Phagolysosome resolution requires contacts with the endoplasmic reticulum and phosphatidylinositol-4-phosphate signalling


Phosphoinositides have a pivotal role in the maturation of nascent phagosomes into microbicidal phagolysosomes. Following degradation of their contents, mature phagolysosomes undergo resolution, a process that remains largely uninvestigated. Here we studied the role of phosphoinositides in phagolysosome resolution. Phosphatidylinositol-4-phosphate (PtdIns(4)P), which is abundant in maturing phagolysosomes, was depleted as they tubulated and resorbed. Depletion was caused, in part, by transfer of phagolysosomal PtdIns(4)P to the endoplasmic reticulum, a process mediated by oxysterol-binding protein-related protein 1L (ORP1L), a RAB7 effector. ORP1L formed discrete tethers between the phagolysosome and the endoplasmic reticulum, resulting in distinct regions with alternating PtdIns(4)P depletion and enrichment. Tubules emerged from PtdIns(4)P-rich regions, where ADP-ribosylation factor-like protein 8B (ARL8B) and SifA- and kinesin-interacting protein/pleckstrin homology domain-containing family M member 2 (SKIP/PLEKHM2) accumulated. SKIP binds preferentially to monophosphorylated phosphoinositides, of which PtdIns(4)P is most abundant in phagolysosomes, contributing to their tubulation. Accordingly, premature hydrolysis of PtdIns(4)P impaired SKIP recruitment and phagosome resolution. Thus, resolution involves phosphoinositides and tethering of phagolysosomes to the endoplasmic reticulum.

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Fig. 1: Phagosomal resolution is characterized by tubular fission events.
Fig. 2: Dynamics and distribution of PtdIns(4)P during initiation of phagosome resolution.
Fig. 3: ORP1L accumulates in phagosomes during late maturation.
Fig. 4: ORP1L expression accelerates late PtdIns(4)P disappearance in maturing phagosomes.
Fig. 5: ORP1L binds and transports PtdIns(4)P to the ER.
Fig. 6: ORP1L-mediated PtdIns(4)P degradation is dependent on its FFAT motif.
Fig. 7: PtdIns(4)P directly binds SKIP and recruits it to the phagolysosome.
Fig. 8: Phagosome resolution is mediated by contacts with the ER and by PtdIns(4)P.

Data availability

Source data for Figs. 17 and Supplementary Figs. 2, 3, 6 and 7 have been provided as Supplementary Table 2. All other data sets generated during and/or analysed during this study are available from the corresponding authors on reasonable request.


  1. 1.

    Stuart, L. M. & Ezekowitz, R. A. B. Phagocytosis. Immunity 22, 539–550 (2005).

    CAS  Article  Google Scholar 

  2. 2.

    Flannagan, R. S., Jaumouillé, V. & Grinstein, S. The cell biology of phagocytosis. Annu. Rev. Pathol. 7, 61–98 (2012).

    CAS  Article  Google Scholar 

  3. 3.

    Levin, R., Grinstein, S. & Canton, J. The life cycle of phagosomes: formation, maturation, and resolution. Immunol. Rev. 273, 156–179 (2016).

    CAS  Article  Google Scholar 

  4. 4.

    Elliott, M. R. & Ravichandran, K. S. Clearance of apoptotic cells: implications in health and disease. J. Cell Biol. 189, 1059–1070 (2010).

    CAS  Article  Google Scholar 

  5. 5.

    Bianconi, E. et al. An estimation of the number of cells in the human body. Ann. Hum. Biol. 40, 463–471 (2013).

    Article  Google Scholar 

  6. 6.

    Elliott, M. R. & Ravichandran, K. S. The dynamics of apoptotic cell clearance. Dev. Cell 38, 147–160 (2016).

    CAS  Article  Google Scholar 

  7. 7.

    Swanson, J. A. Shaping cups into phagosomes and macropinosomes. Nat. Rev. Mol. Cell Biol. 9, 639–649 (2008).

    CAS  Article  Google Scholar 

  8. 8.

    Underhill, D. M. Phagosome maturation: steady as she goes. Immunity 23, 343–344 (2005).

    CAS  Article  Google Scholar 

  9. 9.

    Kinchen, J. M. & Ravichandran, K. S. Phagosome maturation: going through the acid test. Nat. Rev. Mol. Cell Biol. 9, 781–795 (2008).

    CAS  Article  Google Scholar 

  10. 10.

