pH of endophagosomes controls association of their membranes with Vps34 and PtdIns(3)P levels

Naufer, Amriya; Hipolito, Victoria E. B.; Ganesan, Suriakarthiga; Prashar, Akriti; Zaremberg, Vanina; Botelho, Roberto J.; Terebiznik, Mauricio R.

doi:10.1083/jcb.201702179

Cited by 45 publications

(60 citation statements)

References 88 publications

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“…Importantly, HEK293 cells treated only with BafA1 (15 nM, 100 min) + vehicle (last 60 min) showed unaltered steady-state levels of PtdIns3P vs. those found in control cells that received only vehicle (0.61% vs. 0.64%, mean, ± SEM; P = 0.89, n = 3; S1 Fig ). Consistent with our observations with BafA1, PtdIns3P levels also remained unaltered by slight alkalinization via treatment with weak bases such as concanavalin A1 or NH 4 Cl of RAW macrophages [ 51 ]. Together, this data indicate that BafA1 attenuated only elevated PtdIns3P by apilimod treatment but did not affect the basal PtdIns3P in untreated cells.…”

Section: Resultssupporting

confidence: 91%

Apilimod, a candidate anticancer therapeutic, arrests not only PtdIns(3,5)P2 but also PtdIns5P synthesis by PIKfyve and induces bafilomycin A1-reversible aberrant endomembrane dilation

Sbrissa¹,

Naisan²,

Ikonomov³

et al. 2018

PLoS ONE

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PIKfyve, an evolutionarily conserved kinase synthesizing PtdIns5P and PtdIns(3,5)P2, is crucial for mammalian cell proliferation and viability. Accordingly, PIKfyve inhibitors are now in clinical trials as anti-cancer drugs. Among those, apilimod is the most promising, yet its potency to inhibit PIKfyve and affect endomembrane homeostasis is only partially characterized. We demonstrate here for the first time that apilimod powerfully inhibited in vitro synthesis of PtdIns5P along with that of PtdIns(3,5)P2. HPLC-based resolution of intracellular phosphoinositides (PIs) revealed that apilimod triggered a marked reduction of both lipids in the context of intact cells. Notably, there was also a profound rise in PtdIns3P resulting from arrested PtdIns3P consumption for PtdIns(3,5)P2 synthesis. As typical for PIKfyve inhibition and the concomitant PtdIns(3,5)P2 reduction, apilimod induced the appearance of dilated endomembrane structures in the form of large translucent cytoplasmic vacuoles. Remarkably, bafilomycin A1 (BafA1) fully reversed the aberrant cell phenotype back to normal and completely precluded the appearance of cytoplasmic vacuoles when added prior to apilimod. Inspection of the PI profiles ruled out restoration of the reduced PtdIns(3,5)P2 pool as a molecular mechanism underlying BafA1 rescue. Rather, we found that BafA1 markedly attenuated the PtdIns3P elevation under PIKfyve inhibition. This was accompanied by profoundly decreased endosomal recruitment of fusogenic EEA1. Together, our data demonstrate that apilimod inhibits not only PtdIns(3,5)P2 but also PtdIns5P synthesis and that the cytoplasmic vacuolization triggered by the inhibitor is precluded or reversed by BafA1 through a mechanism associated, in part, with reduction in both PtdIns3P levels and EEA1 membrane recruitment.

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Section: Resultssupporting

confidence: 91%

Apilimod, a candidate anticancer therapeutic, arrests not only PtdIns(3,5)P2 but also PtdIns5P synthesis by PIKfyve and induces bafilomycin A1-reversible aberrant endomembrane dilation

Sbrissa¹,

Naisan²,

Ikonomov³

et al. 2018

PLoS ONE

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“…However, on endosomes and at least on phagosomes enclosing apoptotic bodies, Rab5 then recruits the Mon1/Ccz1 complex to stimulate GTP-loading of the Rab7 GTPase (Kinchen and Ravichandran, 2010;Yasuda et al, 2016;Rink et al, 2005). Moreover, within 10 minutes of maturation, the phagosomal lumen acidifies to pH 6, causing the Vps34 complex to dissociate from early phagosomes (Naufer et al, 2018). This, combined with the conversion of PtdIns(3)P to phosphatidylinositol-3,5bisphosphate [PtdIns(3,5)P 2 ] by PIKfyve, terminates PtdIns(3)P signaling (Kim et al, 2014;Hazeki et al, 2012;Naufer et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, within 10 minutes of maturation, the phagosomal lumen acidifies to pH 6, causing the Vps34 complex to dissociate from early phagosomes (Naufer et al, 2018). This, combined with the conversion of PtdIns(3)P to phosphatidylinositol-3,5bisphosphate [PtdIns(3,5)P 2 ] by PIKfyve, terminates PtdIns(3)P signaling (Kim et al, 2014;Hazeki et al, 2012;Naufer et al, 2018). In addition, phosphatidylinositol-4-phosphate [PtdIns(4)P] is also synthesized on late phagosomes and may mediate phagosome maturation as well (Levin et al, 2017;Jeschke and Haas, 2018).…”

