324 nature chemical biology | vol 6 | may 2010 | www.nature.com/naturechemicalbiology

brief communicationpublished online: 4 april 2010 | doi: 10.1038/nchembio.348

Phosphatidylinositol 3-phosphate (PtdIns(3)P) is a phospho-lipid residing on early endosomes, where it is proposed to be involved in endosomal fusion. We synthesized membrane-permeant derivatives of PtdIns(3)P, including a caged version that is to our knowledge the first photoactivatable phospho-inositide derivative developed so far. In living cells, photo-activation of caged PtdIns(3)P induced rapid endosomal fusion in an EEA1-dependent fashion, thus providing in vivo evidence that PtdIns(3)P is a sufficient signal for driving this process.

Phospholipids are principal components of cellular membranes, and many of them also participate in intracellular signal transduc-tion networks. Some of the best studied lipids in this respect are the phosphoinositides, a group of eight isomers with varying phospho-rylation patterns on the D-3, D-4 and D-5 positions of the inositol moiety. Phosphoinositides phosphorylated at the D-3 position by a special class of lipid kinases, the phosphatidylinositol-3-OH kinases (PI(3)Ks), play a major role in signal transduction and membrane trafficking1–3. PtdIns(3)P (1; Scheme 1a) is a product of the type III PI(3)K (hVps34)4 and is the first phosphoinositide shown to have a role in membrane trafficking4. It is predominantly found on early endosomes5 and has been implicated in endosomal fusion3,6, protein trafficking and sorting4,7, pathogen trafficking8,9 and autophagy10–12.

In order to facilitate the investigation of PI(3)K signaling and the function of the various phosphoinositides, methods are needed that artificially but nondisruptively increase the concentration of a specific phospholipid. In the past, membrane-permeant phospho-inositide derivatives using bioactivatable protecting groups have been successfully developed13–15. These compounds have the negative charges masked with acetoxymethyl ester groups that permit passive cell entry. Once inside cells, endogenous esterases and lipases effi-ciently remove these groups, thereby regenerating negative charges and biological activity. ‘Caged’ derivatives are even more sophisticated— they are similar derivatives carrying a photoactivatable protecting group that prevents biological activity until its removal by a flash of light. By using these tools, the cellular machinery has sufficient time to remove all enzymatically cleavable masking groups until a light flash liberates the biologically active compound. This technique has frequently been applied to nucleotides such as ATP16 and cyclic AMP17 as well as some of the inositol polyphosphates18,19. However, of these photoactivatable tools, only inositol trisphosphate and cAMP were rendered membrane-permeant, and caging groups have never been applied to phosphoinositides17,20,21.

Here we synthesized an enantiomerically pure membrane- permeant derivative of PtdIns(3)P (2, Scheme 1a) as well as its regioisomer PtdIns(4)P in 7.5% and 24% overall yield, respectively, from enantiopure starting materials (for structures and full synthetic

activation of membrane-permeant caged ptdins(3)p induces endosomal fusion in cellsdevaraj subramanian1,2, Vibor laketa1,2, rainer müller1, christian tischer1, sirus Zarbakhsh1, rainer pepperkok1 & carsten schultz1*

1European molecular Biology Institute, Cell Biology and Biophysics Unit, Heidelberg, Germany. 2These authors contributed equally to this work. *e-mail: schultz@embl.de

Scheme 1 | Membrane-permeant PtdIns(3)P derivatives and their chemical synthesis. (a) PtdIns(3)P (1) and its membrane-permeant derivatives are shown. In cells, bioactivatable protecting groups of the membrane-permeant PtdIns(3)P derivative 2 are removed by endogenous esterases to release active PtdIns(3)P. (b) Synthesis of the caged membrane-permeant phosphoinositide PtdIns(3)P/am (3). (i) TBDPSCl, imidazole, pyridine, −10 °C; (ii) Bt2o, DmaP, pyridine, 95%; (iii) TFa/CHCl3, 76%; (iv) C3H7C(ome)3, PPTS resin, CH2Cl2, H2o, 80%; (v) 9, dicyanoimidazole, CH2Cl2, 3 d, then −20 °C, t-butylhydroperoxide, 70%; (vi) Et3N*3HF, meCN/CH2Cl2 (7:3), 44%; (vii) 1,2-di-O-octanoylglyceryl(Fmo)PNiPr2 (12), DCI, meCN, then −20 °C, t-butylhydroperoxide, 40%; (viii) EtNiPr2, meCN, 0 °C→25 °C, 15 h, then acoCH2Br, 30%. TBDPSCl, t-butyldiphenylsilyl chloride; DmaP, 4-(dimethylamino)pyridine; TFa, trifluoroacetic acid; PPTS, pyridinium toluene-4-sulfonate; Et3N*3HF, triethylamine trihydrofluoride; DCI, 4,5-dicyanoimidazole; Bt2o, butyric anhydride; ac, acetyl; Fm, 9-fluorenylmethyl; DoG, dioctanoylglyceryl.