    Fairn, G. D. & Grinstein, S. How nascent phagosomes mature to become phagolysosomes. Trends Immunol. 33, 397–405 (2012).

    CAS  Article  Google Scholar 

  11. 11.

    Gray, M. & Botelho, R. J. Phagocytosis: hungry, hungry cells. Methods Mol. Biol. 1519, 1–16 (2017).

    CAS  Article  Google Scholar 

  12. 12.

    Blander, J. M. & Medzhitov, R. On regulation of phagosome maturation and antigen presentation. Nat. Immunol. 7, 1029–1035 (2006).

    CAS  Article  Google Scholar 

  13. 13.

    Mantegazza, A. R., Magalhaes, J. G., Amigorena, S. & Marks, M. S. Presentation of phagocytosed antigens by MHC class I and II. Traffic 14, 135–152 (2013).

    CAS  Article  Google Scholar 

  14. 14.

    Gillooly, D. J., Simonsen, A. & Stenmark, H. Phosphoinositides and phagocytosis. J. Cell Biol. 155, 15–18 (2001).

    CAS  Article  Google Scholar 

  15. 15.

    Deretic, V. et al. Phosphoinositides in phagolysosome and autophagosome biogenesis. Biochem. Soc. Symp. 74, 141–148 (2007).

    CAS  Article  Google Scholar 

  16. 16.

    Levin, R., Grinstein, S. & Schlam, D. Phosphoinositides in phagocytosis and macropinocytosis. Biochim. Biophys. Acta 1851, 805–823 (2015).

    CAS  Article  Google Scholar 

  17. 17.

    Araki, N., Johnson, M. T. & Swanson, J. A. A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages. J. Cell Biol. 135, 1249–1260 (1996).

    CAS  Article  Google Scholar 

  18. 18.

    Botelho, R. J. et al. Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis. J. Cell Biol. 151, 1353–1368 (2000).

    CAS  Article  Google Scholar 

  19. 19.

    Marshall, J. G. et al. Restricted accumulation of phosphatidylinositol 3-kinase products in a plasmalemmal subdomain during Fcɣ receptor-mediated phagocytosis. J. Cell Biol. 153, 1369–1380 (2001).

    CAS  Article  Google Scholar 

  20. 20.

    Cox, D., Tseng, C. C., Bjekic, G. & Greenberg, S. A requirement for phosphatidylinositol 3-kinase in pseudopod extension. J. Biol. Chem. 274, 1240–1247 (1999).

    CAS  Article  Google Scholar 

  21. 21.

    Rohatgi, R., Ho, H. Y. & Kirschner, M. W. Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4,5-bisphosphate. J. Cell Biol. 150, 1299–1310 (2000).

    CAS  Article  Google Scholar 

  22. 22.

    Fratti, R. A., Backer, J. M., Gruenberg, J., Corvera, S. & Deretic, V. Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest. J. Cell Biol. 154, 631–644 (2001).

    CAS  Article  Google Scholar 

  23. 23.

    Vieira, O. V. et al. Distinct roles of class I and class III phosphatidylinositol 3-kinases in phagosome formation and maturation. J. Cell Biol. 155, 19–25 (2001).

    CAS  Article  Google Scholar 

  24. 24.

    Levin, R. et al. Multiphasic dynamics of phosphatidylinositol 4-phosphate during phagocytosis. Mol. Biol. Cell 28, 128–140 (2017).

    CAS  Article  Google Scholar 

  25. 25.

    Jeschke, A. et al. Phosphatidylinositol 4-phosphate and phosphatidylinositol 3-phosphate regulate phagolysosome biogenesis. Proc. Natl Acad. Sci. USA 112, 4636–4641 (2015).

    CAS  Article  Google Scholar 

  26. 26.

    Krajcovic, M., Krishna, S., Akkari, L., Joyce, J. A. & Overholtzer, M. mTOR regulates phagosome and entotic vacuole fission. Mol. Biol. Cell 24, 3736–3745 (2013).

    CAS  Article  Google Scholar 

  27. 27.

    Hammond, G. R. V., Machner, M. P. & Balla, T. A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi. J. Cell Biol. 205, 113–126 (2014).

    CAS  Article  Google Scholar 

  28. 28.

    Johansson, M., Lehto, M., Tanhuanpää, K., Cover, T. L. & Olkkonen, V. M. The oxysterol-binding protein homologue ORP1L interacts with Rab7 and alters functional properties of late endocytic compartments. Mol. Biol. Cell 16, 5480–5492 (2005).

