© 2018. Published by The Company of Biologists Ltd | Journal of Science (2018) 131, jcs213587. doi:10.1242/jcs.213587

RESEARCH ARTICLE enlargement during inhibition of the kinase PIKfyve proceeds through lysosome coalescence Christopher H. Choy1,2,*, Golam Saffi1,2,*, Matthew A. Gray1, Callen Wallace3, Roya M. Dayam1,2, Zhen-Yi A. Ou1, Guy Lenk4, Rosa Puertollano5, Simon C. Watkins3 and Roberto J. Botelho1,2,‡

ABSTRACT is not limited to molecular degradation. Among other Lysosomes receive and degrade cargo from , roles, lysosomes are an important storage , they mediate and . They also play an important role in , they undergo to repair membrane sensing and instructing cells on their metabolic state. The lipid damage, and are a genuine signalling platform that senses and kinase PIKfyve generates phosphatidylinositol-3,5-bisphosphate to informs the cell of its nutrient and stress conditions (Medina et al., modulate lysosome function. PIKfyve inhibition leads to impaired 2015; Settembre and Ballabio, 2014; Sancak et al., 2010; Delamarre degradative capacity, ion dysregulation, abated autophagic flux and a et al., 2005; Xiong and Zhu, 2016; Reddy et al., 2001; Settembre massive enlargement of lysosomes. Collectively, this leads to et al., 2012; Roczniak-Ferguson et al., 2012). various physiological defects, including embryonic lethality, Strikingly, the lysosome is now considered to be a highly and overt . The reasons for such dynamic and heterogeneous organelle. Not only can individual cells drastic lysosome enlargement remain unclear. Here, we examined host tens to hundreds of lysosomes, lysosomes themselves are whether biosynthesis and/or fusion-fission dynamics contribute to highly heterogeneous in their pH (Johnson et al., 2016), subcellular swelling. First, we show that PIKfyve inhibition activates TFEB, TFE3 position (Li et al., 2016a; Pu et al., 2015; Bagshaw et al., 2006) and and MITF, enhancing lysosome expression. However, this morphology (Swanson et al., 1987; Saric et al., 2016; Vyas et al., did not augment lysosomal protein levels during acute PIKfyve 2007). Lastly, lysosomes can adapt to their environment. For inhibition, and deletion of TFEB and/or related proteins did not impair example, in and dendritic cells, lysosomes are lysosome swelling. Instead, PIKfyve inhibition led to fewer but transformed from individual puncta into a highly tubular network enlarged lysosomes, suggesting that an imbalance favouring (Saric et al., 2016; Vyas et al., 2007; Swanson et al., 1987). In lysosome fusion over fission causes lysosome enlargement. addition, nutrient depletion, protein aggregation and phagocytosis Indeed, conditions that abated fusion curtailed lysosome swelling in activate a family of transcription factors that can enhance expression PIKfyve-inhibited cells. of lysosome and adjust lysosome activity (Gray et al., 2016; Pastore et al., 2016; Sardiello et al., 2009; Tsunemi et al., 2012; KEY WORDS: Transcription factors, Phosphoinositides, Medina et al., 2011; Settembre et al., 2012; Roczniak-Ferguson Lysosomes, , Fusion, Fission et al., 2012). This is best understood for the related transcription factors, TFEB and TFE3, which are recruited to the surface of INTRODUCTION lysosomes and subject to phosphorylation by various kinases, Lysosomes are organelles responsible for the degradation of a large including mTORC1 to modulate their nucleocytoplasmic transport variety of molecules that originate from within and from outside (Li et al., 2016b; Martina et al., 2012; Peña-Llopis et al., 2011; cells. Lysosomes accomplish this by fusing with cargo-containing Martina et al., 2014, 2016; Roczniak-Ferguson et al., 2012). organelles such as autophagosomes, Golgi-derived vesicles, Given the above, lysosomes are highly regulated organelles. A and (Luzio et al., 2007, 2009; Schwake master modulator of lysosomes is the lipid kinase PIKfyve, which et al., 2013). To enable their degradative capacity, lysosomes are converts phosphatidylinositol-3-phosphate [PtdIns(3)P] into equipped with a plethora of hydrolytic that digest proteins, phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] (Sbrissa , nucleic acids and . In addition, the vacuolar et al., 1999). Inactivation of PIKfyve or of its regulators, Vac14/ H+-pump ATPase generates a highly acidic optimal for ArPIKfyve and /Sac3, causes a variety of physiological hydrolytic activity (Mindell, 2012; Schwake et al., 2013; Xiong and problems, including embryonic lethality, neurodegeneration and Zhu, 2016; Luzio et al., 2007). Nonetheless, the function of immune malfunction (Cai et al., 2013; Chow et al., 2007; Jin et al., 2008; Ho et al., 2012; Mccartney et al., 2014; Sbrissa et al., 2007; Ferguson et al., 2010; Min et al., 2014; Lenk et al., 2016; Ikonomov 1Department of Chemistry and , Ryerson University, Toronto, ON, Canada, et al., 2011). At the cellular level, these defects relate to impaired M5B2K3. 2The Graduate Program in Molecular Science, Ryerson University, 3 autophagic flux, altered delivery to lysosomes, dysregulation of Toronto, ON, Canada, M5B2K3. Department of , University of 2+ Pittsburgh, Pittsburgh, PA 15261, USA. 4Department of Human Genetics, University lysosomal Ca transport and massive enlargement of lysosomes of Michigan, Ann Arbor, MI 48109, USA. 5Cell Biology and Physiology Center, (Ferguson et al., 2009; Dong et al., 2010; de Lartigue et al., National Heart, Lung, and Blood Institute, National Institutes of Health, 50 South 2009; Ferguson et al., 2010; Kim et al., 2014, 2016; Ho et al., 2012; Drive, Building 50, Room 3537, Bethesda, MD 20892, USA. *These authors contributed equally to this work Mccartney et al., 2014). Remarkably, it remains unclear how lysosomes enlarge in cells with defective PIKfyve activity. It has ‡ Author for correspondence ([email protected]) been suggested that PIKfyve, or loss of the yeast orthologue Fab1 R.J.B., 0000-0002-7820-0999 impairs recycling from endosomes, lysosomes or from the yeast , thus causing an influx of membrane into lysosomes and

Received 28 November 2017; Accepted 10 April 2018 their enlargement (Rutherford et al., 2006; Bryant et al., 1998; Dove Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs213587. doi:10.1242/jcs.213587 et al., 2004). This recycling process is assumed to occur through vesicular-tubular transport intermediates like those induced by clathrin, adaptor proteins and retromer (Currinn et al., 2016; de Lartigue et al., 2009; Ho et al., 2012; Phelan et al., 2006), but remains speculative. This type of membrane fission is distinct from the ‘kiss-and-run’ process, where lysosomes undertake transient homotypic and heterotypic fusion events that are followed by fission to avoid coalescence of lysosomes (Bright et al., 2005). Membrane fission can also occur as organelle splitting, rather than budding, as is the case of mitochondrial fission (Chan, 2006). Recently, impairment of PIKfyve was shown to cause nuclear accumulation of TFEB, suggesting that increased lysosomal biogenesis may contribute to lysosome swelling (Gayle et al., 2017; Wang et al., 2015). Here, we examined the role of TFEB and the related proteins TFE3 and MITF in lysosome enlargement and conclude that biosynthesis does not contribute to lysosome enlargement, at least acutely. Instead, we observed that PIKfyve inhibition causes a significant reduction in lysosome number concurrent with lysosome enlargement. We propose that lysosome swelling proceeds through lysosome coalescence, probably as a result of reduced ‘run’ or fission events during lysosome kiss-and- run and/or full fusion and fission cycles.

