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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Stuart, L. M. & Ezekowitz, R. A. B. Phagocytosis. Immunity 22, 539–550 (2005).
Flannagan, R. S., Jaumouillé, V. & Grinstein, S. The cell biology of phagocytosis. Annu. Rev. Pathol. 7, 61–98 (2012).
Levin, R., Grinstein, S. & Canton, J. The life cycle of phagosomes: formation, maturation, and resolution. Immunol. Rev. 273, 156–179 (2016).
Elliott, M. R. & Ravichandran, K. S. Clearance of apoptotic cells: implications in health and disease. J. Cell Biol. 189, 1059–1070 (2010).
Bianconi, E. et al. An estimation of the number of cells in the human body. Ann. Hum. Biol. 40, 463–471 (2013).
Elliott, M. R. & Ravichandran, K. S. The dynamics of apoptotic cell clearance. Dev. Cell 38, 147–160 (2016).
Swanson, J. A. Shaping cups into phagosomes and macropinosomes. Nat. Rev. Mol. Cell Biol. 9, 639–649 (2008).
Underhill, D. M. Phagosome maturation: steady as she goes. Immunity 23, 343–344 (2005).
Kinchen, J. M. & Ravichandran, K. S. Phagosome maturation: going through the acid test. Nat. Rev. Mol. Cell Biol. 9, 781–795 (2008).
Fairn, G. D. & Grinstein, S. How nascent phagosomes mature to become phagolysosomes. Trends Immunol. 33, 397–405 (2012).
Gray, M. & Botelho, R. J. Phagocytosis: hungry, hungry cells. Methods Mol. Biol. 1519, 1–16 (2017).
Blander, J. M. & Medzhitov, R. On regulation of phagosome maturation and antigen presentation. Nat. Immunol. 7, 1029–1035 (2006).
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).
Gillooly, D. J., Simonsen, A. & Stenmark, H. Phosphoinositides and phagocytosis. J. Cell Biol. 155, 15–18 (2001).
Deretic, V. et al. Phosphoinositides in phagolysosome and autophagosome biogenesis. Biochem. Soc. Symp. 74, 141–148 (2007).
Levin, R., Grinstein, S. & Schlam, D. Phosphoinositides in phagocytosis and macropinocytosis. Biochim. Biophys. Acta 1851, 805–823 (2015).
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).
Botelho, R. J. et al. Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis. J. Cell Biol. 151, 1353–1368 (2000).
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).
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).
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).
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).
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).
Levin, R. et al. Multiphasic dynamics of phosphatidylinositol 4-phosphate during phagocytosis. Mol. Biol. Cell 28, 128–140 (2017).
Jeschke, A. et al. Phosphatidylinositol 4-phosphate and phosphatidylinositol 3-phosphate regulate phagolysosome biogenesis. Proc. Natl Acad. Sci. USA 112, 4636–4641 (2015).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
Balla, T. Phosphoinositides: tiny lipids with giant impact on cell regulation. Physiol. Rev. 93, 1019–1137 (2013).
Rohde, H. M. et al. The human phosphatidylinositol phosphatase SAC1 interacts with the coatomer I complex. J. Biol. Chem. 278, 52689–52699 (2003).
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).
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).
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).
Moser von Filseck, J. et al. Phosphatidylserine transport by ORP/Osh proteins is driven by phosphatidylinositol 4-phosphate. Science 349, 432–436 (2015).
Chung, J. et al. PI4P/phosphatidylserine countertransport at ORP5- and ORP8-mediated ER-plasma membrane contacts. Science 349, 428–432 (2015).
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).
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).
Miyamoto, T. et al. Rapid and orthogonal logic gating with a gibberellin-induced dimerization system. Nat. Chem. Biol. 8, 465–470 (2012).
de Saint-Jean, M. et al. Osh4p exchanges sterols for phosphatidylinositol 4-phosphate between lipid bilayers. J. Cell Biol. 195, 965–978 (2011).
Maeda, K. et al. Interactome map uncovers phosphatidylserine transport by oxysterol-binding proteins. Nature 501, 257–261 (2013).
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).
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).
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).
Rosa-Ferreira, C. & Munro, S. Arl8 and SKIP act together to link lysosomes to kinesin-1. Dev. Cell 21, 1171–1178 (2011).
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).
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).
Lemmon, M. A. Phosphoinositide recognition domains. Traffic 4, 201–213 (2003).
Hammond, G. R. V. & Balla, T. Polyphosphoinositide binding domains: key to inositol lipid biology. Biochim. Biophys. Acta 1851, 746–758 (2015).
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).
Rong, Y. et al. Clathrin and phosphatidylinositol-4,5-bisphosphate regulate autophagic lysosome reformation. Nat. Cell Biol. 14, 924–934 (2012).
Du, W. et al. Kinesin 1 drives autolysosome tubulation. Dev. Cell 37, 326–336 (2016).
Rai, A. et al. Dynein clusters into lipid microdomains on phagosomes to drive rapid transport toward lysosomes. Cell 164, 722–734 (2016).
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).
Marquer, C. et al. Arf6 controls retromer traffic and intracellular cholesterol distribution via a phosphoinositide-based mechanism. Nat. Commun. 7, 11919 (2016).
Dong, R. et al. Endosome–ER contacts control actin nucleation and retromer function through VAP-dependent regulation of PI4P. Cell 166, 408–423 (2016).
Montaño, F., Grinstein, S. & Levin, R. Quantitative phagocytosis assays in primary and cultured macrophages. Methods Mol. Biol. 1784, 151–163 (2018).
Hirano, K. et al. GID1-mediated gibberellin signaling in plants. Trends Plant Sci. 13,192–199 (2008).
Chen, B.-C. et al. Lattice light-sheet microscopy: imaging molecules to embryos at highspatiotemporal resolution. Science 346, 1257998 (2014).
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.
The authors declare no competing interests.
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Integrated supplementary information
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).
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.
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.
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.
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.
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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). https://doi.org/10.1038/s41556-019-0394-2
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