    CAS  Article  Google Scholar 

  29. 29.

    Loewen, C. J. R., Roy, A. & Levine, T. P. A conserved ER targeting motif in three families of lipid binding proteins and in Opi1p binds VAP. EMBO J. 22, 2025–2035 (2003).

    CAS  Article  Google Scholar 

  30. 30.

    Loewen, C. J. R. & Levine, T. P. A highly conserved binding site in vesicle-associated membrane protein-associated protein (VAP) for the FFAT motif of lipid-binding proteins. J. Biol. Chem. 280, 14097–14104 (2005).

    CAS  Article  Google Scholar 

  31. 31.

    Rocha, N. et al. Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7–RILP–p150 Glued and late endosome positioning. J. Cell Biol. 185, 1209–1225 (2009).

    CAS  Article  Google Scholar 

  32. 32.

    Suchanek, M. et al. The mammalian oxysterol-binding protein-related proteins (ORPs) bind 25-hydroxycholesterol in an evolutionarily conserved pocket. Biochem. J. 405, 473–480 (2007).

    CAS  Article  Google Scholar 

  33. 33.

    Vihervaara, T. et al. Sterol binding by OSBP-related protein 1L regulates late endosome motility and function. Cell. Mol. Life Sci. 68, 537–551 (2011).

    CAS  Article  Google Scholar 

  34. 34.

    van der Kant, R. et al. Late endosomal transport and tethering are coupled processes controlled by RILP and the cholesterol sensor ORP1L. J. Cell Sci. 126, 3462–3474 (2013).

    Article  Google Scholar 

  35. 35.

    Wijdeven, R. H. et al. Cholesterol and ORP1L-mediated ER contact sites control autophagosome transport and fusion with the endocytic pathway. Nat. Commun. 7, 11808 (2016).

    CAS  Article  Google Scholar 

  36. 36.

    Zhao, K. & Ridgway, N. D. Oxysterol-binding protein-related protein 1L regulates cholesterol egress from the endo-lysosomal system. Cell Rep. 19, 1807–1818 (2017).

    CAS  Article  Google Scholar 

  37. 37.

    Dong, J. et al. Allosteric enhancement of ORP1-mediated cholesterol transport by PI(4,5)P2/PI(3,4)P2. Nat. Commun. 10, 829 (2019).

    Article  Google Scholar 

  38. 38.

    Balla, T. Phosphoinositides: tiny lipids with giant impact on cell regulation. Physiol. Rev. 93, 1019–1137 (2013).

    CAS  Article  Google Scholar 

  39. 39.

    Rohde, H. M. et al. The human phosphatidylinositol phosphatase SAC1 interacts with the coatomer I complex. J. Biol. Chem. 278, 52689–52699 (2003).

    CAS  Article  Google Scholar 

  40. 40.

    Zewe, J. P., Wills, R. C., Sangappa, S., Goulden, B. D. & Hammond, G. R. V. SAC1 degrades its lipid substrate PtdIns4P in the endoplasmic reticulum to maintain a steep chemical gradient with donor membranes. eLife 7, e35588 (2018).

    Article  Google Scholar 

  41. 41.

    Mesmin, B. et al. A four-step cycle driven by PI(4)P hydrolysis directs sterol/PI(4)P exchange by the ER-Golgi tether OSBP. Cell 155, 830–843 (2013).

    CAS  Article  Google Scholar 

  42. 42.

    von Filseck, J. M., Vanni, S., Mesmin, B., Antonny, B. & Drin, G. A phosphatidylinositol-4-phosphate powered exchange mechanism to create a lipid gradient between membranes. Nat. Commun. 6, 6671 (2015).

    CAS  Article  Google Scholar 

  43. 43.

    Moser von Filseck, J. et al. Phosphatidylserine transport by ORP/Osh proteins is driven by phosphatidylinositol 4-phosphate. Science 349, 432–436 (2015).

    CAS  Article  Google Scholar 

  44. 44.

    Chung, J. et al. PI4P/phosphatidylserine countertransport at ORP5- and ORP8-mediated ER-plasma membrane contacts. Science 349, 428–432 (2015).

    CAS  Article  Google Scholar 

  45. 45.

    Sohn, M. et al. PI(4,5)P2 controls plasma membrane PI4P and PS levels via ORP5/8 recruitment to ER–PM contact sites. J. Cell Biol. 217, 1797–1813 (2018).

