdIns(3,5)P2 represents 0.0001% of total membrane lipids (Stephens et al., 2000) and it is usually only 0.1% or less of total cellular PIs in unstressed mammalian cells (Michell, Heath, Lemmon, & Dove, 2006). It is generally accepted that PtdIns(3,5)P2 derives from phosphorylation of PtdIns3P by a member of the family of type III PIPKs (Dove, Dong, Kobayashi, Williams, & Michell, 2009), mainly PI kinase for five positions containing a FYVE finger (PIKfyve) whose activation involves formation of a complex with the activator ArPIKfyve (Shisheva, 2008). PtdIns(3,5)P2 dephosphorylation can be mediated by different phosphatases including the 5-phosphatase FIG4/Sac3 and the scaffolding protein VAC14 although Sac3 can also associate with the complex PIKfyve/ArPIKfyve and promote maximal PtdIns(3,5)P2 synthesis. It has also been suggested that inactive MTMs can dimerize with active MTMs and change their specificity towards this PI rather than PtdIns3P (Lecompte, Poch, & Laporte, 2008). Other 5-phosphatases have been proposed to be able to dephosphorylate PtdIns(3,5)P2 including 72 kDa 5-phosphatase/INPP5E or SHIP2 (Ooms et al., 2009), but it is not known whether the endogenous enzymes have a role in modulating the levels of PtdIns(3,5)P2 in vivo.
3. ANALYSIS OF PtdIns3P LEVELS 3.1. Metabolic labeling and HPLC analysis The classical experimental procedure to quantitatively measure the intracellular levels of PtdIns3P involves labeling of cells with myo-[3H] inositol, lipid extraction, and HPLC analysis. Alternatively, cells can be labeled with [32P] orthophosphate.
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3.1.1 Cellular labeling Incorporation of myo-[3H] inositol is performed in inositol-free media for the time required to reach isotopic equilibrium, which can vary between cellular types and therefore needs to be optimized for the specific cell system to be used. Labeling can be performed in the presence or absence of serum depending on the cell types and the efficiency of incorporation. Similarly, optimization is required to determine the amount of myo-[3H] inositol, which allows detection of the PIs of interest. In our studies, we typically used 5 mCi myo-[3H] inositol/ml (Falasca et al., 2007; Maffucci et al., 2005). The following protocol refers to cells plated in a 6-well plate.
3.1.2 Lipid extraction Following incorporation, cells are lysed and lipids are extracted by phase separation using a mix of water/hydrochloric acid/methanol/chloroform. [3H]PIs containing two acyl chains accumulate in the organic phase, whereas the water-soluble [3H]inositol phosphates accumulate in the aqueous phase. 1. Lyse cells with 500 ml ice-cold HCl 1 M þ 1 mM tetrabutylammonium hydrogen sulfate/well, scrape, and collect the lysates in glass Chromacol vials. 2. Wash each well with 670 ml ice-cold CH3OH, collect, and transfer to the corresponding vial. 3. Prepare a mix of CHCl3:PIs from bovine brain (1.33 ml CHCl3 þ 2 mg PIs/sample). 4. Add 1.332 ml of mix/sample. 5. Vortex. 6. Centrifuge (1500 rpm, 5 min, room temperature). 7. Separate aqueous (upper) from organic (lower) phase. 8. Prepare washing mix (3.545 ml HCl 1 M þ 18.75 ml tetrabutylammonium hydrogen sulfate 1 M þ 187.5 ml EDTA 0.5 M, 5 ml CH3OH, and 10 ml CHCl3). 9. Add 1 ml of aqueous washing phase (upper) to the sample organic phase. 10. Add 1.5 ml of organic (lower) washing phase to the sample aqueous phase. 11. Vortex. 12. Centrifuge (1500 rpm, 5 min, room temperature). 13. Separate aqueous (upper) from organic (lower) phase.