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nature chemical biology | vol 6 | may 2010 | www.nature.com/naturechemicalbiology 325

brief communicationNAturE chEMIcAl bIology doi: 10.1038/nchembio.348

procedures, see Supplementary Methods). In addition, we prepared membrane-permeant PtdIns(3)P as a photoactivatable derivative with 7-diethylamino-4-hydroxymethylcoumarin masking the D3-phosphate (3, Scheme 1a). For membrane permeability, we chose acetoxymethyl (AM) esters to mask the remaining charged phosphate groups and butyrates to cover the hydroxy groups in order to prevent phosphate migration during the AM hydrolysis process. The synthe-sis (Scheme 1b) started from commercially available enantiomeri-cally pure 2,3-O-cyclohexylidene-myo-inositol (4). The position for lipid coupling was regioselectively protected by using the sterically demanding t-butyldiphenylsilyl (TBDPS) group, which attached almost exclusively to the 1-OH group to give triol 5. Subsequent exhaustive butyrylation and cleavage of the ketal afforded diol 7. The required regioselectivity for the last protecting step was achieved by

reacting 7 with butyric acid trimethylorthoester and careful subse-quent hydrolysis of the cyclic intermediates to give exclusively the axial 2-O-butyrate 8. We then prepared the phosphoramidite reagent 9 (see Supplementary Methods) containing the light-sensitive diethyl-aminocoumarin and a fluorenylmethyl group. The latter is later easily cleaved in the presence of piperidine or Hünig’s base with little loss of coumarin. Phosphitylation of 8 with 9 gave the fully protected inositol 3-phosphate 10 as a pair of diastereomers in 70% yield. The TBDPS group was carefully removed with Et3N*3HF, but some loss of the coumarin could not be avoided. To introduce the lipid moiety, alcohol 11 was reacted with 1,2-di-O-octanoylglyceryl-3-O-(fluorenylmethyl)-N,N-diiso-propylphosphoramidite (12) followed by oxidation with t-butyl hydroperoxide in nonane to give the fully protected PtdIns(3)P derivative 13. Deprotection

and introduction of acetoxymethyl esters was achieved in a one-pot reaction, where acetoxymethyl bromide was added to the diisopro pylethylamine solution after 15 h. The presence of four diastereomers made NMR anal-ysis complex (see Supplementary Methods). The overall yield of the desired photoactivatable PtdIns(3)P derivative 3 was 1.2%.

In living cells, PtdIns(3)P is found predomi-nantly on early endosomes5, and in vitro experi-ments have established its involvement in early endosome vesicular fusion3,6. The molecular details of the endosome fusion process are still largely unsolved; however, a current model sug-gests that PtdIns(3)P-binding proteins assemble on early endosomes, tether incoming vesicles and induce their fusion3. It was shown in vitro that PI(3)K inhibitors block6 endosome fusion and that constitutively active PI(3)K induces endosome fusion22. Recently, researchers dem-onstrated that adding PtdIns(3)P to the reaction mixture can substitute for the PI(3)K require-ment in in vitro fusion reactions23. Similar experiments have not been performed in living cells, mainly because the tools to acutely increase PtdIns(3)P levels were missing. Therefore, we wanted to test the biological activity of newly synthesized PtdIns(3)P analogs and to assess their effect on endosome fusion in living cells. HeLa Kyoto cells were treated with PtdIns(3)P/AM, fixed and stained with anti-EEA1 antibody (a known marker of early endosomes). We observed a 2.5-fold increase in the average endo-some size in cells treated with PtdIns(3)P/AM compared to nontreated cells (Fig. 1a, see quantification in Supplementary Fig. 1). Compounds that are chemically identical to PtdIns(3)P/AM but structurally slightly differ-ent, such as PtdIns(4)P/AM and the enantiomer of PtdIns(3)P/AM, were unable to induce early endosome fusion (Supplementary Fig. 1), which demonstrates the high structural speci-ficity. Within the same time period, the number of early endosomes per cell was reduced by 46 ± 8%, indicating that endosome fusion was tak-ing place. To accurately characterize the changes in endosome size and morpho logy, we exam-ined the early endosomes of PtdIns(3)P/AM- treated cells using quantitative electron micro-scopy. We measured a threefold increase in the average early endosome cross-section area in PtdIns(3)P/AM-treated cells compared to

