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Inhibition of lipid kinase PIKfyve reveals a role for phosphatase Inpp4b in the regulation of PI(3)P-mediated lysosome dynamics through VPS34 activity

Open AccessPublished:June 24, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102187
      Lysosome membranes contain diverse phosphoinositide (PtdIns) lipids that coordinate lysosome function and dynamics. The PtdIns repertoire on lysosomes is tightly regulated by the actions of diverse PtdIns kinases and phosphatases; however, specific roles for PtdIns in lysosomal functions and dynamics are currently unclear and require further investigation. It was previously shown that PIKfyve, a lipid kinase that synthesizes PtdIns(3,5)P2 from PtdIns(3)P, controls lysosome “fusion-fission” cycle dynamics, autophagosome turnover, and endocytic cargo delivery. Furthermore, INPP4B, a PtdIns 4-phosphatase that hydrolyzes PtdIns(3,4)P2 to form PtdIns(3)P, is emerging as a cancer-associated protein with roles in lysosomal biogenesis and other lysosomal functions. Here, we investigated the consequences of disrupting PIKfyve function in Inpp4b-deficient mouse embryonic fibroblasts. Through confocal fluorescence imaging, we observed the formation of massively enlarged lysosomes, accompanied by exacerbated reduction of endocytic trafficking, disrupted lysosome fusion-fission dynamics, and inhibition of autophagy. Finally, HPLC scintillation quantification of 3H-myo-inositol labeled PtdIns and PtdIns immunofluorescence staining, we observed that lysosomal PtdIns(3)P levels were significantly elevated in Inpp4b-deficient cells due to the hyperactivation of phosphatidylinositol 3-kinase catalytic subunit VPS34 enzymatic activity. In conclusion, our study identifies a novel signaling axis that maintains normal lysosomal homeostasis and dynamics, which includes the catalytic functions of Inpp4b, PIKfyve, and VPS34.

      Keywords

      Abbreviations:

      FBS (fetal bovine serum), IF (immunofluorescence), INPP4B (inositol polyphosphate 4-phosphatase type II), LY (Lucifer yellow), MEF (mouse embryonic fibroblast), PIKfyve (Phosphoinositide Kinase, FYVE-Type Zinc Finger Containing), PtdIns(3)P (phosphatidylinositol-3-monophosphate), PtdIns(3,5)P2 (phosphatidylinositol-3,5-bisphosphate), PtdIns (phosphoinositide), TFEB (Transcription Factor EB)
      Lysosomes are membrane-bound organelles that serve as a cell’s main degradative center (
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      ). PtdIns exist in seven different forms which are defined by the phosphorylation status of the 3, 4, and/or 5 hydroxyl groups of their inositol head, a process controlled by a number of cellular kinases and phosphatases with PtdIns specificity (
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      Of notable importance on lysosomal membranes is phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2], generated through 5`-phosphorylation of phosphatidylinositol-3-monophosphate [PtdIns(3)P] by Phosphoinositide Kinase, FYVE-Type Zinc Finger Containing (PIKfyve) (
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      ). Pharmacological or genetic inhibition of PIKfyve and subsequent depletion of PtdIns(3,5)P2 has been demonstrated to disrupt lysosomal-related processes including autophagic flux, endocytic and phagocytic cargo delivery to lysosomes, substrate export from lysosome, and calcium release (
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      ). Notably, inhibition of PIKfyve also leads to a dramatic enlargement of lysosomes, a phenomenon explained in part by defective membrane recycling from endosomes and/or lysosomes (
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      ). PtdIns(3,5)P2 depletion and lysosome enlargement in various cell lines can be effectively induced by the pharmacological PIKfyve inhibitor apilimod (
      • Saffi G.T.
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      • Mamand S.
      • Inpanathan S.
      • Fountain A.
      • Salmena L.
      • et al.
      Reactive oxygen species prevent lysosome coalescence during PIKfyve inhibition.
      ,
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      • Dayam R.M.
      • Ou Z.-Y.A.
      • et al.
      Lysosome enlargement during inhibition of the lipid kinase PIKfyve proceeds through lysosome coalescence.
      ). Specifically, apilimod interacts with the amino acid asparagine (N1939) predicted to be located within the ATP-binding pocket of the catalytic kinase domain of PIKfyve (
      • Gayle S.
      • Landrette S.
      • Beeharry N.
      • Conrad C.
      • Hernandez M.
      • Beckett P.
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      Identification of apilimod as a first-in-class PIKfyve kinase inhibitor for treatment of B-cell non-Hodgkin lymphoma.
      ). More recently, Choy et al. attributed apilimod-induced lysosome enlargement to the disruption of lysosome fusion-fission cycling and/or disruption of “kiss-and-run”, a term which describes a transient membrane fusion event followed by rapid fission event that permits content exchange between lysosomes (
      • Bissig C.
      • Hurbain I.
      • Raposo G.
      • van Niel G.
      PIKfyve activity regulates reformation of terminal storage lysosomes from endolysosomes.
      ,
      • Choy C.H.
      • Saffi G.
      • Gray M.A.
      • Wallace C.
      • Dayam R.M.
      • Ou Z.-Y.A.
      • et al.
      Lysosome enlargement during inhibition of the lipid kinase PIKfyve proceeds through lysosome coalescence.
      ,
      • Bright N.A.
      • Gratian M.J.
      • Luzio J.P.
      Endocytic delivery to lysosomes mediated by concurrent fusion and kissing events in living cells.
      ,
      • Duclos S.
      • Corsini R.
      • Desjardins M.
      Remodeling of endosomes during lysosome biogenesis involves “kiss and run” fusion events regulated by rab5.
      ). In their model, PIKfyve inhibition compromises lysosomal fission (the “run” event) and thus promotes lysosome coalescence which results in increased individual lysosome volume and reduced lysosome numbers (
      • Choy C.H.
      • Saffi G.
      • Gray M.A.
      • Wallace C.
      • Dayam R.M.
      • Ou Z.-Y.A.
      • et al.
      Lysosome enlargement during inhibition of the lipid kinase PIKfyve proceeds through lysosome coalescence.
      ,
      • Saffi G.T.
      • Botelho R.J.
      Lysosome fission: planning for an exit.
      ).
      Emerging evidence suggests that other PtdIns including PtdIns(3)P, PtdIns(4)P, and PtdIns(3,4)P2 may also play important roles in regulating lysosomal homeostasis. For instance, PtdIns(3)P has been implicated in regulating lysosomal positioning and lysosomal mTORC1 activity (
      • Hong Z.
      • Pedersen N.M.
      • Wang L.
      • Torgersen M.L.
      • Stenmark H.
      • Raiborg C.
      PtdIns3P controls mTORC1 signaling through lysosomal positioning.
      ). VPS34, a class III PtdIns 3-kinase, is recruited by active GTP-bound Rab5 and Rab7 in complex with VPS15, toward early and late endosomes. VPS34-induced PtdIns(3)P generation regulates early and late endosomal morphology (
      • Law F.
      • Seo J.H.
      • Wang Z.
      • DeLeon J.L.
      • Bolis Y.
      • Brown A.
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      The VPS34 PI3K negatively regulates RAB-5 during endosome maturation.
      ), intraluminal vesicle formation within late endosomes (
      • Giridharan S.S.P.
      • Luo G.
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      • Tronchere H.
      • Singla A.
      • et al.
      Lipid kinases VPS34 and PIKfyve coordinate a phosphoinositide cascade to regulate retriever-mediated recycling on endosomes.
      ), and endosomal assembly of cargo recycling complex (
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      • Luo G.
      • Rivero-Rios P.
      • Steinfeld N.
      • Tronchere H.
      • Singla A.
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      Lipid kinases VPS34 and PIKfyve coordinate a phosphoinositide cascade to regulate retriever-mediated recycling on endosomes.
      ). Additionally, mTOR regulation of VPS34 activity controls lysosome tubular morphology (
      • Munson M.J.
      • Allen G.F.
      • Toth R.
      • Campbell D.G.
      • Lucocq J.M.
      • Ganley I.G.
      mTOR activates the VPS34-UVRAG complex to regulate autolysosomal tubulation and cell survival.
      ). PtdIns(4)P has demonstrated important functions in lysosomal membrane fusion in late endocytic trafficking (
      • Jeschke A.
      • Haas A.
      Sequential actions of phosphatidylinositol phosphates regulate phagosome-lysosome fusion.
      ). PtdIns(3,4)P2 was also demonstrated to promote repression of mTORC1 activity and cell growth through functions on lysosomal membranes (
      • Marat A.L.
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      • Müller R.
      • Norata G.D.
      • Falasca M.
      • et al.
      mTORC1 activity repression by late endosomal phosphatidylinositol 3,4-bisphosphate.
      ). Notably, an emerging role for Inositol polyphosphate 4-phosphatase type II (INPP4B), a PtdIns phosphatase that dephosphorylates PtdIns(3,4)P2 to form PtdIns(3)P, in lysosome homeostasis has been reported in diverse cancer settings (
      • Rodgers S.J.
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      • Oorschot V.M.J.
      • Schittenhelm R.B.
      • Nguyen E.V.
      • Hamila S.A.
      • et al.
      INPP4B promotes PI3Kα-dependent late endosome formation and Wnt/β-catenin signaling in breast cancer.
      ,
      • Woolley J.F.
      • Chen K.
      • Genc G.E.
      • Lee D.K.C.
      • Dzneladze I.
      • He R.
      • et al.
      INPP4B promotes leukemia by restricting leukemic stem cell differentiation through regulation of lysosomal functions.
      ). Overexpression of INPP4B leads to depleted intracellular PtdIns(3,4)P2 and promoted endosomal trafficking of cargo toward lysosomes in breast cancer (
      • Rodgers S.J.
      • Ooms L.M.
      • Oorschot V.M.J.
      • Schittenhelm R.B.
      • Nguyen E.V.
      • Hamila S.A.
      • et al.
      INPP4B promotes PI3Kα-dependent late endosome formation and Wnt/β-catenin signaling in breast cancer.
      ), and our recent study implicates INPP4B in lysosomal biogenesis in leukemia cells (
      • Woolley J.F.
      • Chen K.
      • Genc G.E.
      • Lee D.K.C.
      • Dzneladze I.
      • He R.
      • et al.
      INPP4B promotes leukemia by restricting leukemic stem cell differentiation through regulation of lysosomal functions.
      ). Overall, roles for PtdIns(3,4)P2 and PtdIns(3)P in lysosomal function and dynamics are currently unclear and require further investigation.
      To better understand a role for INPP4B in lysosome homeostasis, we investigated the consequences of PIKfyve inhibition in Inpp4b+/+ and Inpp4b−/− mouse embryonic fibroblasts (MEFs). Surprisingly, PIKfyve inhibition with apilimod in Inpp4b−/− fibroblasts leads to the formation of very massively enlarged lysosomes, compared to the typically enlarged lysosomes produced in Inpp4b+/+ fibroblasts. Our results suggest that exacerbated lysosomal enlargement was, at least in part, a result of aberrantly elevated levels of lysosomal PtdIns(3)P generated by increased activity of the Class III PtdIns 3-kinase VPS34 in Inpp4b−/− cells. Together, these point to a novel role for Inpp4b in suppressing VPS34 activity and the existence of coordinated functions for Inpp4b, PIKfyve, and VPS34 in maintaining normal lysosomal homeostasis, dynamics, and function.