RESULTS PIKfyve inhibition activates the transcription factor TFEB independently of mTOR PIKfyve inhibition causes lysosome enlargement (Ikonomov et al., 2001; Mccartney et al., 2014) but the mechanism by which this happens remains unclear. There is a growing body of literature that links TFEB and related transcription factors to enhanced lysosome and protein expression (Settembre and Ballabio, 2014; Raben and Puertollano, 2016). Thus, we set out to test if TFEB activation might contribute to lysosome swelling. Indeed, PIKfyve inhibition using apilimod, an exquisitely specific inhibitor of PIKfyve (Cai et al., 2013; Gayle et al., 2017), in RAW cells triggered a rapid and robust nuclear translocation of GFP-tagged TFEB and endogenous TFEB (Fig. 1A,B). To complement our pharmacological treatment, we also demonstrated that siRNA-mediated silencing of PIKfyve significantly boosted the number of RAW cells with nuclear TFEB-GFP relative to control cells (Fig. 1C). In addition, HeLa cells treated with apilimod also exhibited nuclear TFEB relative to resting cells, showing that activation of TFEB in the absence of PIKfyve occurs across distinct cell types (Fig. 1D). Lastly, we showed that GFP fusions of TFE3 and MITF, which are related to TFEB (Ploper and De Robertis, 2015; Raben and Puertollano, Fig. 1. PIKfyve inhibition causes nuclear translocation of TFEB. (A,B) RAW cells expressing TFEB-GFP (A) or stained for endogenous TFEB (B) 2016), also translocated to the nucleus upon PIKfyve abrogation in treated with vehicle or 20 nM apilimod for 1 h. (C) RAW cells silenced for RAW cells (Fig. S1A,B). Overall, our data are consistent with recent PIKfyve or mock-silenced and expressing TFEB-GFP. (D) HeLa cells stained reports that PIKfyve activity robustly controls nuclear localization for endogenous TFEB treated with vehicle or 20 nM apilimod for 1 h. DAPI was of TFEB and related factors (Gayle et al., 2017; Wang et al., 2015). used to stain the nuclei. Nuclear translocation of TFEB was quantified either as TFEB is controlled by several factors including mTOR, which percentage of cells containing nuclear GFP-tagged TFEB (A,C) or as the phosphorylates and maintains TFEB in the (Martina et al., nuclear-to-cytosol fluorescence intensity ratio for endogenous TFEB (B,D). 2012; Roczniak-Ferguson et al., 2012; Settembre et al., 2012). In Shown is the mean and s.e.m. from at least three independent experiments and based on 50-300 cells per condition per experiment. *P<0.05 compared addition, PIKfyve has been suggested to control mTORC1 in with respective control conditions using one-way ANOVA and Tukey’s post hoc adipocytes (Bridges et al., 2012), although others have noted that test or with an unpaired Student’s t-test, as appropriate. Scale bars: 5 µm. mTOR activity is sustained in PIKfyve-inhibited cells (Wang et al., 2015; Krishna et al., 2016). To better assess the possibility that PIKfyve controls TFEB by coordinating with mTOR in our cells, we We also wondered whether mTOR could control TFEB by regulating examined the phosphorylation state of S6K, a major mTORC1 target, PIKfyve activity instead. To test this, we exposed cells to torin1, an in cells treated with apilimod. Strikingly, we could not observe a inhibitor of mTOR. First, we showed that apilimod reduced significant difference in the phosphorylation levels of S6K between PtdIns(3,5)P2 by >70% with a concomitant increase in its precursor control and apilimod-treated cells (Fig. 2A). This suggests that phosphatidylinositol-3-phosphate (Fig. 2B). In comparison, we did mTORC1 retains its activity in PIKfyve-inhibited RAW macrophages. not observe a decrease in the levels of either phosphatidylinositol-3- Journal of Cell Science

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(Fig. 2C). Importantly, lysates from Tfeb−/− RAW cells lacked most bands resolved by Phos-tag gels and detected with anti-TFEB antibodies, identifying the signal as specific to TFEB (Fig. 2C). In addition, pre-incubation of lysates with λ-phosphatase collapsed the signal into a single fast-running TFEB band (Fig. 2C), suggesting that we specifically detected a variety of phospho-TFEB isoforms. Overall, these data suggest that PIKfyve regulates kinases or phosphatases that impinge on TFEB, but these are seemingly in parallel and/or independent of mTOR.

PIKfyve inhibition boosts lysosome gene expression but not lysosomal protein levels Our observations suggest that PIKfyve may stimulate lysosomal gene and protein expression by activating TFEB and related factors. To test this, we measured mRNA levels of genes encoding selected lysosomal proteins, including LAMP1, MCOLN1, D and the V-ATPase H and D subunits using qRT-PCR. As expected, we observed augmented mRNA levels for these genes after incubation with apilimod for 3 h (Fig. 3A). We next examined if the enhanced mRNA levels corresponded to an increase in protein levels. Surprisingly, the levels of the V-ATPase H subunit, LAMP1 and in apilimod-treated RAW macrophages were similar to control cells after 3 or 6 h of PIKfyve inhibition, although the small increase in LAMP1 protein levels after treatment for 6 h was statistically higher than that in resting cells (Fig. 3B,C). The decoupling between enhanced mRNA levels and unchanged protein levels may be due to the role of PIKfyve in targeting lysosomal proteins to lysosomes (Ikonomov et al., 2009a; Rutherford et al., 2006; Min et al., 2014). Given this, we suspected that TFEB activation may not contribute to lysosome swelling in cells acutely suppressed for PIKfyve.

PIKfyve inhibition induces lysosome swelling in cells deleted Fig. 2. mTOR and PIKfyve function independently. (A) Western blot for TFEB and related proteins detection of phosphoThr389-p70S6K and total p70S6K in PIKfyve-inhibited Next, we tested whether expression of TFEB and/or TFE3 is cells treated as indicated. The ratio of phospho-S6K to total S6K was quantified dispensable for lysosome swelling during PIKfyve inhibition. To do from three independent blots. Shown is the mean ratio and s.d. normalized to this, RAW macrophages deleted for TFEB, TFE3 or both (Pastore DMSO-treated cells. (B) Quantification of PtdIns(3)P and PtdIns(3,5)P2 levels 3 et al., 2016) were treated with vehicle or apilimod for 1 h and using H-myo-inositol incorporation and HPLC-coupled flow scintillation. manually assessed for the average diameter and number of swollen Results are shown as the mean and s.d. from three independent experiments, normalized to their PtdInsP levels and to the respective DMSO-treated using differential interference contrast (DIC) optics. controls. *P<0.05 compared with respective control conditions using multiple As suspected, RAW cells carrying deletions for TFEB and/or Student’s t-test with the Bonferroni correction. (C) Western blot showing TFEB TFE3 were not impaired in apilimod-induced vacuolation, with expression in whole-cell lysates resolved on a Phos-tag gel. Shown in a vacuole number and size comparable to values in wild-type RAW representative blot from three independent experiments showing differential cells (Fig. S2A). migration of TFEB in lysates from control relative to torin1- and apilimod-treated We then followed manual assessment with a more robust cells. Anti-TFEB antibody specificity was confirmed by the loss of most bands in Tfeb−/− RAW whole-cell lysates. In addition, most bands represent volumetric analysis to quantify the size and number of endo/ phosphorylated isoforms of TFEB since pre-treatment of lysates with λ protein lysosomes by decorating their luminal volume with fluid-phase phosphatase led to a single fast-running band. pinocytic tracers fluorescent-tagged dextrans and Lucifer Yellow. Notably, our labelling method likely decorates a collection of late endosomes, lysosomes and endolysosomes, a term that describes phosphate or PtdIns(3,5)P2 in torin1-treated cells relative to control hybrid -lysosome organelles (Huotari and Helenius, cells; in fact, there was a statistically significant increase in 2011). Thus, we use the term endo/lysosome to reflect the PtdIns(3,5)P2 levels in torin1-treated cells (Fig. 2B). heterogeneous mixture of endosomes and lysosomes likely to be Since TFEB is regulated by phosphorylation at various sites labelled here and to differentiate from endolysosomes. First, we (Dephoure et al., 2008; Mayya et al., 2009), we decided to assess showed that fluorescent-dextran colocalized to LAMP1-positive whether PIKfyve activity might govern TFEB through structures similarly well between all RAW strains, confirming that phosphorylation. To achieve this, we examined changes in TFEB deletion of TFEB and/or TFE3 did not disrupt trafficking of the phosphorylation using Phos-tag gels, which can resolve multiple label to endo/lysosomes (Fig. S2B). Second, we defined parameters phospho-isoforms of a target protein. Notably, apilimod treatment such as thresholding and minimum particle size to estimate endo/ caused a change in the phosphorylation of TFEB when resolved on a lysosome number and volume (see Materials and Methods and