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Figure 1 | PtdIns(3)P/AM-induced endosome fusion. (a) antibodies against EEa1 stained early endosomes in nontreated cells (upper image) and PtdIns(3)P/am-treated cells (100 µm, 40 min; lower image). Typical fused endosomes are marked by arrows. Scale bar, 10 µm. (b) Rab5-CFP–labeled endosomes after treatment with PtdIns(3)P/am (upper panel) and caged PtdIns(3)P/am (middle panel) and in nontreated control cells (lower panel). The two large images show endosomes before and after treatment. The sequence of eight small images is a blowup of the area (white square) where fusion events are most apparent (see arrows). The time is given as mins. Scale bar, 10 µm. (c) Changes in the average endosome size (y axis, fold change) over time. Blue line, PtdIns(3)P/am; red line, caged PtdIns(3)P/am; orange line, PtdIns(4)P/am treatments; green line, control cells treated with the Uv flash only. Time point 0 indicates the time of the compound addition, except for caged PtdIns(3)P/am, where time point 0 indicates Uv flash. Error bars are omitted for clarity; s.d. of the mean varies from 0.1 to 0.5 calculated from at least 3 different cells from 3 different experiments. (d) Downregulation of EEa1 blocks PtdIns(3)P/am-mediated endosome fusion. Shown are changes in endosome size (y axis) depending on the PtdIns(3)P/am concentration in nontargeting siRNa–treated cells or EEa1 siRNa–treated cells. Bars represent average change in endosome size ± s.e.m. over a minimum of 20 cells.

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control nontreated cells (control endosomes were 5,531 ± 778 nm2, n = 224; PtdIns(3)P/AM-treated endosomes were 16,506 ± 1,605 nm2, n = 213; where n is the number of counted endosomes) (Supplementary Fig. 2a,b). Additionally, early endosomes in the control cells were mostly of spherical or elliptical morphology, whereas the endosomes in PtdIns(3)P/AM-treated cells exhibited more convoluted morpho-logy (‘T’ or ‘flower’ shapes) (Supplementary Fig. 2a,b). We also observed autophagosome-like structures in PtdIns(3)P/AM-treated cells (Supplementary Fig. 2c,d). No changes in vesicle size or organ-elle morphology were observed when other membrane systems were examined, such as the Golgi complex, the endoplasmic reticulum, lyso-somes and COPI vesicles (which are endoplasmic reticulum and Golgi trafficking vesicles) (Supplementary Fig. 3a–d). This demonstrated that PtdIns(3)P/AM specifically induced fusion of early endosomes while keeping other membrane systems intact, which is consistent with the specific endosomal role of PtdIns(3)P.