    CAS  Article  Google Scholar 

  46. 46.

    Johansson, M. et al. The two variants of oxysterol binding protein-related protein-1 display different tissue expression patterns, have different intracellular localization, and are functionally distinct. Mol. Biol. Cell 14, 903–915 (2003).

    CAS  Article  Google Scholar 

  47. 47.

    Miyamoto, T. et al. Rapid and orthogonal logic gating with a gibberellin-induced dimerization system. Nat. Chem. Biol. 8, 465–470 (2012).

    CAS  Article  Google Scholar 

  48. 48.

    de Saint-Jean, M. et al. Osh4p exchanges sterols for phosphatidylinositol 4-phosphate between lipid bilayers. J. Cell Biol. 195, 965–978 (2011).

    Article  Google Scholar 

  49. 49.

    Maeda, K. et al. Interactome map uncovers phosphatidylserine transport by oxysterol-binding proteins. Nature 501, 257–261 (2013).

    CAS  Article  Google Scholar 

  50. 50.

    Tong, J., Yang, H., Yang, H., Eom, S. H. & Im, Y. J. Structure of Osh3 reveals a conserved mode of phosphoinositide binding in oxysterol-binding proteins. Structure 21, 1203–1213 (2013).

    CAS  Article  Google Scholar 

  51. 51.

    Downey, G. P. et al. Phagosomal maturation, acidification and inhibition of bacterial growth in nonphagocytic cells transfected with FcɣRIIA receptors. J. Biol. Chem. 274, 28436–28444 (1999).

    CAS  Article  Google Scholar 

  52. 52.

    Johansson, M. et al. Activation of endosomal dynein motors by stepwise assembly of Rab7–RILP–p150Glued, ORP1L, and the receptor βlll spectrin. J. Cell Biol. 176, 459–471 (2007).

    CAS  Article  Google Scholar 

  53. 53.

    Rosa-Ferreira, C. & Munro, S. Arl8 and SKIP act together to link lysosomes to kinesin-1. Dev. Cell 21, 1171–1178 (2011).

    CAS  Article  Google Scholar 

  54. 54.

    Fazeli, G., Stetter, M., Lisack, J. N. & Wehman, A. M. C. elegans blastomeres clear the corpse of the second polar body by LC3-associated phagocytosis. Cell Rep. 23, 2070–2082 (2018).

    CAS  Article  Google Scholar 

  55. 55.

    Boucrot, E., Henry, T., Borg, J.-P., Gorvel, J.-P. & Méresse, S. The intracellular fate of Salmonella depends on the recruitment of kinesin. Science 308, 1174–1178 (2005).

    CAS  Article  Google Scholar 

  56. 56.

    Lemmon, M. A. Phosphoinositide recognition domains. Traffic 4, 201–213 (2003).

    CAS  Article  Google Scholar 

  57. 57.

    Hammond, G. R. V. & Balla, T. Polyphosphoinositide binding domains: key to inositol lipid biology. Biochim. Biophys. Acta 1851, 746–758 (2015).

    CAS  Article  Google Scholar 

  58. 58.

    Mantegazza, A. R. et al. TLR-dependent phagosome tubulation in dendritic cells promotes phagosome cross-talk to optimize MHC-II antigen presentation. Proc. Natl Acad. Sci. USA 111, 15508–15513 (2014).

    CAS  Article  Google Scholar 

  59. 59.

    Rong, Y. et al. Clathrin and phosphatidylinositol-4,5-bisphosphate regulate autophagic lysosome reformation. Nat. Cell Biol. 14, 924–934 (2012).

    CAS  Article  Google Scholar 

  60. 60.

    Du, W. et al. Kinesin 1 drives autolysosome tubulation. Dev. Cell 37, 326–336 (2016).

    CAS  Article  Google Scholar 

  61. 61.

    Rai, A. et al. Dynein clusters into lipid microdomains on phagosomes to drive rapid transport toward lysosomes. Cell 164, 722–734 (2016).

    CAS  Article  Google Scholar 

  62. 62.

    Niu, Y. et al. PtdIns(4)P regulates retromer–motor interaction to facilitate dynein–cargo dissociation at the trans-Golgi network. Nat. Cell Biol. 15, 417–429 (2013).

    CAS  Article  Google Scholar 

  63. 63.