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3.1.3 PIs deacylation Extracted PIs are then deacylated using a mix of monomethylamine/methanol/water/butanol to avoid that acyl chains affect the binding to the HPLC column. 1. Dry samples (speed vac). 2. Add 500 ml of monomethylamine solution (CH3NH2:CH3OH:H2O: C4H9OH 5:4:3:1). 3. Vortex. 4. Incubate at 53 C, 45 min. 5. Cool down. 6. Centrifuge samples (2500 rpm, 1 min, room temperature). 7. Dry samples (speed vac) for at least 1 h. 3.1.4 Glycerophosphoinositide extraction A mix butanol/petroleum ether/ethyl acetate is used to separate the organic phase containing the acyl chains from the aqueous phase containing the [3H] glycerophosphoinositides (gPIs). 1. Prepare a mix C4H9OH:C6H14:C4H8O2 (20:4:1). 2. Add 600 ml mix/sample. 3. Add 500 ml of distilled water/sample. 4. Vortex. 5. Centrifuge (2500 rpm, 1 min, room temperature). 6. Discard upper phase. 7. Add 600 ml mix/sample. 8. Vortex. 9. Centrifuge (2500 rpm, 1 min, room temperature). 10. Discard upper phase. 11. Dry samples (speed vac). 12. Resuspend pellet of gPIs in 600 ml H2O. 3.1.5 HPLC analysis gPIs are separated by HPLC using PartiSphere™ 5 mm strong anion exchange column (25 4.6 mm). We optimized a protocol to better separate PtdIns3P using a nonlinear gradient of 1 mM EDTA (buffer A) and 1.3 M (NH4)2HPO4 þ 1 mM EDTA (pH 3.8) (buffer B): 0–1 min, 0% buffer B; 1–40 min, 0–5% buffer B; 40–41 min, 5–15% buffer B; 41–75 min, 15–24% buffer B; 75–76 min, 24–33% buffer B; 76–95 min, 33–60% buffer B; 95–96 min, 60–100% buffer B; 96–100 min, 100% buffer B;
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100–101 min, 100% to 0% buffer B; and 101–121 min, 0% buffer B wash. Fractions are collected (1 ml/min) and radioactivity determined using appropriate scintillation liquids. 3.1.6 Pro&Con This method allows an accurate and quantitative analysis of PtdIns3P levels in cell lines and in some primary cells. One of the main limitations of this approach is the fact that many primary cells cannot be maintained in culture for a sufficient length of time to allow equilibrium radiolabeling. In addition, this technique cannot be used to directly quantify PtdIns3P levels in samples such as biopsies. Because of the many steps of the experimental procedures, it can also be less sensitive to detect trace amounts of PIs or to monitor subtle changes in specific intracellular compartments.
3.2. Liquid chromatography–mass spectrometry An experimental protocol to analyze all PIs in total lipid extract was developed a few years ago based on liquid chromatography coupled directly to a mass spectrometry detector (Pettitt, Dove, Lubben, Calaminus, & Wakelam, 2006). This approach provides a very sensitive analysis of all PIs without prior fractionation and modification. However, the experimental procedure and protocol requires specific instrument and expertise.
3.3. Novel assay to measure PtdIns3P levels Recently, a novel mass assay has been described to quantify PtdIns3P, which can potentially overcome the difficulties of primary cells radiolabeling and also allow the quantification of PtdIns3P in various biological samples such as biopsies. The method is based on the use of recombinant PYKfyve and [g-32P]ATP and the quantification of radiolabeled PtdIns(3,5)P2 from PtdIns3P (Chicanne et al., 2012). In this protocol, lipids are extracted from the sample of interest and resolved by Thin Layer Chromatography (TLC). PIs are then scraped off, extracted from silica, and then combined with phosphatidylethanolamine to allow vesicle formation. Lipid kinase assay is then performed using recombinant GST-PIKfyve and [g-32P]ATP and phosphorylated lipids are extracted and separated by TLC. The radioactive bisphosphates are then scraped off, deacylated, and analyzed by HPLC.