Phos-tag gel to a pattern comparable to that in torin1-treated cells Fig. S3 for optimization testing). Using this approach, we conclude Journal of Cell Science

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apilimod-induced lysosome swelling (Fig. 4B). As a control for the effectiveness of cycloheximide inhibition of protein synthesis, we assayed for translation activity with puromycylation and steady- state levels of p53, which is a high turnover protein. As shown in Fig. 4C, resting cells exhibited a high rate of puromycylation, which is a marker for -mediated protein synthesis, whereas cycloheximide ablated this. In addition, cycloheximide rapidly depleted p53 levels, demonstrating that protein synthesis was arrested (Fig. 4D). Overall, our data suggest that protein biosynthesis and lysosome biogenesis controlled by TFEB and related transcription factors do not contribute substantially to endo/ lysosome swelling during acute PIKfyve inhibition.

PIKfyve inhibition concurrently increases endo/lysosome volume while decreasing endo/lysosome number To better understand the process of endo/lysosome enlargement during PIKfyve inhibition, we further employed quantitative volumetric image analysis. Using this approach, we observed that the average volume of individual endo/lysosomes in RAW cells treated with apilimod increased with incubation period relative to vehicle-only cells (Fig. 5A,B). Remarkably, the average number of endo/lysosomes per cell in apilimod-treated cells decreased considerably with incubation period and relative to vehicle- exposed cells (Fig. 5A,C). Despite the decrease in lysosome number, the total lysosome volume per cell remained unperturbed (Fig. 5A,D). As suggested above, we also observed similar trends with HeLa cells exposed to apilimod and Fig4−/− mouse embryonic fibroblasts; there was an increase in the average volume of individual endo/lysosomes, a concurrent decline in their number, whereas total lysosome volume per cell was not significantly altered (Fig. S4, Fig. 5D-H). Finally, and as implied above, there was no significant difference in the average size, number and total volume of endo/lysosomes between wild-type RAW and HeLa cells and counterpart strains deleted for TFEB, TFE3 and/or MITF before and after PIKfyve suppression (Fig. 4; Fig. S4). Interestingly, replacing Fig. 3. Acute PIKfyve inhibition enhances lysosomal gene transcription apilimod-containing medium with fresh medium reversed the but not protein levels. (A) Expression of selected lysosomal genes quantified effects on endo/lysosome volume and number per cell (Fig. 6). by qRT-PCR and normalized against Abt1. Shown is the mean±s.e.m. from Overall, our results collectively suggest that acute PIKfyve seven independent experiments. (B) Western blot of selected lysosomal inhibition not only enlarges endo/lysosomes, but also decreases proteins in RAW cells treated as indicated. Numbers on the right represent their numbers without disturbing the total endo/lysosome volume. relative molecular mass (kDa). (C) Quantification of indicated proteins demonstrated in B and shown as the mean±s.e.m. intensity from four Imbalanced fusion-fission cycles may underpin endo/ independent blots. *P<0.05 compared with respective control conditions using lysosome swelling in PIKfyve-inhibited cells multiple Student’s t-test with the Bonferroni correction. It is unlikely that endo/lysosome number is reduced during PIKfyve inhibition via lysosome lysis because the total lysosome volume per that the average number and volume of individual endo/lysosomes cell was unchanged relative to control cells. Instead, the reduction in were similar between apilimod-treated wild-type and Tfeb−/−, endo/lysosome number coupled to their enlargement suggests that Tfe3−/− and Tfeb−/− Tfe3−/− RAW macrophages (Fig. 4A), there is a shift towards endo/lysosome homotypic fusion relative to consistent with the manual analysis (Fig. S2A). Nevertheless, fission in PIKfyve-abrogated cells. Hence, we predicted that Tfeb−/− Tfe3−/− RAW macrophages retain expression of the related conditions that abate homotypic lysosome fusion would lessen MITF protein, which becomes nuclear upon addition of apilimod enlargement and maintain higher lysosome numbers in PIKfyve- (Fig. S1); this raised the possibility that MITF may be sufficiently inhibited cells. redundant with TFEB and TFE3 to promote lysosome expansion To test this hypothesis, we disrupted the system and during PIKfyve inhibition. To test this, we opted to compare HeLa motor activity using nocadozole and ciliobrevin, respectively, to cells to counterpart HeLa cells deleted for all three proteins, impair lysosome-lysosome fusion (Rosa-Ferreira and Munro, 2011; previously described by Nezich et al. (2015). Despite deletion of Deng and Storrie, 1988; Jordens et al., 2001). Indeed, cells treated genes encoding TFEB, TFE3 and MITF, endo/lysosome with these compounds resisted apilimod-induced swelling and enlargement caused by apilimod was indistinguishable between retained higher endo/lysosome number relative to results in the wild-type and mutant HeLa strains (Fig. S4). Finally, we then asked apilimod-only condition (Fig. 7A-C for nocadozole, Fig. S5A-C for whether protein biosynthesis was necessary for lysosomes to ciliobrevin). Conversely, endo/lysosome shrinkage was accelerated enlarge during PIKfyve repression. Strikingly, cycloheximide, upon washing apilimod in microtubule-disrupted cells (Fig. 7D-F). which arrests protein biosynthesis, did not interfere with To complement the pharmacological approach, we transfected cells Journal of Cell Science

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Fig. 4. Volumetric analysis of endo/lysosomes in PIKfyve-curtailed wild- type, Tfeb−/−, Tfe3−/− and Tfeb−/− Tfe3−/− RAW cells. (A) Cells were pre- labelled with Lucifer Yellow and then treated with apilimod to induce lysosome vacuolation. Lysosome number and lysosome volume were quantified from 100 cells from three independent experiments. (B) Lysosome morphology in wild-type RAW cells treated as indicated. Lysosome volume and number were quantified. In all cases, data shown are the mean±s.e.m. from three independent experiments, with at least 100 cells per condition per experiment. (C) Puromycylation of proteins in the absence and presence of cycloheximide. Puromycin is incorporated into elongating proteins by the ribosome, which can be detected by anti-puromycin antibodies during western blotting. Cycloheximide treatment eradicated puromycylation of proteins by blocking protein synthesis. The first two lanes resolved lysates unexposed to puromycin and thus accounts for non-specific bands detected by anti-puromycin antibodies. Representative blot shown from three independent experiments. (D) Cycloheximide reduces p53 abundance relative to β-actin. Representative blot shown from three independent experiments. Numbers on the right represent relative molecular mass (in kDa). Data are mean±s.e.m. abundance by measuring p53 signal as a ratio against β-actin. *P<0.05 compared with respective control conditions using Student’s t-test. Scale bars: 5 µm.