    Marquer, C. et al. Arf6 controls retromer traffic and intracellular cholesterol distribution via a phosphoinositide-based mechanism. Nat. Commun. 7, 11919 (2016).

    CAS  Article  Google Scholar 

  64. 64.

    Dong, R. et al. Endosome–ER contacts control actin nucleation and retromer function through VAP-dependent regulation of PI4P. Cell 166, 408–423 (2016).

    CAS  Article  Google Scholar 

  65. 65.

    Montaño, F., Grinstein, S. & Levin, R. Quantitative phagocytosis assays in primary and cultured macrophages. Methods Mol. Biol. 1784, 151–163 (2018).

    Article  Google Scholar 

  66. 66.

    Hirano, K. et al. GID1-mediated gibberellin signaling in plants. Trends Plant Sci. 13,192–199 (2008).

  67. 67.

    Chen, B.-C. et al. Lattice light-sheet microscopy: imaging molecules to embryos at highspatiotemporal resolution. Science 346, 1257998 (2014).

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This work was supported by the following grants and scholarships: Connaught International Scholarship for Doctoral Students from the University of Toronto and the National Council for Science and Technology/Consejo Nacional de Ciencia y Tecnología (CONACYT) of Mexico (to R.L.-K.) and FDN-143202 from the Canadian Institutes of Health Research (to S.G.). We thank J. Heddleston, S. Khuon and T. Leong-Chew from the AIC for training and support for LLSM. The AIC is a jointly-funded venture of the Gordon and Betty Moore Foundation and the Howard Hughes Medical Institute.

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R.L.-K., S.G. and G.D.F. conceived the project. R.L.-K., F.M.-R., S.G. and G.D.F. designed the experiments and analysed the data. R.L.-K. and S.G. wrote the manuscript. R.L.-K. and F.M.-R. performed the experimental work with the following exceptions: T.K.-K. and J.S.B. designed and analysed experiments involving SKIP purification and lipid overlay assays. T.K.-K. performed the protein purification, lipid overlay assays and SKIP cloning. B.E. and M.C.B. generated the CRISPR cell lines. B.E. validated gene editing. R.L. performed the transmission electron microscopy. S.M. performed western blot replicates. J.E.D. performed and analysed the phagosome resolution characterization by electron microscopy and quantification. F.M.-R., J.S.B., T.K.-K., B.E., W.S.T. and G.D.F. reviewed and edited the manuscript.

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Correspondence to Gregory D. Fairn or Sergio Grinstein.

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Supplementary Figure 1 Dispersed compartments during phagosome resolution are of phagosomal origin.

a) Transmission electron micrographs of RAW macrophages during stage II of phagosome resolution of IgG-SRBC pre-labeled with ferritin; experiment was repeated independently 3 times with similar observations; scale bars = 1 µm b) Confocal micrograph of RAW macrophages after 30 min of phagocytosis of IgG-SRBC (blue); cells were expressing LAMP1-PAGFP; micrographs were acquired before (left) and after activation of LAMP1-PAGFP in the membrane of an individual phagosome (right); similar observations were made in 5 independent experimental replicates. c) Time-lapse micrographs of RAW macrophages expressing LAMP1-PAGFP during the three stages of phagosome resolution; the phagosomal membrane was photo-activated after 30 min of phagocytosis; similar results were observed in 3 independent experiments; scale bars in b-c = 10 µm.

Supplementary Figure 2 PtdIns4P dynamics and metabolism during the transition from phagolysosome biogenesis to phagosome resolution.

a) Time-course gallery of maximum intensity projections of lattice light-sheet micrographs of RAW macrophages expressing GFP-2xP4M during late stages of phagocytosis of SRBC; similar observations were made in 2 independent experimental repeats. b) Representative confocal micrographs of RAW cells expressing GFP-PI4K2A during the transition from late phagosome maturation to early phagosome resolution at 45, 60 and 80 min of phagocytosis; similar results were obtained from 10 independent experimental replicates. c) Time-course gallery of representative confocal micrographs of RAW macrophages co-expressing GFP-SAC2 and CFP-RAB5, and immunostained with an anti-LAMP1 antibody. The main micrographs show GFP-SAC2; insets show magnifications of phagosomes in dashed boxes; similar results were observed in 3 independent replicates; scale bars = 10 µm. d) Means and individual values of 3 independent replicate determinations of the percentage of SAC2-positive phagosomes at the indicated times of phagocytosis. e) Means and individual values of 3 independent replicate determinations of the percentage of SAC2-positive phagosomes that are also positive for RAB5 (blue circles) or LAMP1 (red squares).