      Results

      PIKfyve inhibition in Inpp4b-null MEF leads to formation of massively enlarged LAMP1+ vacuoles

      Treatment of Inpp4b+/+ and Inpp4b−/− MEF with the specific PIKfyve inhibitor apilimod generated the formation of enlarged cellular vacuoles at 48 h of apilimod treatment in Inpp4b+/+ MEF by light microscopy; strikingly, Inpp4b−/− MEF demonstrated an exacerbated vacuolation phenotype with many more massively enlarged vacuoles (Fig. 1A). Ectopic expression of the lysosomal specific membrane protein (LAMP1) tagged to mCherry (LAMP1-mCherry) followed by fluorescence microscopy revealed that nearly all the enlarged vacuoles in Inpp4b+/+ and Inpp4b−/− MEF were membrane positive for LAMP1 indicating a lysosomal origin for enlarged vacuoles (Figs. 1B and S1A). Quantitation of LAMP1 immunofluorescence (IF) confirmed that enlarged vacuoles were average significantly larger and significantly more abundant in apilimod-treated Inpp4b−/− MEFs than in Inpp4b+/+ MEF (Fig. 1, CE). Total LAMP1 staining demonstrated that vehicle-treated Inpp4b−/− MEF had less lysosomal content compared to Inpp4b+/+ MEF (Fig. 1F). Apilimod treatment significantly increased total LAMP1 staining in both Inpp4b+/+ and Inpp4b−/− MEF in a proportional manner, indicating that induction of lysosomal content was not compromised in Inpp4b−/− MEF (Fig. 1, F, S1, E and F). These findings were corroborated with Lysotracker Red (LTR; Fig. S1, DF) and ectopic expression of a LAMP1-mCherry in Inpp4b+/+ and Inpp4b−/− MEF (Fig. S1, A–C).
      Figure thumbnail gr1
      Figure 1PIKfyve inhibition in Inpp4b-deficient cells leads to altered lysosome homeostasis. A, Inpp4b+/+ or Inpp4b−/− MEF cells were incubated with vehicle or 10 nM apilimod for 48 h followed by imaging with DIC optics. B, scoring for percentage of LAMP1-mCherry membrane positive vacuoles formed upon apilimod treatment. C, fluorescence micrographs of LAMP1 immunostaining for Inpp4b+/+ or Inpp4b−/− MEFs treated with vehicle or 10 nM apilimod for 48 h. D, mean vacuole diameter (μm) positive for LAMP1 membrane immunostain, (E) LAMP1 positive vacuole number (>1.5 μm in diameter) per cell across indicated treatments, and (F) LAMP1 puncta intensity. The scale bar represents 20 μm, zoomed inset: 5 μm. Data represent ± SD from three independent experiments with 25 to 30 cells assessed per treatment condition per experiment for (BF). Statistical significance was measured by ANOVA and multiple Student’s t test and represented as ∗ (p < 0.05). INPP4B, inositol polyphosphate 4-phosphatase type II; MEF, mouse embryonic fibroblast; PIKfyve, Phosphoinositide Kinase, FYVE-Type Zinc Finger Containing.
      To confirm that the aberrant vacuolation response upon apilimod treatment was indeed a result of Inpp4b deficiency, we transduced Inpp4b−/− MEF with constructs that express Inpp4b-EGFP, catalytically dead Inpp4bC845A-EGFP, and control EGFP and treated with apilimod or vehicle and measured vacuole size and number (Fig. S1, GI). Inpp4b-EGFP expression in Inpp4b−/− MEF generated significantly reduced numbers and average size of enlarged vacuoles upon apilimod treatment compared to nontransduced and EGFP negative controls (Fig. S1, GI). Notably, transduction of Inpp4bC845A-EGFP in Inpp4b−/− MEF was unable to rescue the number and size of enlarged vacuoles upon apilimod treatment (Fig. S1, G–I). Exacerbated lysosomal enlargement observed with INPP4B deficiency was also generalizable to human cells, as determined in U2OS cells upon INPP4B knockdown with RNAi (Fig. S2, AE). We also evaluated the effect of Inpp4b suppression on late endosomes. Inpp4b−/− MEF displayed reduced CD63+ late endosome puncta number (Fig. S3). Notably, apilimod treatment did not alter CD63+ late endosome levels, indicating that late endosomes do not reflect the changes observed in lysosomal levels. This suggests that CD63+ late endosomes are not affected by apilimod, and that PIKfyve inhibition may selectively disrupt lysosomal homeostasis and dynamics (Fig. S3). Overall, apilimod treatment in Inpp4b−/− MEF demonstrate an exacerbated lysosomal vacuolation phenotype compared to Inpp4b+/+ MEF that can be rescued by WT Inpp4b, but not a catalytically dead Inpp4b. These data suggest an important role for Inpp4b phosphatase function in maintaining lysosomal dynamics and homeostasis.

      Inpp4b deficiency exacerbates apilimod-mediated inhibition of endocytic trafficking to lysosomes

      We attempted to measure lysosome dynamics using Lucifer yellow (LY), a membrane impermeable fluorescent dye that is internalized by endocytosis and accumulates within lysosomes. LY fluorescence provides an effective tool to specifically delineate lysosomes for accurate measurement of lysosome number and volume (
      • Baluska F.
      • Samaj J.
      • Hlavacka A.
      • Kendrick-Jones J.
      • Volkmann D.
      Actin-dependent fluid-phase endocytosis in inner cortex cells of maize root apices.
      ,
      • Chaurra A.
      • Gutzman B.M.
      • Taylor E.
      • Ackroyd P.C.
      • Christensen K.A.
      Lucifer Yellow as a live cell fluorescent probe for imaging water transport in subcellular organelles.
      ). MEF were treated with vehicle or apilimod prior to LY pulses of 1, 2, and 4 h. Flow cytometry was used to measure LY uptake which demonstrated that apilimod elevated total intracellular LY-fluorescence similarly in both Inpp4b+/+ and Inpp4b−/− MEF for up to 4 h (Fig. S4). To monitor specific LY trafficking to lysosomes, we labeled lysosomes with ectopic expression of LAMP1-mCherry and measured colocalization with LY (Fig. S5A). Apilimod treatment demonstrated a markedly reduced accumulation of LY to lysosomes in Inpp4b+/+ MEF. Comparatively, Inpp4b−/− MEF has significantly less LY accumulation at lysosomes compared to Inpp4b+/+ MEF for up to 2 h, indicating a slower colocalization of LY with lysosomes with Inpp4b deficiency (Fig. S5, A and B). We performed the same assay but replaced LY with DQ-BSA, bovine serum albumin labeled with a self-quenched BODIPY TR-X dye that upon lysosomal delivery is cleaved by lysosomal hydrolases, resulting in bright green fluorescent signal (
      • Marwaha R.
      • Sharma M.
      DQ-red BSA trafficking assay in cultured cells to assess cargo delivery to lysosomes.
      ,
      • Gray M.A.
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      • Ospina-Escobar E.
      • Somerville A.
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      • et al.
      Phagocytosis enhances lysosomal and bactericidal properties by activating the transcription factor TFEB.
      ). We observed that DQ-BSA activation was delayed but not inhibited in apilimod-treated Inpp4b−/− MEF (Fig. S5C), suggesting that degradation of lysosomal cargo was not compromised. These findings indicate that PIKfyve inhibition reduces the ability of endocytosed cargo to reach terminal lysosomes, with no major effects on lysosomal proteolysis. Interestingly, like the vacuolation phenotype, apilimod-mediated accumulation was exacerbated in Inpp4b−/− MEF, further indicating a role for Inpp4b in lysosomal trafficking (
      • Bissig C.
      • Hurbain I.
      • Raposo G.
      • van Niel G.
      PIKfyve activity regulates reformation of terminal storage lysosomes from endolysosomes.
      ,
      • Zhang Y.
      • McCartney A.J.
      • Zolov S.N.
      • Ferguson C.J.
      • Meisler M.H.
      • Sutton M.A.
      • et al.
      Modulation of synaptic function by VAC14, a protein that regulates the phosphoinositides PI(3,5)P₂ and PI(5)P.
      ).