Lysosomes undergo constant rounds of ‘kiss-and-run’ fusion and fission (Duclos et al., 2003; Bright et al., 2005; Pryor et al., 2000). We propose a model in which PIKfyve, probably through synthesis of PtdIns(3,5)P2, controls endo/lysosome fission. Consequently, in PIKfyve-inhibited cells, lysosomes fail to perform fission and/or ‘kiss-and-marry’, resulting in endo/lysosome coalescence, enlargement and a drop in total lysosome number. To support this model, we tried to visualize endo/lysosome swelling during apilimod exposure in RAW cells by using live-cell spinning disc confocal microscopy to obtain z-stacks at high temporal resolution. However, endo/lysosomes under observation failed to swell when acquiring z-stacks even as infrequently as every 2 min (Fig. S6, Movies 1 and 2). In addition, lysosomes also appeared to become less dynamic. Indeed, when we moved to a different field of view within the same coverslip after the period of observation, endo/ lysosomes were engorged (Fig. S6). This suggests that photo- toxicity impairs apilimod-dependent endo/lysosome swelling. We could observe dynamic endo/lysosome movement and enlargement when imaging single planes at 1 frame/2 min (Fig. 8A,B, Movies 3 and 4). Unfortunately, this made tracking of individual endo/ lysosomes difficult due to their highly dynamic nature. To bypass the photo-toxicity issue, we switched to swept-field confocal microscopy, which employs dramatically less light energy. Using this technology, we could reconstruct cells at high temporal resolution and using z-stacks. Indeed, we could track the dynamics of endo/lysosomes in control cells and those undergoing vacuolation in PIKfyve-inhibited cells over 2 h (Fig. 8C,D, Movies 5 and 6). When imaging untreated cells, endo/lysosomes proved to be highly dynamic structures undertaking significant number of collisions, splitting and ‘tubulation’ events (Fig. 8C, Movie 5). After adding apilimod, endo/lysosomes underwent enlargement and became fewer in number through coalescence (Fig. 8D, Movie 6). However, it is possible to see that even enlarged endo/lysosome vacuoles in PIKfyve-suppressed cells retain their dynamic nature. To try to quantify fission and ‘run’ events, we used particle tracking and splitting functions in Imaris as a proxy. with the p50 dynamitin subunit or dominant negative kinesin-1 to Although this analysis cannot distinguish between fission and respectively impair and kinesin. We observed reduced separation of overlapping but independent particles, we reasoned swelling and increased endo/lysosome numbers during apilimod that this could estimate differences in fission rates if the defect was exposure relative to untransfected cells (Fig. S5D-I). Overall these sufficiently large. Indeed, while ∼85% of endo/lysosomes ‘split’ in data suggest that endo/lysosomes swell in PIKfyve-inhibited cells control cells over a period of 30 min, this was reduced to ∼50% in through endo/lysosome coalescence. apilimod-treated cells (Fig. 8E). Altogether, we propose that Journal of Cell Science

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Fig. 5. Volumetric analysis of endo/lysosomes during acute and chronic PIKfyve suppression. (A) RAW cells were pre-labelled with Lucifer Yellow and exposed to vehicle only or to 20 nM apilimod for the indicated times. Fluorescence micrographs are z-projections of 45-55 z-planes acquired by spinning disc confocal microscopy. Scale bar: 5 µm. (B-D) Quantification of individual endo/lysosome volume (B), endo/lysosome number per RAW (C) and total endo/lysosome volume (D). (E) Wild-type and Fig4−/− mouse embryonic fibroblasts labelled with Lucifer Yellow. Scale bar: 30 µm. (F-H) Analysis of individual endo/lysosome volume (F), endo/lysosome number per cell (G) and total endo/lysosome volume per cell (H) in wild-type and Fig4−/− mouse embryonic fibroblasts. In all cases, data shown are the mean±s.e.m. from three independent experiments, with at least 15-20 cells per condition per experiment. *P<0.05 compared with respective control conditions using one-way ANOVA and Tukey’s post hoc test for B-D, and unpaired Student’s t-test for F-H.

PIKfyve loss of function causes lysosome enlargement by reducing lysosome and vacuole swelling (McCartney et al., 2014; Ho et al., the rate of lysosome fission/‘run’ events relative to lysosome fusion/ 2012). Arguably, this is assumed to proceed through tubulo- ‘kiss’ events, leading to lysosome coalescence. vesicular transport intermediates formed during membrane fission catalysed by coat proteins like the retromer (Bryant et al., 1998; de DISCUSSION Lartigue et al., 2009; Rutherford et al., 2006; Zhang et al., 2007). Disruption of PIKfyve activity causes several major physiological Here, we explored two other possibilities that could contribute to defects, including neurological decay, lethal embryonic endo/lysosome enlargement: enhanced endo/lysosome biosynthesis development and inflammatory disease, among others (Ikonomov and/or an imbalance in endo/lysosome fusion-fission dynamics. et al., 2011; Min et al., 2014; Chow et al., 2007; McCartney et al., Collectively, our work intimates that enhanced lysosome 2014). It is unclear how these defects arise, but they are likely to biosynthesis does not drive endo/lysosome swelling. Instead, our relate to disruption of endo/lysosome homeostasis, including work suggests that endo/lysosomes coalescence during PIKfyve impaired hydrolytic function, reduced rates of autophagic flux and inhibition, resulting in concurrent enlargement of endo/lysosomes endo/lysosome swelling (Martin et al., 2013; de Lartigue et al., and abatement in their numbers. We provide evidence that 2009; Min et al., 2014; Kim et al., 2016). Endo/lysosome coalescence is due to a shift away from fission during endo/ vacuolation remains the most dramatic and visible defect in lysosome fusion-fission cycling (Fig. S7). Our observations are PIKfyve-abrogated cells. Yet, the mechanism by which endo/ consistent with recent work by Bissig et al. (2017), who suggest a lysosomes become engorged remains poorly defined. The role for PIKfyve in reforming terminal lysosomes from prevailing model is that recycling from endo/lysosomes is endolysosomes, a term they use to define a subset of lysosomes impaired in PIKfyve/Fab1-deficient cells, thus causing endo/ that are acidic and proteolytic. Journal of Cell Science

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Fig. 6. Volumetric analysis of endo/lysosome number and volume during PIKfyve reactivation. (A) Cells were pre-labelled with Lucifer Yellow as before and then exposed to vehicle only or to 20 nM apilimod for 1 h, followed by removal and chase in fresh medium for the indicated times. Fluorescence micrographs are z-projections of 45-55 z-planes acquired by spinning disc confocal microscopy. Scale bar: 5 µm. (B-D) Volumetric quantification of individual endo/lysosome volume (B), endo/lysosome number per cell (C) and total endo/lysosome volume per cells (D). Data shown are the mean±s.e.m. from three independent experiments, with at least 15-20 cells per condition per experiment. *P<0.05 between data indicated by lines using one-way ANOVA and Tukey’s post hoc test.