Supplementary Figure 3 ORP1L localizes to phagosomes and mediates contacts with the ER.

a) Representative immunoblot showing ORP1L expression in primary human monocytes and macrophages, and in murine RAW macrophages; similar results were obtained from 2 independent replicates. b) Endomembrane localization of endogenous ORP1L in primary human macrophages (left) and of GFP-ORP1L expressed in RAW macrophages (right); similar observations were made in 3 independent experimental replicates. c) Top: endogenous localization of ORP1L in primary human macrophages during phagocytosis of IgG-opsonized RBC; yellow lines show the cell periphery; magnification of phagosome within red box (right). Bottom: localization of ectopically expressed GFP-ORP1L during phagocytosis; other details as above; similar observations were made in 3 independent replicates. d) Kinetics of accumulation of ORP1L in maturing phagosomes of RAW macrophages; means of 6 experimental replicates ± SEM. e) Immunolocalization of endogenous VAPA (left) or VAPB (right) in primary human macrophages after 40 min of phagocytosis of RBC; insets show magnification of the phagosome in the red box; representative of 3 independent replicate experiments. f) Confocal micrographs showing RAW macrophages co-expressing GFP-ORP1L and mCh-SEC61 (top), or GFP-ORP1L-FFAT* and mCh-SEC61 (bottom); representative of 5 independent experimental replicates. g) Confocal micrographs showing COS-2A cells co-expressing GFP-ORP1L and mCh-Sec61; representative results of 3 independent experiments. h) Transmission electron micrographs of RAW macrophages after 40 min of phagocytosis showing close appositions between the ER and phagosomal membranes. Bottom panels highlights ER membranes (coloured in green) that are in contact with the phagosomal membrane (colored in magenta); similar observations were made in 3 independent experimental replicates. Scale bars = 10 µm except for h, where scale bars = 1 µm.

Supplementary Figure 4 ORP1L mediates ER contact sites through its FFAT motif.

a) Schematic representation of the gibberellin-induced dimerization system. ORP1L variants (WT and FFAT*) lacking ankyrin repeats were rendered soluble (ORP1L747). These variants were linked to the smallest recruitable fragment of the gibberellin-insensitive protein GAI (GAI92)46 and tagged with mTurquoise2. A second construct consists of YFP-tagged gibberellin-insensitive dwarf1 (GID1) linked to the 11 amino acid tail of Lyn kinase that localizes to the plasma membrane. The plant hormone gibberellin binds GID1 causing a conformational change. The alternative GID1 conformation recruits the gibberellin-insensitive protein GAI. After addition of a cell permeant gibberellin analogue (GA3-AM), the soluble mTq2-GAI92-ORP1L747 is recruited to the plasma membrane. b, c) Representative confocal micrographs of 3 independent experimental replicates showing HeLa cells expressing mCh-VAPB, YFP-GID1-Lyn11 and either mTq2-GAI92-ORP1L747 (b) or its FFAT* variant (c), after the addition of GA3-AM.

Supplementary Figure 5 PtdIns4P and ORP1L localize to mutually exclusive phagosomal microdomains.

a) Representative time-lapse gallery of confocal micrographs showing mutual exclusion between PtdIns4P (detected with mCh-2xP4M) and GFP-ORP1L. b) Confocal slice of RAW macrophages expressing either soluble GFP (left) or GFP-ORP1L (right) after 4.5 h of phagocytosis of TAMRA-labeled RBC; insets show magnifications of the green channel of the dotted white boxes. Scale bars = 10 µm.

Supplementary Figure 6 Impairment of PtdIns4P disappearance by ORP1L KO clones and ORP1L variants.