      Inpp4b deficiency exacerbates apilimod-mediated lysosome fusion-fission dynamics

      Given that apilimod blocks lysosomal accumulation of endocytic cargo such as LY, we adjusted our treatment conditions so that MEF were first pulsed with LY followed by acute treatment with apilimod (Fig. 2A). In these experiments, quantitation of LY-labeled lysosomes revealed that individual basal lysosome size was similar in Inpp4b+/+ or Inpp4b−/− MEF. Apilimod induced significant lysosomal enlargement compared to vehicle, and lysosome enlargement in Inpp4b−/− MEF was significantly larger than Inpp4b+/+ MEF (Fig. 2, A and B). Inpp4b−/− MEF have fewer total lysosomes than Inpp4b+/+ MEF; and apilimod treatment reduced lysosome numbers in both Inpp4b+/+ or Inpp4b−/− MEF (Fig. 2, A and C). Notably, total lysosomal content as measured by total cellular LY accumulation was significantly lower in Inpp4b−/− MEF and remained unchanged upon apilimod treatment (Fig. 2, A and D). These data conform to a model whereby apilimod treatment promotes lysosomal coalescence, which is manifested as increased individual lysosome volume and reduced total lysosome numbers (
      • Saffi G.T.
      • Tang E.
      • Mamand S.
      • Inpanathan S.
      • Fountain A.
      • Salmena L.
      • et al.
      Reactive oxygen species prevent lysosome coalescence during PIKfyve inhibition.
      ,
      • Choy C.H.
      • Saffi G.
      • Gray M.A.
      • Wallace C.
      • Dayam R.M.
      • Ou Z.-Y.A.
      • et al.
      Lysosome enlargement during inhibition of the lipid kinase PIKfyve proceeds through lysosome coalescence.
      ,
      • Saffi G.T.
      • Tang E.
      • Fountain A.
      • Botelho R.J.
      Reactive oxygen species rescue lysosome coalescence during PIKfyve inhibition.
      ). Notably, Inpp4b deficiency exacerbates apilimod-induced lysosomal enlargement, thus providing further evidence of a role for Inpp4b in lysosomal homeostasis and dynamics which is revealed upon apilimod treatment.
      Figure thumbnail gr2
      Figure 2Lysosome volume and numbers are altered upon PIKfyve inhibition in Inpp4b-deficient cells. A, lysosomes of Inpp4b+/+ or Inpp4b−/− MEFs prelabeled with Lucifer Yellow and exposed to vehicle or apilimod at indicated concentrations for 1 h. Fluorescence micrographs represent Z-projections of 25 to 30 Z-planes acquired through spinning disc confocal microscopy. Image analysis performed for individual lysosome volume (B), lysosome number per cell (C), and total lysosome volume per cell (D). The scale bar represents 25 μm. Data represent mean ± SD from three independent experiments, with 25 to 30 cells assessed per treatment condition per experiment. Statistical significance was measured by ANOVA and multiple Student’s t test and represented as ∗ (p < 0.05). INPP4B, inositol polyphosphate 4-phosphatase type II; MEF, mouse embryonic fibroblast; PIKfyve, Phosphoinositide Kinase, FYVE-Type Zinc Finger Containing.
      Inhibition of lysosomal fission has been proposed as an explanation for apilimod-induced lysosome enlargement (
      • Saffi G.T.
      • Tang E.
      • Mamand S.
      • Inpanathan S.
      • Fountain A.
      • Salmena L.
      • et al.
      Reactive oxygen species prevent lysosome coalescence during PIKfyve inhibition.
      ,
      • Choy C.H.
      • Saffi G.
      • Gray M.A.
      • Wallace C.
      • Dayam R.M.
      • Ou Z.-Y.A.
      • et al.
      Lysosome enlargement during inhibition of the lipid kinase PIKfyve proceeds through lysosome coalescence.
      ,
      • Saffi G.T.
      • Tang E.
      • Fountain A.
      • Botelho R.J.
      Reactive oxygen species rescue lysosome coalescence during PIKfyve inhibition.
      ). To investigate the specific consequence of Inpp4b deficiency on “kiss-and-run” and/or “fusion-and-fission” cycles, live-imaging of LY-labeled lysosomes was recorded. Inpp4b+/+ and Inpp4b−/− MEF treated with either vehicle or apilimod were monitored for up to 15 min and lysosomal fission events were recorded. Despite presenting similar fission rates in vehicle-treated Inpp4b+/+ and Inpp4b−/− MEF, apilimod significantly reduced lysosome fission rates in Inpp4b+/+ MEF, and remarkably fission was nearly abrogated in Inpp4b−/− MEF (Fig. 3, A and B and Movies S1–S4). This suggests that the enhanced lysosomal coalescence observed in Inpp4b−/− MEF upon apilimod treatment is due to an exacerbated inhibition of lysosomal fission and Inpp4b deficiency alters lysosome dynamics such that they are sensitized to the lysosomal fission-inhibiting effects of apilimod.
      Figure thumbnail gr3
      Figure 3Lysosome “kiss-and-run” dynamics are disrupted upon PIKfyve inhibition in Inpp4b-deficient cells. A, Inpp4b+/+ or Inpp4b−/− MEFs were prelabeled with Lucifer Yellow followed by treatment with vehicle or 10 nM apilimod. Time in sec (s) refers to time post treatment. See for full capture imaging videos. Inset represents an enlarged section from the field of view tracking individual lysosome particle dynamics over time. The scale bar represents 20 μm, zoomed inset: 10 μm. B, lysosome splitting frequency over 15 min for WT or Null MEFs treated with vehicle or apilimod demonstrate reduced splitting in PIKfyve inhibited WT MEFs and to a greater extent in PIKfyve inhibited Null MEFs, indicative of possible reduced lysosome fission. Data represent mean ± SD from three independent experiments, where at least a total of 1300 lysosomes were tracked for 1 to 3 cells over 15 min. Statistical significance was measured by ANOVA and multiple Student’s t test and represented as ∗ (p < 0.05). INPP4B, inositol polyphosphate 4-phosphatase type II; MEF, mouse embryonic fibroblast; PIKfyve, Phosphoinositide Kinase, FYVE-Type Zinc Finger Containing.

      Apilimod treatment induces differential effects on gene expression and lysosome function in Inpp4b+/+ and Inpp4b−/− MEF

      To shed light on the exacerbated vacuolation phenotypes observed with Inpp4b deficiency, we assessed the effects of apilimod treatment on lysosomal biogenesis in Inpp4b+/+ and Inpp4b−/− MEF. Firstly, evaluation of nuclear translocation of Transcription Factor EB (TFEB) (
      • Sardiello M.
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      • di Ronza A.
      • Medina D.L.
      • Valenza M.
      • Gennarino V.A.
      • et al.
      A gene network regulating lysosomal biogenesis and function.
      ), a key regulator of lysosomal gene transcription that undergoes nuclear translocation upon apilimod treatment, did not reveal any difference between Inpp4b+/+ and Inpp4b−/− MEF (Fig. 4, A and B). Next, transcript levels of representative lysosomal genes including LAMP1, MCOLN1, CTSB, CTSD, ATP6V1D, and ATP6V1H were measured by TaqMan qPCR after vehicle or apilimod treatment. In vehicle-treated cells, Inpp4b−/− MEF demonstrated modest, but significantly reduced expression of all lysosomal genes, except for CTSD (Fig. 4C). Strikingly, apilimod treatment led to a ∼3-fold increase of all lysosomal transcripts tested in Inpp4b−/− MEF, whereas Inpp4b+/+ MEF showed no significant changes under the same conditions (Fig. 4C). Immunoblotting of representative lysosomal proteins demonstrated that Inpp4b−/− MEF have reduced steady state expression of LAMP1, pre- and mature-cathepsin B, and V-ATPase V1H, similar to transcript expression profiles (Fig. 4, D and E). However, apilimod treatment of Inpp4b+/+ MEF demonstrated elevated levels of the lysosomal membrane-bound LAMP1 and V-ATPase V1H proteins, but no significant change in cathepsin proteins. By contrast, apilimod treatment demonstrated significantly elevated expression of all lysosomal proteins tested in Inpp4b−/− MEF (Fig. 4, D and E). To quantify the differential effects of apilimod on lysosomal proteolytic function, we used the membrane permeable cathepsin B substrate-Magic Red which upon hydrolysis forms membrane impermeable fluorescent cresyl-violet and accumulates within functional lysosomes (
      • Bright N.A.
      • Davis L.J.
      • Luzio J.P.
      Endolysosomes are the principal intracellular sites of acid hydrolase activity.
      ). The results of this assay paralleled the transcript and protein expression levels of cathepsin B, where apilimod treatment had no effect in Inpp4b+/+ MEF, and in Inpp4b−/− MEF, despite having a reduced steady state cathepsin B activity, apilimod treatment led to a more than 3-fold induction of cathepsin B activity (Fig. 4, F and G). Although activation of nuclear translocation of TFEB by apilimod was no different in Inpp4b+/+ and Inpp4b−/− MEF, lysosomal gene expression, protein expression, and proteolytic function of lysosomes are differentially impacted by apilimod treatment in Inpp4b+/+ and Inpp4b−/− MEF.
      Figure thumbnail gr4
      Figure 4PIKfyve inhibition in Inpp4b-deficient cells disrupts lysosome gene expression. A, Inpp4b+/+ or Inpp4b−/− MEFs transiently expressing pEGFP-TFEB and treated with vehicle or apilimod 10 nM for 48 h. DNA was stained with Hoechst 33342. B, TFEB nuclear translocation measured as percentage of cells demonstrating nuclear EGFP-TFEB for (A). The scale bar represents 25 μm. C, Inpp4b+/+ or Inpp4b−/− MEFs treated with vehicle or apilimod 10 nM for 48 h followed by qRT-PCR analysis of lysosome gene expression normalized against Actb. Shown is mean ± SD from three independent experiments. D, immunoblot of Inpp4b+/+ or Inpp4b−/− MEFs treated with vehicle or apilimod 10 nM for 48 h followed by assessment of select lysosomal proteins as indicated. E, analysis of protein levels from (D) shown as mean ± SD from three independent experiments. Data represent ± SD from five independent experiments with 30 to 35 cells assessed per treatment condition per experiment for (A and B). F, Inpp4b+/+ or Inpp4b−/− MEF treated with vehicle or apilimod 10 nM for 48 h followed by Magic red incubation for 1 h. The scale bar represents 25 μm. G, quantification of Magic red intensity from (F) as measured through flow cytometry and mean ± SD from three independent experiments. Statistical significance was measured by ANOVA and multiple Student’s t test and represented as ∗ (p < 0.05). INPP4B, inositol polyphosphate 4-phosphatase type II; MEF, mouse embryonic fibroblast; PIKfyve, Phosphoinositide Kinase, FYVE-Type Zinc Finger Containing; TFEB, Transcription Factor EB.