PIKfyve and TFEB function MITF or treated with cycloheximide, we argue that biosynthesis does Inhibition of PIKfyve caused rapid and robust nuclear translocation of not significantly contribute to lysosome enlargement in cells acutely TFEB, TFE3 and MITF. Indeed, others have recently observed that inhibited for PIKfyve, although we do not exclude a role in other TFEB shuttles into the nucleus in PIKfyve-inhibited cells (Gayle et al., PIKfyve-mediated functions. 2017; Wang et al., 2015). Consistent with nuclear translocation, we also observed increased mRNA levels of selected lysosomal genes PIKfyve and TFEB regulation including LAMP1, V-ATPase subunits and . This increase TFEB and TFE3 are maintained in the cytosol via phosphorylation is in linewith a recent study that looked at transcriptome-wide changes on specific residues that form docking sites for the 14-3-3 in mRNA levels in B-cell non-Hodgkins lymphoma cells treated with scaffolding protein (Roczniak-Ferguson et al., 2012; Martina apilimod (Gayle et al., 2017). This suggests that PIKfyve regulates a et al., 2012). We now know that TFEB and related factors are negative-feedback loop to control lysosome biogenesis through subjected to multiple kinases including mTORC1, PKC, PKD, suppression of TFEB and related factors. However, acute inhibition GSK3β and phosphatases such as the Ca2+-dependent calcineurin of PIKfyve led either to no change, or in the case of LAMP1, to a (Li et al., 2016b; Martina et al., 2014; Roczniak-Ferguson et al., small increase in protein levels after 6 h of PIKfyve inhibition, despite 2012; Settembre et al., 2012; Najibi et al., 2016; Ploper et al., the augmented mRNA levels. The decoupling between enhanced 2015; Marchand et al., 2015; Wang et al., 2015). However, transcription and constant protein levels may result from pleiotropic mTORC1 regulation of TFEB remains the best-understood effects of PIKfyve inhibition. For example, PIKfyve is known to regulatory circuit. Given that PIKfyve is needed for mTORC1 govern trafficking of newly synthesized lysosomal genes by activity in adipocytes (Bridges et al., 2012), we examined whether controlling recycling of mannose-6-phosphate receptors (Gayle there could be a PIKfyve-mTORC1-TFEB axis. However, S6K et al., 2017; Ikonomov et al., 2009a; Rutherford et al., 2006; Zhang remained phosphorylated in apilimod-treated cells, suggesting that et al., 2007). By contrast, cells chronically impaired for PIKfyve have mTORC1 was active. These observations are consistent with the augmented LAMP1 levels (Ferguson et al., 2009, 2010). However, work of Wang et al. (2015) and Krishna et al. (2016) who also this LAMP1 accumulation is thought to occur through impaired showed that mTORC1 retains its activity in PIKfyve-abrogated autophagosome turnover rather than an increase in biosynthesis of cells. We considered the possibility that mTORC1 and starvation LAMP1 (Ferguson et al., 2009; Kim et al., 2016). This may be control PIKfyve activity, which could then modulate TFEB, consistent with the small increase in LAMP1 after prolonged perhaps by PtdIns(3,5)P2-dependent localization of TFEB to inhibition of PIKfyve (Fig. 3C). Overall, given that endo/lysosome lysosomes, as has been shown for Tup1, a transcription factor in enlargement was unaffected in cells deleted for TFEB, TFE3 and/or yeast that controls galactose metabolism (Han and Emr, 2011). Journal of Cell Science

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lipid that is directly or indirectly synthesized by PIKfyve (Zolov et al., 2012; Sbrissa et al., 2012). Overall, our data suggest that mTORC1 and PIKfyve regulate TFEB in parallel. We speculate that PIKfyve may regulate other modulators of TFEB such as PKD, PKC and GSK3β.

Enlargement of lysosomes in PIKfyve-suppressed cells Loss of PIKfyve causes massive lysosome swelling. This has been partially explained through a defect in membrane recycling from endosomes and/or lysosomes (Ho et al., 2012; McCartney et al., 2014). This model arose through experiments done in yeast expressing an engineered ALP protein carrying a Golgi retrieval sequence (RS-ALP). RS-ALP traffics to the vacuole, where it is subjected to proteolytic maturation. Mature RS-ALP appears in the Golgi membrane fraction in wild-type yeast cells, but not in cells with defective PtdIns(3,5)P2 activity, suggesting a defect in retrieval from the vacuole (Bryant et al., 1998; Dove et al., 2004). In addition, PIKfyve appears to regulate retrieval of mannose-6-phosphate receptors from endo/lysosomes in mammalian cells, which suggests a link between PIKfyve and coat proteins such as retromer (Ikonomov et al., 2009b; Rutherford et al., 2006; Zhang et al., 2007). Hence, endo/lysosome enlargement may partly occur through an imbalance in membrane influx, caused by unabated anterograde membrane flow and hindered recycling due to defects in forming tubulo-vesicular intermediates (Fig. S7). However, our observations also suggest that the number of endo/lysosomes in PIKfyve-suppressed cells is significantly reduced. The diminished endo/lysosome number is unlikely to be due to lysosome lysis since the total endo/lysosome volume was unchanged in PIKfyve-arrested cells. Instead, we propose that PIKfyve suppression causes an imbalance in lysosome fusion-fission cycling, leading to lysosome coalescence and enlargement (Fig. S7). Lysosomes constantly interact with each other through full fusion and fission, or through ‘kiss-and-run’ events. During the ‘kiss’ event, at least two lysosomes dock and form a transient pore to help exchange and homogenize their luminal content (Bright et al., 2005; Luzio et al., 2009; Duclos et al., 2000). However, complete fusion and coalescence is pre-empted by the ‘run’ event, when the two lysosomes separate, thus preventing coalescence. Our work suggests that PIKfyve, probably through PtdIns(3,5)P2, plays a role in mediating lysosome fission to balance fusion. This role may occur during complete fusion-fission cycles and/or by catalysing the ‘run’ event during ‘kiss-and-run’ (Fig. S7). In either case, failure to perform fission would result in overt coalescence, resulting in lysosome enlargement and reduced numbers. This model is Fig. 7. Volumetric analysis of endo/lysosome number and volume in cells consistent with recent work by Bissig et al. (2017), who suggest a disrupted for microtubule function. (A) Cells were pre-labelled with Alexa role for PIKfyve in reforming terminal lysosomes from Fluor 488-conjugated dextran, followed by vehicle alone, 10 µM nocadozole for 60 min, or co-treated with apilimod and vehicle or nocadozole for 1 h. endolysosomes. We do not understand how PIKfyve modulates Fluorescence micrographs are z-projections of 45-55 z-planes acquired by lysosome fission dynamics. However, we speculate that spinning disc confocal microscopy. (B,C) Quantification of individual endo/ mechanisms that offset lysosome coalescence are distinct from lysosome volume (B) and endo/lysosome number per RAW macrophage (C). those that drive vesiculation and recycling (Krishna et al., 2016; (D) Cells were pre-labelled with Alexa Fluor 488-conjugated dextran as before, Bryant et al., 1998; Rutherford et al., 2006). One interesting followed by 20 nM apilimod for 1 h and then replaced with fresh medium or possibility may relate to contact sites between the endo/lysosome medium containing nocadozole for the indicated time period. (E,F) Volumetric quantification of individual endo/lysosome volume (E) and endo/lysosome and the since these have been shown to number per cell (D). In all cases, data shown are the mean±s.e.m. from three demarcate and assemble molecular machines that catalyse independent experiments, with at least 15-20 cells per condition per endolysosome fission (Friedman et al., 2013; Rowland et al., experiment. *P<0.05 statistical difference between conditions indicated by 2014). Thus, it is appealing to speculate that PIKfyve and lines using one-way ANOVA and Tukey’s post hoc test. Scale bars: 5 µm. PtdIns(3,5)P2 may mediate this process or be regulated by these contact sites to drive endo/lysosome fission. However, we did not observe a significant decrease in PtdIns(3,5)P2 Overall, we examined two possible mechanisms that might have levels in mTOR-inhibited cells, although we cannot exclude contributed to endo/lysosome enlargement in PIKfyve-inhibited disruption of a specific pool of PtdIns(3,5)P2 or of PtdIns(5)P, a cells. While TFEB and related factors are controlled by PIKfyve, Journal of Cell Science