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4. MONITORING PtdIns3P INTRACELLULAR LOCALIZATION 4.1. PtdIns3P-binding domains The identification of protein domains specifically able to bind PtdIns3P has allowed the development of protocols to visualize the specific intracellular compartment where this PI is synthesized. Fluorescent-tagged PtdIns3Pbinding domains followed by confocal microscopy analysis and GST-tagged domains and electron microscopy analysis have been extensively used to assess PtdIns3P cellular localization. 4.1.1 Fab1/YOTB/Vac1/EEA1 domain The best characterized and most used PtdIns3P-binding domain is the Fab1/YOTB/Vac1/EEA1 (FYVE) domain, a zinc fingers module of about 60–70 amino acids (Stenmark & Aasland, 1999) consisting of two doublestranded antiparallel b-sheets and a small C-terminal a-helix. The structure is held together by two tetrahedrally coordinated Zn2þ ions (Lemmon, 2008), which are critical for preservation of the structure and function of the domain (Kutateladze, 2006). Specificity of FYVE domain for PtdIns3P seems to derive from hydrogen bonds with the 4-, 5- and 6-hydroxyl groups and with the 1- and 3-phosphates (Lemmon, 2008). Nonspecific electrostatic interaction between basic residues within the FYVE domain and negatively charged phospholipids within the membrane, such as phosphatidylserine and phosphatidic acid, in early endosomes can facilitate membrane recruitment of FYVE domain-containing proteins (Kutateladze, 2006). Dimerization is often required for proper endosomal targeting. Green Fluorescent Protein (GFP)-tagged tandem FYVE domains (GFP–2XFYVE) of specific proteins, such as early endosome antigen 1 (EEA1) and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), have been used to study PtdIns3P intracellular localization. For instance, this tool allowed the visualization of a pool of PtdIns3P in endosomes (Gillooly et al., 2000) and it also allowed to identify the de novo synthesis of PtdIns3P at the PM (Maffucci, Brancaccio, et al., 2003; Maffucci et al., 2005) or in LDCV (Wen et al., 2008). More recently, a distinct approach has been used to control dimerization of the FYVE domain probe intracellularly (Hayakawa et al., 2004; Stuffers et al., 2010). Specifically, the monomeric FYVE domain of Hrs was fused to the rapamycin-binding protein FKBP (Fv). The resulting GFP-Fv-FYVE can homodimerize in the presence of a rapamycin derivative, allowing
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controlled dimerization and endosomal targeting of the probe and more specific analysis of PtdIns3P endosomal localization (Stuffers et al., 2010). 4.1.2 Phox homology domain Phox homology (PX) domains are regions of 130 amino acids named after the two phagocyte NADPH oxidase subunits p40phox and p47phox (Kutateladze, 2007). PX domains show a highly conserved threedimensional structure consisting of three-stranded b-sheet and a subdomain of three to four a-helices. All PX domains found in S. cerevisiae bind PtdIns3P (Lemmon, 2008) although PX domains able to bind PtdIns(3,5) P2 and PtdIns(3,4)P2 have also been found in mammals. Nevertheless, it is still generally accepted that PX domains preferentially bind PtdIns3P (Lemmon, 2008). There are only few examples of PX domains able to bind PtdIns3P with high affinity and whose interaction with this PI is sufficient to target them to membranes. GFP-PX domains were used together with GFP-FYVE to visualize changes in the levels and subcellular localization of PtdIns3P during phagosome formation (Ellson et al., 2001). 4.1.3 Pleckstrin homology domain Pleckstrin homology (PH) domains are modules of about 100 amino acids (Maffucci & Falasca, 2001). Although very different in their primary structure, all PH domains possess a similar tertiary structure, consisting of a 7-stranded b-sandwich structure formed by two near-orthogonal b-sheets (Lemmon, 2008). Most PH domains show either low specificity or low affinity (or both) for PIs and require additional mechanisms to guarantee the specific targeting of the host protein (Maffucci & Falasca, 2001). Examples of PtdIns3P-binding PH domains include the amino terminal PH domain of phospholipase C (PLC)b1 (Razzini, Brancaccio, Lemmon, Guarnieri, & Falasca, 2000) and PH domain of insulin receptor substrate (IRS) 3 (Maffucci, Razzini, et al., 2003). In its tagged form, the PLCb1 PH domain first allowed us to visualize a pool of PtdIns3P specifically localized at the PM, which is important for PLCb1 activation (Razzini et al., 2000). Similarly, comparison of the intracellular localization of tagged full-length IRS3, the isolated PH domain, and mutants unable to bind PtdIns3P led us to the identification of a nuclear pool of PtdIns3P (Maffucci, Razzini, et al., 2003), later also observed using the PtdIns3Pbinding PH domain of casein kinase 2-interacting protein-1 (Safi et al., 2004).