      Inhibition of autophagy by apilimod is potentiated in Inpp4b-deficient cells

      PIKfyve inhibition has been demonstrated to inhibit autophagy in various physiological and pathological settings upon extended PIKfyve inhibition, but not upon acute exposure (
      • Sharma G.
      • Guardia C.M.
      • Roy A.
      • Vassilev A.
      • Saric A.
      • Griner L.N.
      • et al.
      A family of PIKFYVE inhibitors with therapeutic potential against autophagy-dependent cancer cells disrupt multiple events in lysosome homeostasis.
      ,
      • Saffi G.T.
      • Tang E.
      • Mamand S.
      • Inpanathan S.
      • Fountain A.
      • Salmena L.
      • et al.
      Reactive oxygen species prevent lysosome coalescence during PIKfyve inhibition.
      ,
      • de Campos C.B.
      • Zhu Y.X.
      • Sepetov N.
      • Romanov S.
      • Bruins L.A.
      • Shi C.-X.
      • et al.
      Identification of PIKfyve kinase as a target in multiple myeloma.
      ). We evaluated how apilimod treatment affected autophagy in the context of Inpp4b deficiency by first measuring levels of MAP1LC3A/B (LC3), a membrane protein specifically expressed on autophagosomes by IF with anti-LC3 antibodies (
      • Runwal G.
      • Stamatakou E.
      • Siddiqi F.H.
      • Puri C.
      • Zhu Y.
      • Rubinsztein D.C.
      LC3-positive structures are prominent in autophagy-deficient cells.
      ). Total cellular LC3 staining was similar in vehicle-treated MEF. Apilimod treatment induced LC3 levels to significantly higher levels in Inpp4b−/− MEF than in Inpp4b+/+ MEF (Fig. 5, A and B). Notably, the corresponding elevation in autophagosome levels induced by apilimod in Inpp4b−/− MEF was fully rescued by ectopic INPP4B expression, indicating that LC3 induction levels are restricted by Inpp4b (Fig. S6, AC). We also assessed extent of autolysosome formation by quantitating colocalization LAMP1-mCherry fluorescence and LC-3 by IF. Relative to vehicle-treated MEF, colocalization of LAMP1 (lysosomes) and LC3 (autophagosomes) was elevated upon apilimod treatment, and Inpp4b−/− MEF had significantly higher levels of colocalization than Inpp4b+/+ MEF (Fig. 5, A and C). Autophagy was also assessed by measuring LC-3 status by immunoblotting (
      • Klionsky D.J.
      • Abdelmohsen K.
      • Abe A.
      • Abedin M.J.
      • Abeliovich H.
      • Acevedo Arozena A.
      • et al.
      Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).
      ). During autophagy, cytosolic LC3-I (∼16 kDa) is conjugated with phosphatidylethanolamine on the phagophore membrane to form LC3-II (∼14 kDa). LC3-II is absent in vehicle-treated Inpp4b+/+ and Inpp4b−/− MEF (Figs. 5, D and E and S7), however apilimod treatment significantly induced LC3-II levels in Inpp4b+/+ MEF, and even further induction was observed in Inpp4b−/− MEF (Figs. 5, D and E and S7). Similar observations were made in U2OS cells where INPP4B expression was silenced with siRNA (Fig. S2E).
      Figure thumbnail gr5
      Figure 5Autophagic flux is blocked upon PIKfyve inhibition in Inpp4b-deficient cells. A, Inpp4b+/+ or Inpp4b−/− MEFs transiently expressing LAMP1-mCherry to mark lysosomes and treated with vehicle or apilimod 10 nM for 48 h, followed by immunostain against LC3. B, quantifications of LC3 puncta intensity per cell across indicated conditions to measure autophagosome levels and (C) LC3 intensity overlapping on LAMP1-positive lysosomes where increased LC-3/LAMP1 intensity ratio indicate autolysosome formation. D, immunoblot of Inpp4b+/+ or Inpp4b−/− MEFs treated with vehicle or apilimod 10 nM for 48 h and assessed for protein levels of Inpp4b, LC3, and Beta actin as loading control. E, quantification of immunoblot from (D) for LC3-II/LC3-I protein ratio shown as mean ± SD from three independent experiments. F, Inpp4b+/+ or Inpp4b−/− MEF transiently expressing mCherry-EGFP-LC3B and treated with vehicle or apilimod 10 nM for 48 h. G, quantification of LC-3 green puncta over red puncta intensity ratio. The scale bar represents 15 μm, zoomed inset: 5 μm. Data represent mean ± SD from three independent experiments, with 25 to 30 cells assessed per treatment condition per experiment for (AC) and (FG). Statistical significance was measured by ANOVA and multiple Student’s t test and represented as ∗ (p < 0.05). PIKfyve, Phosphoinositide Kinase, FYVE-Type Zinc Finger Containing; INPP4B, inositol polyphosphate 4-phosphatase type II; MEF, mouse embryonic fibroblast.
      Autophagy was further assessed by estimating autophagic flux using a reporter system which expresses a fusion of LC3 protein with the acid-insensitive mCherry protein and the acid-sensitive GFP protein (
      • Zhang X.
      • Chen S.
      • Huang K.
      • Le W.
      Why should autophagic flux be assessed?.
      ,
      • N’Diaye E.-N.
      • Kajihara K.K.
      • Hsieh I.
      • Morisaki H.
      • Debnath J.
      • Brown E.J.
      PLIC proteins or ubiquilins regulate autophagy-dependent cell survival during nutrient starvation.
      ). This reporter permits the estimation of autophagic flux—autophagosome to autolysosome transition—by measuring the ratio of yellow (mCherry+ GFP+) or red (mCherry+ GFP-) puncta, respectively (
      • Sharma G.
      • Guardia C.M.
      • Roy A.
      • Vassilev A.
      • Saric A.
      • Griner L.N.
      • et al.
      A family of PIKFYVE inhibitors with therapeutic potential against autophagy-dependent cancer cells disrupt multiple events in lysosome homeostasis.
      ,
      • Hansen T.E.
      • Johansen T.
      Following autophagy step by step.
      ,
      • Kim S.
      • Choi S.
      • Kang D.
      Quantitative and qualitative analysis of autophagy flux using imaging.
      ). In vehicle-treated Inpp4b+/+ and Inpp4b−/− MEF, we observed predominantly red fluorescence indicating functional transition through autophagy (Fig. 5, F and G). By contrast, apilimod treatment resulted in predominantly yellow puncta in Inpp4b+/+ MEF and an even higher ratio of yellow to red puncta in Inpp4b−/− MEF. (Fig. 5, F and G). Notably, the accumulation of yellow fluorescence (mCherry+ GFP+) in apilimod-treated Inpp4b−/− MEF appear to be on the membranes of enlarged vacuoles. These data demonstrate that Inpp4b deficiency alone does not alter autophagic flux, however, apilimod treatment reveals that Inpp4b regulates autophagic flux in some conditions, including PIKfyve inhibition.

      Inpp4b deficiency exacerbates apilimod-mediated PtdIns(3)P generation

      Given the direct roles for PIKfyve and Inpp4b on PtdIns metabolism, we assessed how PtdIns homeostasis may be differentially affected in Inpp4b+/+ and Inpp4b−/− MEF in response to apilimod. We first measured total cell levels of PtdIns(3)P and PtdIns(3,5)P 2 through 3H-myo-inositol labeling and HPLC scintillation and quantification (
      • Klionsky D.J.
      • Abdelmohsen K.
      • Abe A.
      • Abedin M.J.
      • Abeliovich H.
      • Acevedo Arozena A.
      • et al.
      Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).
      ). Inpp4b deficiency did not alter PtdIns(3,5)P2 levels, but as expected, apilimod treatment significantly reduced PtdIns(3,5)P2 levels in both Inpp4b+/+ and Inpp4b−/− MEF (Fig. 6A).
      Figure thumbnail gr6
      Figure 6PIKfyve inhibition in Inpp4b-deficient cells leads to elevated PtdIns(3)P levels. A, 3H-myo-inositol incorporation and HPLC scintillation quantification of PtdIns(3)P and PtdIns(3,5)P2 for Inpp4b+/+ or Inpp4b−/− MEFs treated with vehicle or apilimod (n = 3). B, Inpp4b+/+ or Inpp4b−/− MEFs transiently expressing LAMP1-mCherry and treated with vehicle or apilimod 10 nM for 48 h. C, total cell PtdIns(3)P or (D) LAMP1-mCherry positive PtdIns(3)P within a cell. The scale bar represents 20 μm, zoomed inset: 5 μm. Data represent ± SD from three independent experiments with 25 to 30 cells assessed per treatment condition per experiment for (BD). Statistical significance was measured by ANOVA and multiple Student’s t test and represented as ∗ (p < 0.05). INPP4B, inositol polyphosphate 4-phosphatase type II; MEF, mouse embryonic fibroblast; PtdIns, phosphoinositide; PtdIns(3,5)P2, phosphatidylinositol-3,5-bisphosphate; PtdIns(3)P, phosphatidylinositol-3-monophosphate; PIKfyve, Phosphoinositide Kinase, FYVE-Type Zinc Finger Containing.
      Total steady state PtdIns(3)P levels were also similar in Inpp4b−/− MEF compared to Inpp4b+/+ MEF; this was unexpected given that PtdIns(3)P is the product of Inpp4b catalysis. Furthermore, apilimod treatment induced a significant ∼1.3 fold increase of PtdIns(3)P in Inpp4b+/+ MEF and a ∼3 fold increase of PtdIns(3)P in Inpp4b−/− MEFs (Fig. 6A). To support these surprising findings, we also measured PtdIns(3)P by IF with an anti-PtdIns(3)P antibody in cells where lysosomes were simultaneously marked with LAMP1-mCherry (Fig. 6B). Firstly, IF confirmed the elevated levels of total cellular PtdIns(3)P observed in apilimod-treated Inpp4b+/+ and Inpp4b−/− MEF (Fig. 6C). Colocalization of PtdIns(3)P with LAMP1-mCherry revealed a similar pattern of PtdIns(3)P levels on lysosomal membranes (Fig. 6D). IF of PtdIns(3,4)P2 revealed greater total and lysosomal levels of PtdIns(3,4)P2, as expected in Inpp4b-deficient MEF (Fig. S8, AC). Apilimod treatment had no effect on total cell or LAMP1-mCherry positive PtdIns(3,4)P2 levels in Inpp4b+/+ MEF, however, there was a small but significant reduction in PtdIns(3,4)P2 levels compared to steady state levels for Inpp4b−/− MEF (Fig. S8, AC). In sum, apilimod treatment led to a significant increase in total and lysosomal PtdIns(3)P in Inpp4b−/− MEF, indicating an unexpected role for Inpp4b in suppressing cellular levels of PtdIns(3)P.

      Apilimod-mediated lysosome enlargement is driven by VPS34-mediated PtdIns(3)P production