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Fig. 8. Live-cell imaging of apilimod-induced lysosome swelling. (A,B) Cells were pre-labelled with Alexa Fluor 546-conjugated dextran, followed by (A) vehicle alone or (B) 20 nM apilimod at time 0 min. Single-plane images were obtained by spinning disc microscopy at 1 frame/2 min. Time in min refers to time after adding vehicle or apilimod. See Movies 3 and 4 for full capture. Scale bars: 5 µm. (C,D) Swep-tfield imaging of RAW cells exposed to (C) vehicleor (D) 20 nM apilimod. See Movies 5 and 6 for full capture. Shown are z-collapsed frames at 30 s intervals. The inset is a magnified portion of the field-of-view tracking a few endo/lysosomes (arrows) undergoing contact and split events, which may represent fusion or fission. Scale bars: 3 µm. (E) Rate of ‘splitting’ events over 30 min in vehicle- or apilimod-treated cells showing a reduction in splitting in PIKfyve-inhibited cells, which may reflect reduced fission. *P<0.05 by Student’s t-test. which probably establishes a negative-feedback loop for lysosome 15% FBS. All cells were authenticated, checked for contamination and biogenesis, we did not obtain evidence to suggest that biosynthesis cultured at 37°C with 5% CO2. Transient transfections were performed with contributes to lysosome swelling during acute PIKfyve inhibition. FuGene HD (Promega, Madison, WI) following the manufacturer’s Instead, we observed that loss of PIKfyve activity caused a instructions, using a 3:1 DNA to transfection reagent ratio. The concomitant reduction in the number and increase in the volume of transfection mixture was replaced with fresh medium after 4-6 h and cells were used 24 h post-transfection. Silencing RNA oligonucleotides were endo/lysosomes, suggesting that endo/lysosomes undergo electroporated into RAW cells with the Neon Electroporation system coalescence through fusion and impaired fission. Future work ( Technologies, Burlington, ON) following the manufacturer’s should delineate the contribution of lysosome coalescence and instructions. Briefly, two rounds of knockdown were performed using vesicular recycling to lysosome enlargement. ON-TARGETplus PIKfyve SMARTpool siRNA (L-040127-00-0005) or ON-TARGETplus non-targeting control pool (GE Healthcare, Mississauga, MATERIALS AND METHODS ON) over 48 h. Cell culture and transfection RAW 264.7 macrophage strains and HeLa cell strains were cultured in Pharmacological manipulation of cells Dulbecco’s Modified Eagle Medium (DMEM; Wisent, St Bruno, QC) Cells were treated with apilimod (Toronto Research Chemicals, Toronto, supplemented with 5% heat-inactivated fetal bovine serum (FBS; Wisent). ON) at indicated concentrations and times. Torin-1 (Tocris Bioscience, Mouse embryonic fibroblast (MEF) cells were cultured in Roswell Park Minneapolis, MN) or rapamycin (BioShop, Burlington, ON) were used as

Memorial Institute medium (RPMI-1640; Gibco, Burlington, ON) with positive controls for TFEB nuclear localization and mTORC1 inhibition. Journal of Cell Science