Next, we investigated the timing of endosome fusion by using time-lapse imaging. HeLa Kyoto cells transfected with Rab5-CFP, a fluorescently tagged marker protein for endosomes, were treated with PtdIns(3)P/AM. An increase in endosome size was observed starting after 10–12 min (Fig. 1b,c and Supplementary Video 1). Furthermore, time-lapse imaging allowed us to directly follow single endosome fusion events (Fig. 1b, blowup). A similar result was observed with U2OS cells (Supplementary Fig. 4). One explana-tion for the late onset is that cells need to remove the bioactivatable protecting groups (AM esters and butyrates) from the molecule to render it biologically active. To uncouple the biological event from the bioactivation procedure, we used a photoactivatable (‘caged’) PtdIns(3)P/AM derivative. Cells incubated with the caged membrane- permeant PtdIns(3)P derivative showed that the protected lipid is located in internal membranes, as evidenced by the fluorescence of the photoactivatable coumarin (Supplementary Fig. 5). This observation indicates successful cell entry and wide distribution in all membranes. No change in endosome size was observed when the cells were treated with caged membrane-permeant PtdIns(3)P for 40 min. However, when the entire dish was illuminated with a UV lamp, an increase in endosome size was observed within 0.5–2 min (Fig. 1b,c and Supplementary Video 2). The much earlier onset in activity is likely based on the removal of the bioactivatable protecting groups before uncaging. As a result of this removal, photolysis of the coumarin leads to the immediate generation of an active PtdIns(3)P species. In control cells, PtdIns(4)P/AM, the enantiomer of PtdIns(3)P/AM and UV illumination by themselves did not affect endosome size (Fig. 1b,c and Supplementary Videos 3 and 4). The large difference in the onset of endosomal fusion between the caged and noncaged membrane-permeant derivatives demonstrates that the kinetics of the biological effect reflect cell entry and intracellular bioactivation of PtdIns(3)P/AM rather than the timing of endosomal fusion.

In order to shed some light on the mechanism of PtdIns(3)P action, we investigated the contribution of the endosomal protein EEA1 (early endosomal antigen 1) and its involvement in PtdIns(3)P/AM- induced endosome fusion. The PtdIns(3)P signal is likely picked up by proteins having PtdIns(3)P binding domains such as FYVE domains. EEA1 has an FYVE domain and has been demonstrated to be important for endosome fusion24,25. We were able to effi-ciently downregulate EEA1 protein levels by an siRNA approach (Supplementary Fig. 6). In siRNA-treated cells, endosomal fusion induced by PtdIns(3)P/AM was entirely blocked (Fig. 1d and Supplementary Fig. 7), suggesting that EEA1 is one of the essential mediators of PtdIns(3)P/AM-induced endosomal fusion.

In conclusion, the new membrane-permeant PtdIns(3)P deriva-tive provides a highly specific way to influence intracellular events with good biological effectiveness. Even more control for influenc-ing signaling is provided through the introduction of a membrane- permeant photoactivatable phosphoinositide derivative. There is a clear advantage of using caged PtdIns(3)P over the noncaged variant, as is

evidenced by the faster onset of the biological response. This approach provides better control and an improved mimicking of natural events, although a quantitative evaluation of PtdIns(3)P levels in cells with spatial resolution would be desirable in the future. Here we provide to our knowledge the first in vivo demonstration that the sheer increase in PtdIns(3)P concentration is a sufficient signal to induce EEA1-dependent endosome fusion. This now qualifies PtdIns(3)P as a true second messenger connecting PI(3)K signaling with the endosomal pathway and acting as a regulator of endosome maturation. The pos-sibility that metabolites of PtdIns(3)P contribute to the biological effect cannot be fully excluded. In the future, local fusion events can be investigated by a spatially restricted release of active PtdIns(3)P. Caged PtdIns(3)P/AM and similar photoactivatable phosphoinositides will help to unravel the multitude of biological functions linked to these phospholipids.

received 6 July 2009; accepted 25 January 2010; published online 4 april 2010

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acknowledgmentsWe thank the Advanced Light Microscopy Facility of the European Molecular Biology Laboratory (EMBL), and we also thank C. Funaya and C. Antony of the EMBL Electron Microscopy Core Facility for EM sample preparation. Funding was provided by the Volkswagen Foundation (I81/597 and I81/797) and the Helmholtz-Initiative on Systems Biology (SBCancer) to C.S.

author contributionsD.S., V.L., S.Z., R.P. and C.S. designed the experiments and wrote the manuscript. D.S., V.L., R.M. and S.Z. performed the experiments and analyzed the data. C.T. developed the analytical software.

competing financial interestsThe authors declare no competing financial interests.

additional informationSupplementary information and chemical compound information is available online at http://www.nature.com/naturechemicalbiology/. Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/. Correspondence and requests for materials should be addressed to C.S.

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