      We sought to gain a further understanding of the mechanisms leading to elevated cellular PtdIns(3)P and its role in the exacerbated lysosomal enlargement upon apilimod treatment in Inpp4b−/− MEF. Given its major role in cellular PtdIns(3)P generation, we explored VPS34 functions (
      • Ikonomov O.C.
      • Sbrissa D.
      • Venkatareddy M.
      • Tisdale E.
      • Garg P.
      • Shisheva A.
      Class III PI 3-kinase is the main source of PtdIns3P substrate and membrane recruitment signal for PIKfyve constitutive function in podocyte endomembrane homeostasis.
      ). No changes in VPS34 protein levels were observed by immunoblot in Inpp4b deficiency nor in vehicle- or apilimod-treated conditions (Fig. 7A). Surprisingly, measurement of the lipid kinase activity of VPS34 demonstrated a significant elevation only in apilimod-treated Inpp4b−/− MEF (Fig. 7B), thereby explaining elevated PtdIns(3)P levels.
      Figure thumbnail gr7
      Figure 7VPS34 activity is elevated upon PIKfyve inhibition in Inpp4b-deficient cells. A, Inpp4b+/+ or Inpp4b−/− MEFs treated with vehicle or apilimod 10 nM for 48 h, followed by immunoblotting against Inpp4b, VPS34, or Beta Actin. B, Inpp4b+/+ or Inpp4b−/− MEFs treated with vehicle or apilimod 10 nM for 48 h, followed by VPS34 immunoprecipitation and kinase assay to monitor VPS34 activity. C, Inpp4b+/+ MEF transiently expressing LAMP1-mCherry and treated with vehicle or 500 nM VPS34-IN1 for 48 h followed by PtdIns(3)P immunostain. D, quantification from (C) of total cell PtdIns(3)P fluorescence signal or (E) PtdIns(3)P fluorescence signal overlayed on LAMP1-mCherry positive regions within a cell. The scale bar represents 20 μm, zoomed inset: 5 μm. F, Inpp4b+/+ or Inpp4b−/− MEF transiently expressing LAMP1-mCherry and treated with vehicle or apilimod 10 nM or VPS34-IN1 500 nM at various combinations for 48 h. G, quantification of LAMP1-positive vacuoles greater than 1.5 μm in diameter per cell and (H) mean vacuole diameter (μm). The scale bar represents 20 μm. Data represent ± SD from three independent experiments with 25 to 30 cells assessed per treatment condition per experiment. Statistical significance was measured by ANOVA and multiple Student’s t test and represented as ∗ (p < 0.05). INPP4B, inositol polyphosphate 4-phosphatase type II; MEF, mouse embryonic fibroblast; PtdIns, phosphoinositide; PtdIns(3)P, phosphatidylinositol-3-monophosphate; PIKfyve, Phosphoinositide Kinase, FYVE-Type Zinc Finger Containing.
      To elucidate a role for PtdIns(3)P in the formation of massively enlarged vacuoles in apilimod-treated Inpp4b−/− MEF, we reasoned that depletion of cellular PtdIns(3)P could attenuate this phenotype. Thus, we treated MEF with both apilimod and the specific VPS34 inhibitor, VPS34-IN1 (
      • Munson M.J.
      • Allen G.F.
      • Toth R.
      • Campbell D.G.
      • Lucocq J.M.
      • Ganley I.G.
      mTOR activates the VPS34-UVRAG complex to regulate autolysosomal tubulation and cell survival.
      ,
      • Bago R.
      • Malik N.
      • Munson M.J.
      • Prescott A.R.
      • Davies P.
      • Sommer E.
      • et al.
      Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals that the phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream target of class III phosphoinositide 3-kinase.
      ). PtdIns(3)P IF performed on VPS34-IN1-treated MEF demonstrated that VPS34 inhibition can effectively diminish total cellular and lysosomal localized PtdIns(3)P levels (Fig. 7, CE). Notably, VPS34 inhibition in apilimod-treated Inpp4b−/− MEF rescued the exacerbated lysosome enlargement but only to levels of enlargement normally observed in Inpp4b+/+ MEF; VPS34-IN1 had little effect on the enlarged lysosome size and number in apilimod-treated Inpp4b+/+ MEF, indicating that induction of PtdIns(3)P levels may not be necessary or sufficient for normal apilimod responses (Fig. 7, FH). In sum, VPS34-IN1 revealed that aberrant hyperactivation of VPS34 in Inpp4b−/− MEF treated with apilimod leads to exacerbated lysosomal enlargement. These findings suggest the existence of a role for Inpp4b in the regulation of VPS34 activation, and thereby PtdIns(3)P levels, in cells undergoing lysosomal stress induced by PIKfyve inhibition.