Section: Introductionmentioning

confidence: 99%

“…In addition, phosphatidylinositol-4-phosphate [PtdIns(4)P] is also synthesized on late phagosomes and may mediate phagosome maturation as well (Levin et al, 2017;Jeschke and Haas, 2018). Collectively, the acquisition of Rab7, termination of PtdIns(3)P, and the synthesis of PtdIns(3,5)P 2 and PtdIns(4)P, all drive phagosome fusion with late endosomes and lysosomes, converting the early phagosome into a phagolysosome, which then possesses lysosomal determinants like the Arf-like GTPase 8 (Arl8), lysosomal membrane proteins like lysosomal-associated membrane proteins 1 and 2 (LAMP1 and LAMP2), a myriad collection of hydrolytic enzymes, and a luminal pH <5 established by the V-ATPase (Gray and Botelho, 2017;Levin et al, 2016;Kinchen and Ravichandran, 2010;Naufer et al, 2018;Kim et al, 2014;Garg et al, 2011;Levin-Konigsberg et al, 2019;Levin et al, 2017). This ensures digestion of particulates into its primordial components like amino acids and sugars, which are exported via transporters such as SLC-36.1 (at least in Caenorhabditis elegans) or by bulk via vesicular-tubular intermediates (Mantegazza et al, 2014;Gan et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

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Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes

Lancaster

Fountain

Somerville

et al. 2020

Preprint

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Abbreviations: Arl 8: Arf-like GTPase 8, CLC-GFP: GFP-fusion of the clathrin-light chain; ConA: concanamycin A; DQ-BSA: dye-quenched bovine serum albumin; IKA: ikarugamycin; LAMP1: lysosomal-associated membrane protein 1; LAMP2: lysosomal-associated membrane protein 2; LB: Luria-Bertani medium; Lp: Legionella pneumophila; mTORC1: mechanistic target of rapamycin complex 1; PDV: phagosome-derived vesicle; PtdIns(3)P: phosphatidylinositol 3-phosphate; PtdIns(3,5)P 2 : phosphatidylinositol 3,5-biphosphate; PtdIns(4)P: phosphatidylinositol 4-phosphate; TFEB: transcription factor EB Summary Phagocytes engulf particles into phagolysosomes for degradation. However, the ultimate fate of phagolysosomes is undefined. Lancaster, Fountain et al. show that phagosomes undergo fragmentation to reform lysosomes in a clathrin-dependent manner. This process is necessary to maintain the degradative capacity of phagocytes during subsequent phagocytosis. AbstractDuring phagocytosis, phagocytes like macrophages engulf and sequester unwanted particles like bacteria into phagosomes. Phagosomes then fuse with lysosomes to mature into phagolysosomes, resulting in the degradation of the enclosed particle. Ultimately, phagosomes must be recycled to help recover membrane resources like lysosomes consumed during phagocytosis, a process referred to as phagosome resolution. Little is known about phagosome resolution, which may proceed through exocytosis or membrane fission. Here, we show that bacteria-containing phagolysosomes in macrophages undergo fragmentation through vesicle budding, tubulation, and constriction. Phagosome fragmentation required cargo degradation, the actin and microtubule 3 cytoskeletons, and clathrin. We provide evidence that lysosome reformation occurs during phagosome resolution since the majority of phagosome-derived vesicles displayed lysosomal properties. Importantly, we showed that the clathrin-dependent phagosome resolution is important to maintain the degradative capacity of macrophages challenged with two waves of phagocytosis. Overall, our work suggests that phagosome resolution contributes to lysosome recovery and to maintain the degradative power of macrophages to handle multiple waves of phagocytosis.

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“…Ellson and colleagues (Ellson et al, 2001) demonstrated that PI3P plays an important role in targeting 228 neutrophil oxidase components to phagosomal membranes and its importance in phagosomal 229 maturation has also been identified in Dictyostelium discoideum (Buckley et al, 2019), murine 230 macrophages, and macrophage-like cell lines (Naufer et al, 2018). However, the role of VPS34 in 231 human neutrophils has previously been inferred indirectly (Anderson et al, 2008), owing to prior lack 232 of selective inhibitors and the difficulties of genetically manipulating human neutrophils.…”

mentioning

confidence: 99%

Complement C5a impairs phagosomal maturation in the neutrophil through phosphoproteomic remodelling

Wood

Vassallo

Ruchaud-Sparagano

et al. 2020

Preprint

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Critical illness is accompanied by the release of large amounts of the anaphylotoxin, C5a. C5a suppresses antimicrobial functions of neutrophils which is associated with adverse outcomes. The signalling pathways that mediate C5a-induced neutrophil dysfunction are incompletely understood. Healthy donor neutrophils exposed to purified C5a demonstrated a prolonged defect (7 hours) in phagocytosis of Staphylococcus aureus. Phosphoproteomic profiling of 2712 phosphoproteins identified persistent C5a signalling and selective impairment of phagosomal protein phosphorylation on exposure to S. aureus. Notable proteins included early endosomal marker ZFYVE16 and V-ATPase proton channel component ATPV1G1. A novel assay of phagosomal acidification demonstrated C5a-induced impairment of phagosomal acidification which was recapitulated in neutrophils from critically ill patients. Examination of the C5a-impaired protein phosphorylation indicated a role for the phosphatidylinositol 3-kinase VPS34 in phagosomal maturation. Inhibition of VPS34 impaired neutrophil phagosomal acidification and killing of S. aureus. This study provides a phosphoproteomic assessment of human neutrophil signalling in response to S. aureus and its disruption by C5a, identifying a defect in phagosomal maturation and new mechanisms of immune failure in critical illness.

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pH of endophagosomes controls association of their membranes with Vps34 and PtdIns(3)P levels

Cited by 45 publications

References 88 publications

Apilimod, a candidate anticancer therapeutic, arrests not only PtdIns(3,5)P2 but also PtdIns5P synthesis by PIKfyve and induces bafilomycin A1-reversible aberrant endomembrane dilation

Apilimod, a candidate anticancer therapeutic, arrests not only PtdIns(3,5)P2 but also PtdIns5P synthesis by PIKfyve and induces bafilomycin A1-reversible aberrant endomembrane dilation

Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes

Complement C5a impairs phagosomal maturation in the neutrophil through phosphoproteomic remodelling

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