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Cells were also incubated with 100 µM ciliobrevin D (EMD Millipore, medium for 1 h before live-cell imaging. This method is likely to label a Toronto, ON) or 10 µM nocodazole (Sigma-Aldrich, Oakville, ON). spectrum of late endosomes, lysosomes and endolysosomes, which are endosome-lysosome hybrids (Huotari and Helenius, 2011). Thus, we refer Western blotting to dextran- or Lucifer Yellow-labelled structures as endo/lysosomes. Whole-cell lysates were generated with 2× Laemmli sample buffer supplemented with protease inhibitor cocktail (Sigma-Aldrich) and Epifluorescence and spinning disc confocal microscopy PhosSTOP phosphatase inhibitor (Roche, Laval, QC) where necessary. Manual quantification of lysosome size and imaging of GFP-based Lysates were passed through a 27-gauge needle six times and heated. transfections were imaged using a Leica DM5000B system outfitted with Puromycylation assays were carried out with the addition of 10 µg/ml a DFC350FX camera and controlled by Leica application suite (Leica puromycin for 15 min before cell lysis. When protein dephosphorylation Microsystems Inc., Concord, ON). TFEB-GFP transfected into PIKfyve was required, cells were lysed with 1% Triton-X100 in PBS supplemented knockout cells and endogenous TFEB in RAW cells were imaged using an with protease inhibitor cocktail, centrifuged at 16,000 g for 10 min, and the Olympus IX83 microscope with a Hamamatsu ORCA-Flash4.0 digital supernatant was subjected to lambda protein phosphatase (NEB, Whitby, camera controlled by CellSens Dimensions software (Olympus Canada Inc., ON) as per the manufacturer’s instruction. Briefly, 20 µg protein was Richmond Hill, ON). Endogenous TFEB was further deconvolved using a incubated with 50 mM HEPES, 100 mM NaCl, 2 mM DTT, 0.01% Brij 25, constrained iterative in CellSens Dimensions. All other imaging of fixed 1 mM MnCl2 and 200 units of phosphatase at 30°C for 60 min. The reaction cells was done with a Quorum DisKovery Spinning Disc confocal was stopped with 5× Laemmli sample buffer and heated. microscope system equipped with a Leica DMi8 microscope connected to Most samples were subjected to SDS-PAGE through a 10% acrylamide Andor Zyla 4.2 Megapixel sCMOS camera and controlled by Quorum Wave resolving gel. To assay for TFEB mobility shift, we used an 8% acrylamide FX Powered by MetaMorph software (Quorum Technologies, Guelph, ON). resolving gel supplemented with 12.5 μM Phos-tag (Wako Chemicals USA, Live-cell imaging by spinning disc confocal microscopy was accomplished Richmond, VA) and 25 μM MnCl2. Phos-tag gels were washed three times with either an Olympus IX81 inverted microscope equipped with a with 100 mM EDTA for 10 min each, followed by a wash with Transfer Hamamatsu C9100-13 EMCCD camera and a 60×1.35 N.A. objective and buffer (25 mM Tris-HCl, 192 mM glycine, 20% methanol). Proteins were controlled with Volocity 6.3.0 (PerkinElmer, Bolton, ON). Alternatively, transferred to PVDF, blocked and incubated with primary and HRP-linked we employed the Quorum DisKovery Spinning Disc confocal microscope secondary antibodies using Tris-buffered saline plus 0.1% Tween-20 with system above but using the iXON 897 EMCCD camera. Cells were imaged either 5% skimmed milk or 3% BSA. Proteins were visualized using Clarity live using cell-specific complete medium in a 5% CO2 chamber at 37°C. All enhanced chemiluminescence (Bio-Rad Laboratories, Mississauga, ON) microscopes were equipped with standard filters appropriate to fluorophores with a ChemiDoc XRS+ or ChemiDoc Touch imaging system (Bio-Rad). used in this study. Quantification of proteins were determined using Image Lab software (Bio- Rad) by normalizing against a loading control and then normalizing against 3H-myo-inositol labelling of phosphoinositides and the vehicle-treated control. Rabbit monoclonal antibodies used were against phosphoinositide measurement by HPLC-coupled flow cathepsin D (1:1000, GTX62603, GeneTex Inc., Irvine, CA), HSP60 scintillation (1:1000, 12165S, Technologies, Danvers, MA), and p70 S6 RAW cells were incubated for 24 h in inositol-free DMEM (MP kinase (1:1000, 2708S, Cell Signaling). Rabbit polyclonal antibodies were Biomedical, CA) with 10 µCi/ml myo-[2-3H(N)] inositol (Perkin Elmer, used against ATP6V1H (1:1000, GTX110778, GeneTex), TFEB (1:2000, MA), 10% dialyzed FBS (Gibco), 4 mM L-glutamine (Sigma Aldrich), 1× A303-673A, Bethyl Laboratories, Montgomery, TX), phosphoThr389-p70 -transferrin-selenium-ethanolamine (Gibco) and 20 mM HEPES S6 kinase (1:1000, 9205S, Cell Signaling) and β-actin (1:1000, 4967S, Cell (Gibco). Cells were then treated with torin1 or apilimod for 1 h. Reactions Signaling). Rat monoclonal antibodies were used against LAMP1 (1:200, were arrested in 600 µl of 4.5% perchloric acid (v/v) on ice for 15 min, 1D4B, Developmental Studies Hybridoma Bank, Iowa City, IO). Mouse scraped, and pelleted at 12,000 g for 10 min. Pellets were washed with 1 ml monoclonal antibodies were used against puromycin (1:1000, 540411, ice-cold 0.1 M EDTA and resuspended in 50 µl water. Phospholipid Millipore, Burlington, MA) and p53 (1:1000, 2524S, Cell Signaling). deacylation was then done in 500 µl of methanol/40% methylamine/1- Secondary antibodies were raised in donkey (Bethyl) and conjugated to butanol [45.7% methanol: 10.7% methylamine: 11.4% 1-butanol (v/v)] for HRP. 50 min at 53°C. Samples were vacuum-dried and washed twice by resuspending in 300 µl water and drying. Dried samples were resuspended Quantitative RT-PCR again in 450 µl water and extracted with 300 µl 1-butanol/ethyl ether/ethyl Total RNA was extracted from RAW cells using the GeneJET RNA formate (20:4:1), vortexed for 5 min and centrifuged at 12,000 g for 2 min. purification (Thermo Fisher Scientific, Mississauga, ON). Equal The bottom aqueous layer was collected and extracted two more times. The aqueous layer was dried in vacuum and resuspended in 50 µl water. quantities of mRNA were reverse transcribed with SuperScript VILO 3 cDNA synthesis kit (Invitrogen) following the manufacturer’s guidelines. Equal counts of H were separated by HPLC (Agilent Technologies, The subsequent cDNA was diluted 1:100 and amplified for quantitative Mississauga, ON) through an anion exchange 4.6×250-mm column PCR using the TaqMan Fast Advanced Master Mix (Applied Biosystems, (Phenomenex, Torrance, CA) with a flow rate of 1 ml/min and subjected Foster City, CA) in the presence of TaqMan assays with a Step One Plus to a gradient of water (buffer A) and 1 M (NH4)2HPO4, pH 3.8 (adjusted Real-Time PCR thermal cycler (Applied Biosystems) with Step One with phosphoric acid) (buffer B) as follows: 0% B for 5 min, 0 to 2% B for software (v.2.2.2; Applied Biosystems). The TaqMan gene expression 15 min, 2% B for 80 min, 2 to 10% B for 20 min, 10% B for 30 min, 10 to assays (Invitrogen) for the reference gene Abt1 (Mm00803824_m1) and 80% B for 10 min, 80% B for 5 min, 80 to 0% B for 5 min. The β target genes Ctsd (Mm00515586_m1), Atp6v1h (Mm00505548_m1), radiolabelled eluate was detected by -RAM 4 (LabLogic, Brandon, FL) ATP6V1D (Mm00445832_m1), LAMP1 (Mm00495262_m1) and with a 1:2 ratio of eluate to scintillant (LabLogic) and analyzed by Laura 4 Mcoln1 (Mm00522550_m1) were done in triplicate. Target gene software. Phosphoinositides were normalized against the parent expression was determined by relative quantification (ΔΔCt method) to phosphatidylinositol peak. Abt1 and the vehicle-treated control sample. Live-cell imaging with swept-field confocal microscopy Cells were seeded into single glass-bottom micro-well dishes (MatTek, Endo/lysosomal labelling Ashland, MA) and grown to 70-80% confluency. Cells were then incubated Endo/lysosomes in RAW cells, HeLa cells and MEFs were labelled by for 2 h in 200 µl DMEM medium supplemented with 10% FBS with a incubating cells with either 200 µg/ml Alexa Fluor 488- or 546-conjugated 200 µg/ml Alexa Fluor 555-conjugated dextran (ThermoFisher). Samples dextran (ThermoFisher) or with 2.5 mg/ml Lucifer Yellow (ThermoFisher) were washed and incubation medium was replaced with fresh complete in cell-specific complete medium for 2 h at 37°C in 5% CO2, followed by DMEM followed by a 90 min post-incubation period. Live-cell imaging was washing in phosphate-buffered saline and replenishing with fresh complete performed with a Nikon Ti inverted microscope equipped with a Prairie Journal of Cell Science