      Discussion

      Lysosomes, the primary catabolic organelles in the cell, play pivotal roles in many cellular processes including cell differentiation, plasma membrane repair, programmed cell death, nutrient sensing, and stress responses (
      • Korolchuk V.I.
      • Saiki S.
      • Lichtenberg M.
      • Siddiqi F.H.
      • Roberts E.A.
      • Imarisio S.
      • et al.
      Lysosomal positioning coordinates cellular nutrient responses.
      ). Thus, exquisite control of lysosome homeostasis, including the dynamic mechanisms that control total lysosomal content, number, and size are critical to maintaining normal lysosomal and cellular functions. Emerging data indicates key roles for PtdIns in maintaining lysosomal homeostasis. In this study, we explored the dynamics of PIKfyve inhibition and resultant PtdIns(3,5)P2 depletion, which leads to disruption of lysosomal “fusion-fission” cycles by compromising “fission”, thereby generating enlarged coalescent lysosomes (
      • Bissig C.
      • Hurbain I.
      • Raposo G.
      • van Niel G.
      PIKfyve activity regulates reformation of terminal storage lysosomes from endolysosomes.
      ,
      • Saffi G.T.
      • Tang E.
      • Mamand S.
      • Inpanathan S.
      • Fountain A.
      • Salmena L.
      • et al.
      Reactive oxygen species prevent lysosome coalescence during PIKfyve inhibition.
      ,
      • Choy C.H.
      • Saffi G.
      • Gray M.A.
      • Wallace C.
      • Dayam R.M.
      • Ou Z.-Y.A.
      • et al.
      Lysosome enlargement during inhibition of the lipid kinase PIKfyve proceeds through lysosome coalescence.
      ). PIKfyve inhibition also disrupts endocytic cargo delivery, autophagosome formation, and autophagic flux, possibly a result of lysosome membrane perturbations where lysosomes fail to resolve from other lysosomes, late endosomes, and autophagosomes (
      • Bissig C.
      • Hurbain I.
      • Raposo G.
      • van Niel G.
      PIKfyve activity regulates reformation of terminal storage lysosomes from endolysosomes.
      ,
      • Sharma G.
      • Guardia C.M.
      • Roy A.
      • Vassilev A.
      • Saric A.
      • Griner L.N.
      • et al.
      A family of PIKFYVE inhibitors with therapeutic potential against autophagy-dependent cancer cells disrupt multiple events in lysosome homeostasis.
      ). PIKfyve has been shown to functionally coordinate with the Class III PI3-Kinase VPS34 through its catalytic product PtdIns(3)P, which serves as a membrane localization target for PIKfyve, as well as a substrate precursor for PtdIns(3,5)P2 synthesis (
      • Ikonomov O.C.
      • Sbrissa D.
      • Venkatareddy M.
      • Tisdale E.
      • Garg P.
      • Shisheva A.
      Class III PI 3-kinase is the main source of PtdIns3P substrate and membrane recruitment signal for PIKfyve constitutive function in podocyte endomembrane homeostasis.
      ). Together, the functions of PIKfyve and VPS34 recruit and regulate components of endosomal and lysosomal recycling (
      • Giridharan S.S.P.
      • Luo G.S.
      • Rivero-Rios P.S.
      • Steinfeld N.S.
      • Tronchere H.
      • Singla A.
      • et al.
      Lipid kinases VPS34 and PIKfyve coordinate a phosphoinositide cascade to regulate Retriever-mediated recycling on endosomes.
      ). This PIKfyve-VPS34 crosstalk suggests the existence of other PIKfyve cross talks with other PtdIns-modifying enzymes.
      Emerging studies present a role for the lipid-phosphatase INPP4B in the regulation of lysosome-associated functions. Our group has demonstrated that INPP4B expression in acute myeloid leukemia regulates lysosomal biogenesis and functions which are crucial for leukemia stem cell maintenance, differentiation, and chemoresistance (
      • Woolley J.F.
      • Chen K.
      • Genc G.E.
      • Lee D.K.C.
      • Dzneladze I.
      • He R.
      • et al.
      INPP4B promotes leukemia by restricting leukemic stem cell differentiation through regulation of lysosomal functions.
      ). Another study demonstrated that INPP4B depletion in triple negative breast cancer resulted in delayed EGFR trafficking from early endosomes to late endosomes/lysosomes (
      • Liu H.
      • Paddock M.N.
      • Wang H.
      • Murphy C.J.
      • Geck R.C.
      • Navarro A.J.
      • et al.
      The INPP4B tumor suppressor modulates EGFR trafficking and promotes triple-negative breast cancer.
      ). A role for INPP4B in lysosomal functions is further supported by the observation that elevated expression of INPP4B in PIK3CA-mutant ER+ breast cancer cells induce formation of late endosomes and lysosomes, increase cargo trafficking toward lysosomes, and promote endosomal sequestration and lysosomal degradation of key signaling proteins (
      • Rodgers S.J.
      • Ooms L.M.
      • Oorschot V.M.J.
      • Schittenhelm R.B.
      • Nguyen E.V.
      • Hamila S.A.
      • et al.
      INPP4B promotes PI3Kα-dependent late endosome formation and Wnt/β-catenin signaling in breast cancer.
      ). Together, these data support a role for INPP4B in the enhancement of function and cellular content of lysosomes. Indeed, this study further supports this notion as Inpp4b-deficient MEF consistently demonstrated reduced total lysosomal content and lysosome numbers as measured by LAMP1 IF, lysotracker labeling, and lysosomal accumulation of LY (Figs. 1, 2 and S1). Furthermore, lysosomal transcript and protein expression levels were reduced in Inpp4b−/− MEF, together suggesting a role for Inpp4b in promoting and/or maintaining lysosomal content by controlling biogenesis through transcriptional mechanisms (Fig. 4). Since INPP4B and PIKfyve both regulate lysosome function, we sought to shed light on putative interactions between these two enzymes.
      Apilimod-mediated PIKfyve inhibition in MEF leads to formation of numerous enlarged translucent cytoplasmic vacuoles readily seen by light microscopy. In previous studies, 20 nM apilimod for 1 h in RAW macrophages led to rapid lysosome enlargement to a volume of ∼10 μm3. HeLa or RPE cells required 100 to 200 nM apilimod to generate lysosome of 3 to 4 um3 (
      • Saffi G.T.
      • Tang E.
      • Mamand S.
      • Inpanathan S.
      • Fountain A.
      • Salmena L.
      • et al.
      Reactive oxygen species prevent lysosome coalescence during PIKfyve inhibition.
      ,
      • Choy C.H.
      • Saffi G.
      • Gray M.A.
      • Wallace C.
      • Dayam R.M.
      • Ou Z.-Y.A.
      • et al.
      Lysosome enlargement during inhibition of the lipid kinase PIKfyve proceeds through lysosome coalescence.
      ). By comparison, apilimod treatment of Inpp4b+/+ MEF at 10 nM or 500 nM for 1 h dilated lysosome volume to 3 um3 or 4.5 um3, respectively. The key findings made in this study stem from the observations that apilimod treatment of Inpp4b−/− MEF generate significantly larger cytoplasmic vacuoles than apilimod-treated Inpp4b+/+. This observation was the first indication of a putative crosstalk between INPP4B and PIKfyve. To gain further insight into the exacerbated vacuolation phenotype in Inpp4b-deficient MEF, we first confirmed that enlarged vacuoles were of lysosomal origin as proposed by Choy et al. (
      • Choy C.H.
      • Saffi G.
      • Gray M.A.
      • Wallace C.
      • Dayam R.M.
      • Ou Z.-Y.A.
      • et al.
      Lysosome enlargement during inhibition of the lipid kinase PIKfyve proceeds through lysosome coalescence.
      ). Loading of MEF with LY prior to apilimod stimulation permitted effective lysosomal accumulation, which then enabled the clear visualization and quantitation of lysosomal features after apilimod treatment. LY labeling confirmed that lysosome size and number were affected to a greater degree in Inpp4b−/− MEF by apilimod; and that apilimod inhibits lysosomal accumulation of cargo, a phenotype which was also exacerbated in Inpp4b−/− MEF. Lysosome dynamics in Inpp4b−/− MEF were exquisitely sensitized to apilimod, as demonstrated by the severely compromised lysosomal fission, relative to the moderately compromised fission rates observed in Inpp4b+/+ MEF.
      In our attempts to further understand the underlying biology responsible for the exaggerated response to apilimod observed in Inpp4b−/− MEF, we observed that the expression of representative lysosomal gene transcripts were either unchanged or decreased in Inpp4b+/+ MEF treated with apilimod. Inpp4b−/− MEF on the other hand demonstrated a significant 2-3-fold increase in the expression of all lysosomal transcripts tested. Elevated lysosomal biogenesis observed in Inpp4b−/− MEF was not explained by the nuclear localization of Tfeb; however, altered Tfeb transactivation activity by phosphorylation and acetylation by the action of factors including mTORC1, PKC, PKD, GSK3β, and phosphatases such as the Ca2+-dependent calcineurin cannot be formally excluded with our data (
      • Zhang J.
      • Wang J.
      • Zhou Z.
      • Park J.-E.
      • Wang L.
      • Wu S.
      • et al.
      Importance of TFEB acetylation in control of its transcriptional activity and lysosomal function in response to histone deacetylase inhibitors.
      ,
      • Brijmohan A.S.
      • Batchu S.N.
      • Majumder S.
      • Alghamdi T.A.
      • Thieme K.
      • McGaugh S.
      • et al.
      HDAC6 inhibition promotes transcription factor EB activation and is protective in experimental kidney disease.
      ,
      • Zhu S.-Y.
      • Yao R.-Q.
      • Li Y.-X.
      • Zhao P.-Y.
      • Ren C.
      • Du X.-H.
      • et al.
      The role and regulatory mechanism of transcription factor EB in health and diseases.
      ). Analysis of lysosomal protein levels showed that lysosomal membrane–embedded proteins including Lamp1 and V-ATPase (V1H) subunit were significantly elevated in Inpp4b+/+ and Inpp4b−/− MEF upon apilimod treatment. On the other hand, cathepsin-B protein levels and activity are elevated only in apilimod-treated Inpp4b−/− MEF, suggesting that the stability of some lysosomal proteins may also be differentially regulated in apilimod-treated Inpp4b+/+ and Inpp4b−/− MEF. Disruption of autophagic flux, which blocks turnover of lysosomal proteins (
      • Sharma G.
      • Guardia C.M.
      • Roy A.
      • Vassilev A.
      • Saric A.
      • Griner L.N.
      • et al.
      A family of PIKFYVE inhibitors with therapeutic potential against autophagy-dependent cancer cells disrupt multiple events in lysosome homeostasis.
      ,
      • de Campos C.B.
      • Zhu Y.X.
      • Sepetov N.
      • Romanov S.
      • Bruins L.A.
      • Shi C.-X.
      • et al.
      Identification of PIKfyve kinase as a target in multiple myeloma.
      ) may explain elevation of some lysosomal proteins (LAMP1 and V-ATPase). Also, it is currently unclear if elevated cathepsin-B protein and activity result due to disrupted feedback regulation from apilimod effects. In sum, the specific consequences of Inpp4b deficiency combined with PIKfyve inhibition on gene expression require further study.
      Our data also show that apilimod can elevate autophagosome levels and promote colocalization with lysosomes to form autolysosomes, however, autolysosome maturation appears to be inhibited as indicated by reduced autophagic flux. Interestingly, enlarged lysosomes in apilimod-treated cells are proteolytically competent as measured by Magic Red and DQ-BSA suggesting that altered lysosomal membrane composition may confer resistance to membrane degradation or other membrane-associated processes required for autolysosome maturation. Our observations are consistent with previous studies regarding the consequences of apilimod (
      • Sharma G.
      • Guardia C.M.
      • Roy A.
      • Vassilev A.
      • Saric A.
      • Griner L.N.
      • et al.
      A family of PIKFYVE inhibitors with therapeutic potential against autophagy-dependent cancer cells disrupt multiple events in lysosome homeostasis.
      ,
      • Qiao Y.
      • Choi J.E.
      • Tien J.C.
      • Simko S.A.
      • Rajendiran T.
      • Vo J.N.
      • et al.
      Autophagy inhibition by targeting pikfyve potentiates response to immune checkpoint blockade in prostate cancer.
      ,
      • Martin S.
      • Harper C.B.
      • May L.M.
      • Coulson E.J.
      • Meunier F.A.
      • Osborne S.L.
      Inhibition of PIKfyve by YM-201636 dysregulates autophagy and leads to apoptosis-independent neuronal cell death.
      ,
      • Hessvik N.P.
      • Øverbye A.
      • Brech A.
      • Torgersen M.L.
      • Jakobsen I.S.
      • Sandvig K.
      • et al.
      PIKfyve inhibition increases exosome release and induces secretory autophagy.
      ), with the exception that Inpp4b−/− MEF display exacerbated versions of this phenotype.
      Finally, we measured intracellular PtdIns levels in apilimod-treated MEF. PtdIns(3)P levels were observed to be unchanged in vehicle-treated MEF, which suggests that other major mechanisms govern PtdIns(3)P levels, and Inpp4b may only be a minor contributor. As previously reported, apilimod leads to moderately elevated levels of PtdIns(3)P, which we also observed in Inpp4b+/+ MEF (
      • Sbrissa D.
      • Naisan G.
      • Ikonomov O.C.
      • Shisheva A.
      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.
      ). Surprisingly, apilimod treatment in Inpp4b−/− MEF led to a dramatic increase in both total cellular and lysosomal levels of PtdIns(3)P. This observation was deemed paradoxical given that Inpp4b deficiency should generate less PtdIns(3)P and suggests that Inpp4b may regulate PtdIns(3)P levels through an indirect mechanism. To explain elevated PtdIns(3)P, our tests revealed that VPS34 activity, but not protein levels, was elevated. Furthermore, VPS34-IN1, a specific inhibitor of VPS34, depleted PtdIns(3)P and reversed the exacerbated lysosomal enlargement observed with Inpp4b deficiency. These data point to a direct role for VPS34 in apilimod-mediated PtdIns(3)P induction (Fig. 8). Furthermore, our results highlight a novel regulatory signaling axis linking PIKfyve, VPS34, and Inpp4b functions.
      Figure thumbnail gr8
      Figure 8Model for lysosome enlargement upon PIKfyve inhibition in Inpp4b deficient cells. A, in the steady state, cycling of phosphoinositides involve VPS34-dependent conversion of phosphatidylinositol (PtdIns) to PtdIns(3)P, PtdIns(3,4)P2 conversion by Inpp4b to PtdIns(3)P, and PtdIns(3)P conversion by PIKfyve to PtdIns(3,5)P2. Apilimod-induced PIKfyve inhibition deplete PtdIns(3,5)P2 to elevate the substrate PtdIns(3)P, and simultaneous PIKfyve and Inpp4b suppression further augment PtdIns(3)P levels. B, PIKfyve inhibition in Inpp4b+/+ MEF induce moderate lysosome enlargement, where lysosomes coalesce to become fewer in number and greater in individual lysosome volume. PIKfyve inhibition in Inpp4b−/− MEF exacerbate lysosome enlargement, where lysosomes are much greater in individual volume and this enlargement is facilitated by enrichment of VPS34-induced membrane PtdIns(3)P. INPP4B, inositol polyphosphate 4-phosphatase type II; MEF, mouse embryonic fibroblast; PtdIns, phosphoinositide; PtdIns(3,5)P2, phosphatidylinositol-3,5-bisphosphate; PtdIns(3)P, phosphatidylinositol-3-monophosphate; PIKfyve, Phosphoinositide Kinase, FYVE-Type Zinc Finger Containing.
      In conclusion, our study demonstrates that Inpp4b deficiency sensitizes cells and lysosomes to the plethora of effects conferred by apilimod-mediated PIKfyve inhibition, the most obvious of which is significantly exacerbated lysosomal enlargement compared to WT cells, most likely due to blocked fission and thus, enhanced lysosome coalescence. Similar exacerbated consequences are observed for various other phenotypes associated with PIKfyve inhibition including disrupted autophagy and reduced cargo delivery to lysosomes (
      • Bissig C.
      • Hurbain I.
      • Raposo G.
      • van Niel G.
      PIKfyve activity regulates reformation of terminal storage lysosomes from endolysosomes.
      ,
      • Sharma G.
      • Guardia C.M.
      • Roy A.
      • Vassilev A.
      • Saric A.
      • Griner L.N.
      • et al.
      A family of PIKFYVE inhibitors with therapeutic potential against autophagy-dependent cancer cells disrupt multiple events in lysosome homeostasis.
      ,
      • Dayam R.M.
      • Saric A.
      • Shilliday R.E.
      • Botelho R.J.
      The phosphoinositide-gated lysosomal Ca(2+) channel, TRPML1, is required for phagosome maturation.
      ,
      • Mironova Y.A.
      • Lenk G.M.
      • Lin J.-P.
      • Lee S.J.
      • Twiss J.L.
      • Vaccari I.
      • et al.
      PI(3,5)P2 biosynthesis regulates oligodendrocyte differentiation by intrinsic and extrinsic mechanisms.
      ). Finally, we have uncovered a novel role for Inpp4b in regulating the activation of VPS34 activity and induction of PtdIns(3)P levels, which was only revealed upon lysosomal stress activated by PIKfyve inhibition. This function is mediated through a novel PIKfyve–VPS34–Inpp4b regulatory signaling axis.

      Experimental procedures

      MEF preparation, cell culture conditions, transfections, drug treatment

      Immortalized Inpp4b+/+ and Inpp4b−/− MEF were generated as previously detailed (
      • Brijmohan A.S.
      • Batchu S.N.
      • Majumder S.
      • Alghamdi T.A.
      • Thieme K.
      • McGaugh S.
      • et al.
      HDAC6 inhibition promotes transcription factor EB activation and is protective in experimental kidney disease.
      ). MEF and U2OS cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS). MEF or U2OS cells were transiently transfected with pTWIST-mCherry, pTWIST-Inpp4b-mCherry, mCherry-Lamp1, mCherry-EGFP-LC3B, pEGFP, GFP-Inpp4b, GFP-Inpp4b (C845A), and pEGFP-TFEB. U2OS cells were stably transfected with mCherry-Lamp1 through selection with 200 μg/ml G418 for 10 days. Transfections of MEF and U2OS cells performed with Fugene HD (Promega) at 3:1 of DNA:Fugene ratio for 24 h followed by washing and supplementation with complete DMEM growth media. siRNA-mediated gene silencing for INPP4B in U2OS cells carried out using DharmaFECT1 Transfection reagent (GE Dharmacon). Briefly, 0.1 nmol of nontargeting or Inpp4b siRNA (GE Dharmacon) mixed with 2 μl of DharmaFECT1 Transfection reagent in DMEM media without FBS was added to U2OS cells for 24 h, followed by washing off the transfection mix with PBS and growth of cells for 48 h with treatment before imaging and Western blot. MEF and U2OS cells treated with apilimod or VPS34-IN1 (Selleck Chemicals) to inhibit PIKfyve or VPS34 functions respectively at doses and durations indicated.