a) TIDE analysis of a homozygous ORP1L CRISPR KO clonal RAW macrophage cell lines. b) Representative western blot showing expression of ORP1L in WT, control (cells stably expressing Cas9 and transduced with control sgRNA) and ORP1L KO RAW macrophage cell lines. c) Representative confocal micrographs of 3 independent replicates showing control or ORP1L KO RAW macrophages expressing GFP-2xP4M at the indicated times during phagosome maturation. Scale bars = 10 µm. d) PtdIns4P dynamics in maturing phagosomes of control cells (black bars), ORP1L KO clone “3” (white bars) and ORP1L KO clone “10” (stippled bars); means of ≥ 39 phagosomes per time point and per condition, from 3 experimental replicates ± SD; significance was determined by two-way ANOVA using Sidak correction, details are in the statistics source data supplementary file. e) Schematic representation of: (from top to bottom) WT ORP1L; ORP1L with a mutated FFAT motif (D478A) incapable of binding VAPA/B; ORP1L with a mutated lipid-binding pocket within the ORD (HH651/652AA) predicted to abolish PtdIns4P binding; ORPSAC1, in which the ORD was replaced with the phosphatase domain of Sac1 from S. cerevisiae. f) Schematic representation of the predicted behaviour of each ORP1L variant at ER-phagosome contacts. g-j) Representative maximum intensity projections of three independent replicates of RAW cells after 30 min of phagocytosis of SRBC expressing: g) GFP-ORP1L-FFAT* and mCh-VAPB; h) mCh-ORP1L-ORD* and GFP-VAPB; i) GFP-ORP1L-FFAT* and mCh-VAPA; and j) GFP-ORP1L-ORD* and mCh-VAPA. Insets show magnifications of each channel and merged image at bottom. Scale bars = 10 µm. k) Phagosomal 2xP4M (normalized to plasma membrane) in control cells, ORP1L KO cells (re-plotted from Fig. 5b) and for ORP1L KO cells expressing GFP-ORP1L-ORD*. Data are means ± SD of the indicated number of determinations from 3 independent experiments. Significance calculated using two-way ANOVA with Sidak correction, assuming statistical independence between groups, details are in the statistics source data supplementary file. l) Relative mRNA levels of control RAW cells treated with scrambled siRNA cells (black bar) or treated with siRNA against Orp1l, measured by qPCR; means ± SD of six independent experiments normalized individually to mRNA levels of paired cells treated with scrambled siRNA.

Supplementary Figure 7 PtdIns4P implications with the association of SKIP – ARL8B to the phagosomal membrane.

a, b) Representative confocal micrographs of 3 independent experimental replicates showing phagosomes in RAW macrophages transfected with (a) GFP-SKIP and mTq2-2xP4M or (b) mCh-ARL8B and GFP-2xP4M. c) Protein overlay assay; a hydrophobic membrane spotted with the indicated lipids and overlaid with purified recombinant GST-SKIP (0.6 µg/mL); bound protein was detected by immunoblotting; similar results were observed in 3 independent replicates. d) Representative confocal micrographs of 3 independent experimental replicates showing RAW macrophages expressing mCh-ARL8B after 45 min of phagocytosis of IgG-SRBC under: control conditions (left panel); overexpression of mCh-ORP1L (middle panel); and ORP1L KO (right panel). e) Quantitation of ARL8B recruitment to phagosomes under the conditions shown in f. Means ± SEM of the number of determinations indicated in each bar, from 3 independent experiments. Significance determined by two-tailed unpaired t tests; ****p ≤ 0.0001.

Supplementary Figure 8 PtdIns4P-positive phagosomal membrane domains are enriched in actin and WASH and do not contain PtdIns(4,5)P2.

a) Representative confocal micrographs of 3 independent replicate experiments showing RAW macrophages expressing GFP-2xP4M, fixed after 45 min of phagocytosis and stained with phalloidin conjugated to Alexa Fluor 568. Each row shows different cells under the same conditions. b) Representative confocal micrographs of 3 independent replicate experiments showing RAW macrophages expressing GFP-PH-PLCδ, fixed after 45 min of phagocytosis and stained with phalloidin conjugated to Alexa Fluor 568. Insets are magnifications of individual phagosomes. Scale bars in (a) and (b) = 10 µm. c) Representative confocal micrographs of 3 independent replicate experiments showing of phagosomes of RAW macrophages expressing GFP-2xP4M that were fixed and immunostained for endogenous WASH and labeled with phalloidin 45 min after phagocytosis. Scale bars = 2.5 µm.

Supplementary Figure 9 Unprocessed images of all gels and blots.

Unprocessed western blot from Fig. 6a, nprocessed western blot from Supplementary Fig. 3a, Unprocessed western blot from Supplementary Fig. 6B.

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Levin-Konigsberg, R., Montaño-Rendón, F., Keren-Kaplan, T. et al. Phagolysosome resolution requires contacts with the endoplasmic reticulum and phosphatidylinositol-4-phosphate signalling. Nat Cell Biol 21, 1234–1247 (2019).

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