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Technologies swept-field slit scanning confocal scan head and a 100×1.49 Acknowledgements N.A. objective (Prairie Technologies, Sioux Falls, SD). Nikon NIS Plasmid constructs expressing the kinesin-1 dominant negative and of the p50 Elements software equipped with a Photometrics Prime 95B back dynamitin subunit were obtained from Addgene (Cambridge, MA). Plasmids illuminated sCMOS camera was used for image acquisition while high- encoding GFP fusion proteins of TFEB, TFE3 and MITF were kind gifts from Dr Shawn Ferguson at Yale University. The Tfeb−/− Tfe3−/− Mitf−/− HeLa cells were a speed triggered acquisition was controlled by a National Instruments DAQ kind gift from Dr Richard Youle at the National Institutes of Health. card and Mad City Labs Piezo Z stage controller. Sample volumes were acquired at 100 ms per z-slice at 30 s interval over a 30 min observation Competing interests period before adding 20 nM apilimod and then for 2 h in the presence of The authors declare no competing or financial interests. apilimod. Author contributions Immunofluorescence Conceptualization: R.J.B.; Methodology: C.H.C., G.S., M.A.G., Z.-Y.A.O., G.L., R.P.; Following treatment, cells were fixed with 4% paraformaldehyde (v/v) for Software: G.S.; Formal analysis: C.H.C., G.S., S.C.W., R.J.B.; Investigation: C.H.C., 15 min. Cells were blocked and permeabilized with 0.2% (v/v) Triton X-100 G.S., M.A.G., C.W., R.M.D., R.J.B.; Resources: G.L., R.P., S.C.W., R.J.B.; Data curation: C.H.C., G.S., M.A.G., C.W., R.M.D., Z.-Y.A.O.; Writing - original draft: and 2% (v/v) BSA for 10 min, then subjected to rabbit polyclonal antibody C.H.C., G.S., R.J.B.; Writing - review & editing: R.J.B.; Visualization: C.H.C., G.S., against TFEB (1:250; Bethyl) and Dylight-conjugated donkey polyclonal M.A.G., C.W., R.M.D., Z.-Y.A.O., S.C.W.; Supervision: S.C.W., R.J.B.; Project antibody against rabbit (1:500; Bethyl). Nuclei were counterstained with administration: R.J.B.; Funding acquisition: R.J.B. 0.4 μg/ml DAPI. For LAMP1-specific immunostaining, cells were fixed and permeabilized with 100% ice-cold methanol for 5 min, followed by a 2% Funding BSA block, incubation with Dylight-conjugated rat monoclonal antibody The research performed in the lab of R.J.B. was funded by a Discovery Grant from against LAMP1 (1:200, Clone 1D4B; Developmental Studies Hybridoma the Natural Sciences and Engineering Research Council of Canada, by the Early Bank) and polyclonal donkey anti-rat IgG antibodies (1:500; Bethyl). Researcher Award program from the Government of Ontario, by a Tier II Canada Research Chairs Award and by contributions from Ryerson University. Research by C.H.C. was made possible through support from an Ontario Graduate Scholarship Imaging analysis and volumetrics and Queen Elizabeth II Graduate Scholarship. G.L. was funded through the National To quantify the proportion of cells with nuclear TFEB-GFP (including Institutes of Health (GM24872). Deposited in PMC for release after 12 months. TFE3-GFP or MiTF-GFP), RAW cells were visually scored as cytosolic if the cytosol had equal or greater staining intensity compared with the Supplementary information nucleus. Alternatively, the nuclear:cytosolic ratio of endogenous TFEB was Supplementary information available online at measured as the ratio of the mean intensity in the nucleus over the mean http://jcs.biologists.org/lookup/doi/10.1242/jcs.213587.supplemental intensity in the cytosol using ImageJ. To manually quantify vacuole diameter in DIC images, vacuoles were first defined as having a diameter References greater than 1.5 µm and then the diameter was measuring using line plot in Bagshaw, R. D., Callahan, J. W. and Mahuran, D. J. (2006). The Arf-family protein, Arl8b, is involved in the spatial distribution of lysosomes. Biochem. Biophys. Res. ImageJ. Commun. 344, 1186-1191. To quantify average endo/lysosome volume and number per cells, we Bissig, C., Hurbain, I., Raposo, G. and van Niel, G. (2017). PIKfyve activity employed volumetric and particle detection tools in Volocity (Volocity regulates reformation of terminal storage lysosomes from endolysosomes. Traffic. 6.3.0) or Icy BioImage Analysis. Z-stack images were imported into the 11, 747-757. software and subject to signal thresholding. First, we tried thresholding at Bridges, D., Ma, J.-T., Park, S., Inoki, K., Weisman, L. S. and Saltiel, A. R. (2012). 1.5×, 2× and 2.5× average cytosolic fluorescence background. This was then Phosphatidylinositol 3,5-bisphosphate plays a role in the activation and subject to particle detection by defining particles as those with a volume subcellular localization of mechanistic target of rapamycin 1. Mol. Biol. Cell 23, 3 2955-2962. >0.3 µm , which helped eliminate noise-derived particles. Regions of Bright, N. A., Gratian, M. J. and Luzio, J. P. (2005). Endocytic delivery to interest were then drawn around each cell for analysis and particles split lysosomes mediated by concurrent fusion and kissing events in living cells. Curr. using the watershed function. Importantly, while increasing thresholding Biol. 15, 360-365. levels reduced absolute number of particles, particle size and total particle Bryant, N. J., Piper, R. C., Weisman, L. S. and Stevens, T. H. (1998). Retrograde volume, (i) the reduction occurred at similar levels between control and traffic out of the yeast vacuole to the TGN occurs via the prevacuolar/endosomal apilimod-treated cells and (ii) the relative trends between control and compartment. J. Cell Biol. 142, 651-663. ́ ́ apilimod-treated cells remained the same (Fig. S3A,B). Hence, we Cai, X., Xu, Y., Cheung, A. K., Tomlinson, R. C., Alcazar-Roman, A., Murphy, L., Billich, A., Zhang, B., Feng, Y., Klumpp, M. et al. (2013). PIKfyve, a class III PI employed a threshold of 2× the average cytosol fluorescence intensity for kinase, is the target of the small molecular IL-12/IL-23 inhibitor apilimod and a the remaining analysis. Analysis of endo/lysosome splitting frequency was player in Toll-like receptor signaling. Chem. Biol. 20, 912-921. performed using Imaris (BitPlane, Concord, MA) using its Chan, Y. H. M., Reyes, L., Sohail, S. M., Tran, N. K. and Marshall, W. F. (2016). ‘ImarisTrackLineage’ module, where endo/lysosome splitting was defined Organelle size scaling of the budding yeast vacuole by relative growth and as the frequency of events producing two particles from a single apparent inheritance. Curr. Biol. 26, 1221-1228. particle. Image adjustments such as enhancing contrast were done with Chow, C. Y., Zhang, Y., Dowling, J. J., Jin, N., Adamska, M., Shiga, K., Szigeti, ImageJ or Adobe Photoshop (Adobe Systems, San Jose, CA) without K., Shy, M. E., Li, J., Zhang, X. et al. (2007). of FIG4 causes neurodegeneration in the pale tremor mouse and patients with CMT4J. Nature altering the relative signals within images. Figures were assembled with 448, 68-72. Adobe Illustrator (Adobe Systems). Currinn, H., Guscott, B., Balklava, Z., Rothnie, A. and Wassmer, T. (2016). APP controls the formation of PI(3,5)P2 vesicles through its binding of the PIKfyve Statistical analysis complex. Cell. Mol. Life Sci. 73, 393-408. de Lartigue, J., Polson, H., Feldman, M., Shokat, K., Tooze, S. A., Urbé, S. and All experiments were repeated at least three independent times. The Clague, M. J. (2009). PIKfyve regulation of endosome-linked pathways. Traffic 10, respective figure legends indicate the total number of experiments and 883-893. number of samples/cells assessed. The mean and measure of variation (s.d.) Delamarre, L., Pack, M., Chang, H., Mellman, I. and Trombetta, E. S. (2005). or error (s.e.m.) are indicated. Differential lysosomal in antigen-presenting cells determines antigen All experimental conditions were statistically analysed with either an fate. Science 307, 1630-1634. unpaired Student’s t-test when comparing two parameters only or with a Deng, Y. P. and Storrie, B. (1988). Animal cell lysosomes rapidly exchange one-way ANOVA test when comparing multiple conditions in non- membrane proteins. Proc. Natl. Acad. Sci. USA 85, 3860-3864. Dephoure, N., Zhou, C., Villen, J., Beausoleil, S. A., Bakalarski, C. E., Elledge, normalized controls. ANOVA tests were coupled to Tukey’s post hoc test S. J. and Gygi, S. P. (2008). A quantitative atlas of mitotic phosphorylation. Proc. to analyse pairwise conditions. For multiple comparisons with normalized Natl. Acad. Sci. USA. 105, 10762-10767. controls, Student’s t-tests were utilized with Bonferroni correction. 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