      Retroviral transduction

      3.0 × 106 HEK 293T cells were grown in a 10 cm dish for 24 followed by calcium phosphate transfection. Briefly, 10 μg of retroviral plasmid pWZL hygro SV40 T-Large was mixed with 5 μg of pCL-Eco retroviral packaging vector and 2M CaCl2 to a final volume of 300 μl in sterile water. The transfection mix was supplemented with equal volume of 2× Hepes-buffered saline (140 mM NaCl, 1.5 mM Na2HPO4) followed by addition to the cells. Following 24 h post transfection, the media was changed and supplemented with complete DMEM growth media. Media was collected 48 h and 72 h post transfection. Virus-enriched media was filtered through a 0.45-micron filter, supplemented with 8 μg/ml protamine sulfate, and added to MEF cells grown in 10 cm dishes. Infections were repeated every 8 h and MEF cells were selected for 4 days with 75 μg/ml hygromycin B.

      Lysosome labeling

      MEF cell lysosomes were labeled with 1 mg/ml LY (Thermo Fisher Scientific) for 2 h in complete growth media at 37 °C and 5% CO2, followed by washing in PBS and supplementation of complete media for 1 h. LTR (Thermo Fisher) was also used to label MEF cell lysosomes by incubation at 1 μM for 30 min in complete growth media. Magic Red (Abcam) was used to assess lysosomal cathepsin B activity in MEF cells by incubation for 1 h according to manufacturer instructions.

      Immunofluorescence

      Immunolabeling of cells following apilimod treatment was performed by fixation with 4% (v/v) paraformaldehyde for 15 min, permeabilization with 100% ice-cold methanol for 5 min, and blocking in 3% bovine serum albumin (v/v) in PBS. Cells were incubated with rabbit monoclonal antibody against mouse LC3B (1:200; Cell Signaling) and Alexa Fluor 488-conjugated goat polyclonal antibody against rabbit IgG (1:1000; Thermo Fisher). Alternatively, immunostaining was performed with rat monoclonal antibody against mouse LAMP1 (1:200, Clone 1D4B; Thermo Fisher) and Dylight 488-conjugated donkey polyclonal antibody against rat IgG (1:1000; Bethyl, Montgomery, TX). For CD63 immunostaining, fixations were performed with 4% (v/v) paraformaldehyde for 15 min, followed by permeabilization with 0.1% Triton X-100 for 10 min at room temperature. Immunostaining was performed with mouse monoclonal antibody against mouse CD63 (1:150; Novus Biologicals) and Dylight 488-conjugated Goat polyclonal antibody against mouse IgG (1:1000; Bethyl). Total cell PtdIns(3,4)P2 or PtdIns(3)P immunostaining was performed by fixation with 4% (v/v) paraformaldehyde for 15 min, permeabilization with 20 μM digitonin (Promega) in buffer A (20 mM Pipes pH 6.8, 137 mM NaCl, 2.7 mM KCl) for 30 min, and blocking with buffer A containing 5% normal goat serum and 50 mM NH4Cl. Immunostaining was performed with anti-PtdIns(3,4)P2 IgG or anti-PtdIns(3)P IgG (Echelon) and Dylight 488-conjugated Goat polyclonal antibody against mouse IgG (1:1000; Bethyl). Samples are mounted onto microscope slides through DAKO fluorescent mounting media and imaged.

      Live- and fixed-cell microscopy

      Manual assessment of vacuole and lysosome size, number, LAMP1 positive vesicles, TFEB-GFP localization, Magic Red, or LTR-stained lysosomes were performed using EVOS-FL fluorescent inverted microscope controlled by EVOS XL core imaging system at 20× 0.4 NA. (Thermo Fisher) objective. Spinning disc confocal microscopy was used to perform live imaging through Olympus IX81 inverted microscope connected to Hamamatsu C9100-13 EMCCD camera with 60× 1.35 NA. objective and controlled by Volocity 6.3.0 (PerkinElmer). Time lapse live imaging performed with an environmental chamber set to 37 °C and 5% CO2 in DMEM complete media. Fixed cells were observed through ZEISS AxioImager M2 Epifluorescence microscope connected to AxioCam MRm CCD camera and controlled by AxioVision Software Version 4.8 at 20× 0.8 NA. or 40× 1.4 NA. objective (Carl Zeiss).

      Image analysis

      To identify vacuoles as LAMP-1+ and of lysosomal origin, first, we set a minimum size threshold exclusion parameter to identify vacuoles as being greater than 1.5 μm in diameter on light microscopy using ImageJ. LAMP-1-mCherry fluorescence was then overlaid on these enlarged vacuoles and percentage LAMP-1-mCherry positivity was calculated. To measure the percentage of cells with nuclear TFEB, cells were scored to have nuclear TFEB if the nucleus had greater intensity than cytosol using ImageJ. To measure the percentage of lysosomes filled with LY per cell, images imported into ImageJ and number of LAMP1-positive vesicles were manually scored for presence of LY signal within the lysosome lumen.
      To quantify LAMP1 or LC3 immunostaining through ImageJ, intensity thresholding was applied to identify fluorescent structures and the mean intensity was obtained for each cell. To quantify LC3 intensity over LAMP1-positive structures, ImageJ was used to threshold for LAMP1-mCherry signal and generating a mask, which was applied to the green (LC3 immunostain) channel to measure LC3 intensity on LAMP1-mCherry positive regions. For MEF cells transiently expressing mCherry-eGFP-LC3B, similar approach was used to determine LC3 green puncta intensity over LC3 red puncta structures where relative intensity ratio greater than 1 indicate formation of autolysosomes due to reduced autophagic flux. Similar image analysis technique was applied for MEF cells transiently expressing LAMP1-mCherry to evaluate PtdIns(3,4)P2 or PtdIns(3)P levels overlayed on LAMP1-mCherry positive regions within a cell. To quantify CD63 puncta number per cell, intensity thresholding was similarly applied to identify fluorescent puncta structures followed by total CD63 puncta number per cell.
      To analyze lysosome-to-cytosol intensity ratio of INPP4B-mCherry, images were imported into ImageJ, and lines were assigned to nucleus-excluded areas of a cell measuring 40-pixel in length and 5-pixel in width. Intensity plot profiles were acquired and exported into excel spreadsheet. Intensity arranged according to values and ratio obtained for highest 10 pixels over lowest 10 pixels (FH/FL fluorescent ratio), where ratio values of approximately 1 represent cytosolic distribution.
      To measure lysosome volume and number per cell, particle detection and volumetric tools from Volocity 6.3.0 were used. Briefly, Z-stack images imported into Volocity and punctate lysosome structures identified by applying a 2× cytosol intensity threshold to exclude cytosol and background. Further criteria to include particles greater than 0.3 μm3 removed noise-derived particles. Each cell was isolated by drawing region interest for individual cell analysis. Quantification of lysosome splitting frequency was performed through Imaris (BitPlane) using ‘ImarisTrackLineage’ module, where lysosome splitting was defined as frequency of events where two particles were produced from a single particle.

      VPS34 immunoprecipitation and VPS34 kinase assay

      Following vehicle or apilimod treatment of Inpp4b+/+ and Inpp4b−/− MEF cells, VPS34 immunoprecipitation was performed using the protocol from Cell Signaling (#4263 from Cell Signaling). Five hundred microgram of total protein lysate was incubated with VPS34 antibody #4263 (1:50) overnight at 4 °C. Precleared protein A magnetic beads (Cell Signaling) was incubated with immunocomplex for 20 min at room temperature on a shaking rotator. The magnetic beads were washed five times with TBST buffer using DynaMag-2 Spin Magnet (Thermo Fisher). Kinase assay performed on immunoprecipitated VPS34 using Class III PI3K ELISA Kit (K-3000, Echelon) through the protocol recommended for beads conjugated to enzyme. Kinase assay and PI3P production was performed at 30 °C for 1 h at 1100 r.p.m. Solution-containing PI3P was isolated from the magnetic beads using DynaMag-2 Spin Magnet and used for colorimetric detection of PI3P on ELISA plates.

      Generation of NeuroMab Inpp4b antibody

      Hybridoma cells that secrete NeuroMab clone N171/17-anti-INPP4b antibody were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco). Cells were passaged every 2 days up until 7 days before collection of antibody containing media by centrifugation at 200g. The antibody-containing media was directly applied to nitrocellulose membrane for Inpp4b detection.

      Western blot

      Whole cell lysates were generated using 1× RIPA buffer supplemented with protease inhibitor. Proteins were immunoblotted with the antibodies anti-LC3B (#3868), beta actin (#4967), INPP4B (#14543), LAMP1 (#3243) from Cell Signaling, anti-vATPase V1H (sc-166227), and cathepsin B (sc-365558) from Santa Cruz. Immunoblotting and immunoprecipitation of VPS34 was performed using the following antibody: Rabbit monoclonal antibody for PI3 Kinase Class III (1:1000 for Western blot or 1:50 for immunoprecipitation, 4263, Cell Signaling). Representative western blots used for figure generation are listed for visualization (Fig. S9).

      Flow cytometry

      MEF cells were incubated with 10 μg/ml DQ-BSA (Invitrogen) or 1 mg/ml LY (Thermo Fisher) for 1 h to 6 h at 37 °C, or LTR 1 μM for 30 min, or Magic red for 1 h. Alternatively, LAMP1-mCherry signal of U2OS cells are recorded through flow cytometry following apilimod treatment. Briefly, cells were washed twice with PBS at each time point and whole cell fluorescence was recorded with the Beckman Coulter Cytoflex flow cytometer (Beckman). A total of 10,000 events was counted per condition per sample using the fluorescein isothiocyanate channel for DQ-BSA and LY or phycoerythrin channel for LTR and LAMP1-mCherry and Magic red. Background signal was determined from nonlabeled cells at time 0.

      PtdIns labeling with 3H-myo-inositol and HPLC-coupled flow scintillation

      MEF cells were incubated for two 24 h cycles with inositol-free media (MP Biomedical), 10% dialyzed FBS (Gibco), 4 mM L-glutamine (Sigma Aldrich), 1× insulin-transferrin-selenium-ethanolamine (Gibco), 20 μCi/ml myo-[2-3-H(N)] inositol (PerkinElmer) and indicated treatment conditions. Cells were washed twice with 1× PBS between each 24 h cycle. Lipid precipitation induced, followed by lipid deacylation, extraction, and PtdIns separation by HPLC (Agilent Technologies) through anion-exchange 4.6 × 250-mm column (Phenomenex) as previously mentioned (CH et al., 2018). β-RAM 4 (LabLogic) and 1:2 ratio of eluate to scintillation fluid (LabLogic) was used to detect radiolabeled eluate, followed by analysis with Laura 4 software (Ho et al., 2016).