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4.2. Immunofluorescence microscopy analysis PtdIns3P-binding PH domain has been used both in their GFP-tagged and GST-tagged forms. GFP-tagged domains allow not only direct immunofluorescence analysis on fixed cells but also in vivo imaging of PtdIns3P dynamics. 4.2.1 Sample preparation 1. Plate cells on glass coverslips at least one day before starting the experiment. If cells are stably transfected with the specific GFP-tagged or GST-tagged PtdIns3P-binding domain, go to Step 3. 2. For transient experiment, transfect cells with cDNA expressing the tagged PtdIns3P-binding domain of choice. Several transfection reagents can be used according to the specific cell lines and this needs to be optimized in order to achieve a high efficiency of transfection. 3. Overexpressed proteins are usually easily detectable 24 h posttransfection. If no cellular stimulation is required, coverslips can be fixed at this stage. Alternatively, cells can be serum-starved for an appropriate length of time (depending on the cell type, usually from 6 to 24 h), stimulated as required, and then fixed. 4. Wash coverslips three times with PBS. 5. Fix coverslips with paraformaldehyde 4% in PBS for 15–30 min at room temperature. 6. For GST domains expressing cells and to perform colocalization analysis with specific intracellular compartments markers (for instance, EEA1 for early endosomes), permeabilize cells with 0.1–0.25% Triton X-100 for 5 min at room temperature. 7. Block coverslips with 1% bovine serum albumin (BSA) in PBS for 30 min at room temperature. 8. Incubate coverslips with the required primary antibodies diluted in PBS/BSA for at least 1 h. Dilution depends on the specific primary antibody and needs to be optimized. 9. Wash coverslips three times with PBS. 10. Incubate with the required specific fluorescent secondary antibodies (for instance, Alexa 488- or Alexa 594-conjugated antibodies) for 1 h. 11. Wash coverslips two times with PBS. 12. If required, incubate with 40 ,6-diamidino-2-phenylindole to stain nuclei in PBS for 5 min. 13. Wash coverslips three times with PBS and once with H2O. 14. Mount coverslips using an antiphotobleaching agent.
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4.3. Immunofluorescence analysis: Pro&Con Coverslips can then be analyzed using an inverted fluorescence microscope. For a more precise, accurate analysis, the use of confocal microscope is suggested. Quantification of the degree of colocalization of PtdIns3P and the distinct intracellular compartments markers can be performed (Wen et al., 2008). Quantification of the number of 2XFYVE-positive vesicles/cell has also been performed in some studies (Biswas et al., 2013; Yoshioka et al., 2012). Similarly, changes in fluorescence intensity of 2XFYVE-positive vesicles have been quantified (Wen et al., 2008). Despite this, it must be noted that the immunofluorescence staining remains mainly a qualitative assay. It is not clear whether these domains are indeed able to bind all PtdIns3P intracellular pools: for instance, no nuclear localization has been detected using tagged-2XFYVE domains, indicating that these probes may selectively recognize distinct PtdIns3P pools. Another complication in data interpretation derives from the fact that overexpression of these domains can affect and impair the normal intracellular functions regulated by PtdIns3P.
4.4. Immunofluorescence using anti-PtdIns3P antibody Specific anti-PI antibodies have been developed and used in some studies to visualize the PIs. For instance, a couple of studies suggested that amino acids are able to stimulate PtdIns3P synthesis in vivo based on the use of an antiPtdIns3P antibody and indirect analysis of fluorescence (Gulati et al., 2008; Nobukuni et al., 2005). This approach was also used to detect a perinuclear PtdIns3P in zebrafish myofibers (Dowling et al., 2009), but it is still not widely accepted.
4.5. Immunoelectron microscopy analysis An alternative method to detect the specific intracellular compartment and structure where PtdIns3P accumulates is based on transfection of cells with GST-tagged FYVE domains and immunoelectron microscopy analysis using anti-GST antibody and protein A-gold (Gillooly et al., 2000; Wen et al., 2008).
5. ENDOSOMAL PtdIns3P 5.1. Regulation of the endosomal pool of PtdIns3P Different pools of PtdIns3P have been detected intracellularly, a “constitutive” pool, already detectable in resting cells, and regulated pools, generated
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upon cellular stimulation (Falasca & Maffucci, 2009). Originally, studies showed that the “constitutive” PtdIns3P is specifically localized in endosomes, mostly on early endosomal membranes and on the internal membranes of multivesicular endosomes (Gillooly et al., 2000). Spatiotemporal analysis of PtdIns3P endosomal distribution, obtained using the “controlled dimerization” of FYVE domain, has revealed that the GFP-Fv-FYVE domain associates very rapidly with EEA1-positive, early endosomes compartment and only later with CD63-containing late endosomes (Stuffers et al., 2010). It is generally accepted that hVps34 is responsible for regulation of this PtdIns3P pool. However, the observation that downregulation of hVps34 affects late endosomal structure but not early endosome morphology and trafficking pathways ( Johnson, Overmeyer, Gunning, & Maltese, 2006) first suggested that hVps34 might not be the only enzyme responsible for the synthesis of the endosomal PtdIns3P. Indeed, it was later shown that PtdIns3P can be generated in early endosomes through an enzymatic cascade involving class I PI3K and the sequential action of PI 5- and PI 4-phosphatases (Shin et al., 2005). More recently, it has been reported that downregulation of PI3K-C2a in endothelial cells decreases the number of PtdIns3P-enriched endosomes (Biswas et al., 2013; Yoshioka et al., 2012), suggesting that class II PI3K(s) can also be involved in regulation of endosomal PtdIns3P. Finally, the endosomal pool of PtdIns3P is also regulated by phosphatases, specifically MTM1, which localizes to Rab5-positive early endosomes (Cao, Laporte, Backer, Wandinger-Ness, & Stein, 2007) where it regulates PtdIns3P levels (Cao, Backer, Laporte, Bedrick, & Wandinger-Ness, 2008), and MTMR2, which localizes to Rab7-positive late endosomes (Cao et al., 2007) where it can regulate late endosomal PtdIns3P levels (Cao et al., 2008).
5.2. Cellular functions regulated by the endosomal pool of PtdIns3P The endosomal pool of PtdIns3P controls membrane transport and membrane dynamics mostly by recruiting key proteins containing FYVE, PX, and PH domains (Lindmo & Stenmark, 2006) including EEA1, which has a critical role in endosome fusion, and Hrs, which regulates the first steps of receptor sorting and internalization within the multivesicular bodies (MVBs). Endosomal pool of PtdIns3P can also contribute to growth factor signaling through regulation of maturation of an early endocytic intermediate (Zoncu et al., 2009).
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It is now becoming increasingly clear that the endosomal PtdIns3P has also a role in the control of autophagy. PtdIns3P has a well-established and key role in various steps of the autophagy process, including autophagosome biogenesis, maturation, and intracellular transport (Dall’Armi, Devereaux, & Di Paolo, 2013). Specifically related to the endosomal PtdIns3P pool is the control of the maturation steps, which involve fusion of the autophagosome with the endosomes, generating the amphisomes that then fuse with the lysosomes (Dall’Armi et al., 2013).
6. PHOSPHATIDYLINOSITOL 3,5-BISPHOSPHATE 6.1. Intracellular levels PtdIns(3,5)P2 was first identified in a study using [3H]inositol-labeled S. cerevisiae grown almost to isotopic equilibrium in an inositol-free and/or low-phosphate minimal medium and HPLC analysis (Dove et al., 1997). Subsequent studies using this technique were performed in yeasts (Dove et al., 2002; Phelan, Millson, Parker, Piper, & Cooke, 2006) and mammalian cell lines ( Jefferies et al., 2008; Sbrissa & Shisheva, 2005).
6.2. Intracellular localization Because specific PtdIns(3,5)P2-binding domains have not been described, information on the intracellular localization of this PI has mostly been derived from the localization of PIKfyve. The endogenous enzyme localizes to discrete peripheral punctae resembling endosomes in 3T3L1 adipocytes (Shisheva, 2008). Endosomal localization (both on early and late endosomes) and localization in MVBs and at the trans-Golgi network was reported for the overexpressed wild-type PIKfyve. However, it has also been suggested that the localization is strongly dependent on the cell type, the level of protein expressed, and on the ratio between PtdIns(3,5)P2 and PtdIns3P (Shisheva, 2008).
6.3. Intracellular roles PtdIns(3,5)P2 has a well-established role in membrane trafficking and it is currently believed that specific cargo molecules can stimulate synthesis of this PI to regulate their correct endosome–lysosome trafficking (Dove et al., 2009). Accumulation of swollen intracellular vacuoles, enlarged late endosomes, and abnormal MVB has been observed in cultured cells, in animal models, and in human tissues as a result of defects in PtdIns(3,5)P2
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synthesis. Recent data have also revealed a critical role for PtdIns(3,5)P2 in autophagy in the central nervous system (Ferguson, Lenk, & Meisler, 2009, 2010).
ACKNOWLEDGMENTS Work in our laboratory is supported by Pancreatic Cancer Research Fund (M. F.), Bowel and Cancer Research (M. F.), Prostate Cancer UK (M. F.), Diabetes UK (T. M.) and Barts and The London Charity-Cancer Fund (T. M.).
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