      Quantitative RT PCR

      RNA isolation from MEF cells was performed through Qiagen RNeasy mini kit (Qiagen). Superscript IV Vilo cDNA synthesis kit (Thermo Fisher) was used to reverse transcribe equal amount of mRNA. The resulting cDNA was amplified through quantitative PCR using TaqMan Fast Advanced Master mix (Applied Biosystems) according to manufacturer instructions in presence of Taqman assays with QuantStudio 3 Real-Time PCR system (Thermo Fisher) controlled by QuantStudio Design and Analysis Software version 1.2 (Thermo Fisher). Taqman assays include Actb (Mm02619580_g1), CtsD (Mm00515586_m1), CtsB (Mm00514443_g1), Atp6v1h (Mm01224350_m1), Atp6v1d (Hs00211133_m1), Lamp1 (Mm01217068_g1), and Mcoln1 (Mm01211241_g1) and were performed in triplicates. Relative quantification (ΔΔCt method) was used to determine gene expression normalized to Actb and vehicle-treated WT MEF.

      Statistical analysis

      All experiments are conducted independently at least three times. All error bars represent SD. Statistical analysis to compare significance between multiple conditions were performed by ANOVA and post hoc analyses were performed with multiple Student’s t test with Bonferroni correction. p values less than 0.05 considered were statistically significant.

      Data availability

      Upon Request

      Supporting information

      This article contains supporting information.

      Conflict of interests

      The authors declare no competing or financial interests.

      Acknowledgments

      We thank all the members of the Salmena lab for thoughtful discussions and constructive criticism. The following plasmids were obtained from Addgene (Cambridge, MA): mCherry-Lysosomes-20 was a gift from Michael Davidson (Addgene plasmid # 55073), pBABE-puro mCherry-EGFP-LC3B was a gift from Jayanta Debnath (Addgene plasmid # 22418), pEGFP-N1-TFEB was a gift from Shawn Ferguson (Addgene plasmid # 38119), and pLJC5-Tmem192-3xHA was a gift from David Sabatini (Addgene plasmid # 102930). This work was supported in part by funds from the Department of Pharmacology and Toxicology and Temerty Faculty of Medicine, University of Toronto and awards from Canada Foundation for Innovation ( CFI-#33505 ); The Natural Sciences and Engineering Research Council of Canada ( NSERC-RGPIN-2015–03984 ) and Cancer Research Society grant ( PIN 24261 ).

      Author contributions

      G. T. S. conceptualization; G. T. S. data curation; G. T. S. formal analysis; G. T. S. and L. S. investigation; G. T. S. and L. S. project administration; G. T. S. methodology; G. T. S. validation; G. T. S. software; G. T. S. visualization; G. T. S., R. J. B., and L. S. writing–original draft; G. T. S., R. J. B., and L. S. writing–review and editing; C. A. W., E. M. M., J. V., R. J. B., and L. S. resources; L. S. supervision; L. S. funding acquisition.

      Funding and additional information

      L. S. is the recipient of a Tier II Canada Research Chair (CRC) and was supported through the Human Frontier Career Development Program (HFSP) Award. R. J. B contributions to this work was funded by Natural Sciences and Engineering Council of Canada (Discovery Grant RGPIN-2020–04343 ), the Canada Research Chairs Program ( 950–232333 ), and contributions from Ryerson University . This work was supported in part by funds from CIHR ( MOP# 123343 ) awarded to J. V.

      Supporting information

      • Supplementary Figure S1

        Vacuole formation and rescue in Apilimod treated Inpp4b−/− MEFs. (A) Inpp4b+/+ or Inpp4b−/− MEFs transiently expressing LAMP1-mCherry to mark lysosomes followed by vehicle or 10 nM apilimod treatment for 48 h. Quantification of (B) Number of vacuoles (>1.5 μm in diameter) per cell across indicated treatments, and (C) Mean vacuole diameter (μm). Scale Bar: 20 μm, zoomed inset: 5 μm. (D-F) Inpp4b+/+ or Inpp4b−/− MEFs treated with vehicle or apilimod 10 nM for 48 h, followed by Lysotracker Red staining to monitor lysosome levels in Apilimod treated cells. Scale bar: 20 μm. (G) Inpp4b−/− MEFs transiently expressing pEGFP, GFP-Inpp4b, or GFP-Inpp4b (C845A) and treated with vehicle or apilimod 10 nM for 48 h. Transfected cells indicated by green rectangular box outline vs. non-transfected cell(s) indicated by black rectangular box outline. Scale bar: 25 μm. Quantification of (H) Number of vacuoles (>1.5 μm in diameter) per cell across indicated treatments, and (I) Mean vacuole diameter (μm). Data represent ± SD from three independent experiments with 25 to 30 cells assessed per treatment condition per experiment for A-C or 10,000 events per treatment condition per experiment for E. Statistical significance was measured by ANOVA and multiple Student’s t test and represented as ∗ (P < 0.05)

      • Supplementary Figure S2

        Effect of INPP4B and PIKfyve suppression on lysosomes and autophagosomes in U2OS cells. (A) U2OS cells stably expressing mCherry-LAMP1 and treated with vehicle or apilimod 10 nM for 48 h in control (siCtrl) and INPP4B-silenced cells (siINPP4B). (B) Quantification of mCherry-LAMP1 positive vacuole number (>1.5 μm in diameter) per cell across indicated treatments, (C) Mean vacuole diameter (μm) positive for mCherry-LAMP1 per cell across apilimod treated conditions and (D) mCherry-LAMP1 intensity per cell across indicated conditions as measured through flow cytometry. Scale bar: 20 μm. (E) U2OS cells from (A) immunoblotted for INPP4B, LC-3 and beta actin. Data represent ± SD from three independent experiments with 40 to 50 cells assessed per treatment condition per experiment for A-C or 10,000 events per treatment condition per experiment for D. Statistical significance was measured by ANOVA and multiple Student’s t test and represented as ∗ (P < 0.05)

      • Supplementary Figure S3

        Effect of INPP4B and PIKfyve suppression on late endosomes. (A) Inpp4b+/+ or Inpp4b−/− MEFs treated with vehicle or apilimod 10 nM for 48 followed by late endosome immunostaining with CD63 antibody. Scale bar: 20 μm. (B) Quantification of late endosome or CD63+ puncta number per cell across indicated conditions. Data represent ± SD from three independent experiments with 25 to 30 cells assessed per treatment condition per experiment. Statistical significance was measured by ANOVA and multiple Student’s t test and represented as ∗ (P < 0.05)

      • Supplementary Figure S4

        Endocytic dynamics of Lucifer Yellow in Inpp4b and PIKfyve suppressed cells. (A) Inpp4b+/+ or Inpp4b−/− MEFs treated with vehicle or apilimod 10 nM for 48 followed by pulsing with Lucifer Yellow for 0, 1, 2 and 4 h. Quantification of Lucifer Yellow intensity as measured through flow cytometry as 10,000 events per treatment condition per experiment. (B-D) Representative flow cytometry plot diagrams of Inpp4b+/+ or Inpp4b−/− MEFs from (A) pulsed with Lucifer Yellow for 1 h or 2 h or 4 h respectively. Statistical significance was measured by ANOVA and multiple Student’s t test and represented as ∗ (P < 0.05)

      • Supplementary Figure S5

        Intracellular tracking of endocytic dynamics in Inpp4b and PIKfyve suppressed cells. (A) Inpp4b+/+ or Inpp4b−/− MEFs transiently expressing mCherry-LAMP1 and treated with vehicle or apilimod 10 nM for 48 h. Lucifer yellow pulsed for 0, 1, 2 and 4 h. (B) Quantification of percentage of mCherry-LAMP1 positive vesicles filled with Lucifer Yellow. Scale bar: 25 μm. (C) Inpp4b+/+ or Inpp4b−/− MEFs treated with vehicle or apilimod 10 nM for 48 followed by pulsing with DQ-BSA for 0, 1, 2 and 4 h. Quantification of DQ-BSA intensity as measured through flow cytometry. Data represent + SD from three independent experiments with 25 to 30 cells assessed per treatment condition per experiment for A-B or 10,000 events per treatment condition per experiment for C. Statistical significance was measured by ANOVA and multiple Student’s t test and represented as ∗ (P < 0.05)

      • Supplementary Figure S6

        INPP4B expression rescue autophagosome defect from PIKfyve suppression. (A) Inpp4b+/+ or Inpp4b−/− MEFs transiently expressing mCherry or INPP4B-mCherry and treated with vehicle or apilimod 10 nM for 48 h. (B) Inset represents a single cell from (A) expanded field of view. (C) Comparison between non-transfected and transfected cells for LC3 puncta intensity per cell across indicated conditions. Scale bar: 10 μm, zoomed inset: 5 μm. Data represent ± SD from three independent experiments with 25 to 30 cells assessed per treatment condition per experiment. Statistical significance was measured by ANOVA and multiple Student’s t test and represented as ∗ (P < 0.05)

      • Supplementary Figure S7

        Total LC3-I and LC3-II protein levels from Fig. 5D. Inpp4b+/+ or Inpp4b−/− MEFs treated with vehicle or apilimod 10 nM for 48 h, followed by immunoblot and measurement of total (A) LC3-I or (B) LC3-II normalized against Beta-actin, or (C) LC3-I and LC3-II protein levels in a single graph to show relative increase of each protein under indicated treatment conditions. Statistical significance was measured by ANOVA and multiple Student’s t test and represented as ∗ (P < 0.05)

      • Supplementary Figure S8

        PtdIns(3,4)P2 phosphoinositide measurements in Inpp4b and PIKfyve suppressed cells. (A) Inpp4b+/+ or Inpp4b−/− MEFs transiently expressing LAMP1-mCherry and treated with vehicle or apilimod 10 nM for 48 h. Quantification of (B) Total cell PtdIns(3,4)P2 or (C) PtdIns(3,4)P 2 fluorescence signal overlayed on LAMP1-mCherry positive regions within a cell. Scale bar: 20 μm, zoomed inset: 5 μm. Data represent ± SD from three independent experiments with 25 to 30 cells assessed per treatment condition per experiment for A-C. Significance measured through multiple Student’s t test represented as ∗ in comparison to indicated conditions (p<0.05)

      • Supplementary Figure S9

        Original uncropped western blot images displayed in the manuscript. Complete western blot images with representative ladders and molecular weight markers used to generate manuscript figures. Western blot bands outlined by black box were used for figure generation

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      Biography

      Golam T. Saffi is a postdoctoral researcher in the Department of Pharmacology & Toxicology at the Temerty Faculty of Medicine, University of Toronto. His research focuses on understanding phosphoinositide signaling and other cellular pathways that control lysosome functions and the implications of these functions in cell biology and disease. This study identifies a novel signaling axis between the catalytic functions of Inpp4b-PIKfyve-VPS34 in regulating lysosome morphology and dynamics.