© 2018. Published by The Company of Biologists Ltd.

Lysosome enlargement during inhibition of the lipid kinase PIKfyve proceeds through

lysosome coalescence

Christopher H. Choy*1,2, Golam Saffi*1,2, Matthew A. Gray1, Callen Wallace3, Roya M.

Dayam1,2, Zhen-Yi A. Ou1, Guy Lenk4, Rosa Puertollano5, Simon C. Watkins3and Roberto J.

Botelho1,2#

1Department of Chemistry and Biology and the 2Graduate Program in Molecular Science,

Ryerson University, Toronto, ON, M5B2K3, Canada

3Department of Cell Biology, University of Pittsburgh, Pittsburgh, PA, 15261, USA

4Department of Human Genetics, University of Michigan, Ann Arbor, MI, 48109 USA

5Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National

Institutes of Health, 50 South Drive, Building 50, Room 3537, Bethesda, MD, 20892, USA

*Equal contribution

# To whom correspondence should be sent:

Address: Department of Chemistry and Biology, Ryerson University, Toronto, ON, M5B2K3,

Canada. E-mail: rbotelho@ryerson.ca

Keywords: transcription factors, phosphoinositides, lysosomes, organelles, fusion, fission

Summary statement

PIKfyve inhibition causes lysosomes to coalesce, resulting in fewer, enlarged lysosomes. We

also show that TFEB-mediated lysosome biosynthesis does not contribute to swelling.

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JCS Advance Online Article. Posted on 16 April 2018

Abstract

Lysosomes receive and degrade cargo from endocytosis, phagocytosis and autophagy. They also

play an important role in sensing and instructing cells on their metabolic state. The lipid kinase

PIKfyve generates phosphatidylinositol-3,5-bisphosphate to modulate lysosome function.

PIKfyve inhibition leads to impaired degradative capacity, ion dysregulation, abated autophagic

flux, and a massive enlargement of lysosomes. Collectively, this leads to various physiological

defects including embryonic lethality, neurodegeneration and overt inflammation. While being

the most dramatic phenotype, the reasons for lysosome enlargement remain unclear. Here, we

examined whether biosynthesis and/or fusion-fission dynamics contribute to swelling. First, we

show that PIKfyve inhibition activates TFEB, TFE3 and MITF enhancing lysosome gene

expression. However, this did not augment lysosomal protein levels during acute PIKfyve

inhibition and deletion of TFEB and/or related proteins did not impair lysosome swelling.

Instead, PIKfyve inhibition led to fewer but enlarged lysosomes, suggesting that an imbalance

favouring lysosome fusion over fission causes lysosome enlargement. Indeed, conditions that

abated fusion curtailed lysosome swelling in PIKfyve-inhibited cells.

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Introduction

Lysosomes are organelles responsible for the degradation of a large variety of molecules that

originate from within and from outside cells. Lysosomes accomplish this by fusing with cargo-

containing organelles such as autophagosomes, Golgi-derived vesicles, endosomes and

phagosomes (Luzio et al., 2007, 2009; Schwake et al., 2013). To enable their degradative

capacity, lysosomes are equipped with a plethora of hydrolytic enzymes that digest proteins,

lipids, nucleic acids, and carbohydrates. In addition, the vacuolar H+-pump ATPase generates a

highly acidic lumen optimal for hydrolytic activity (Mindell, 2012; Schwake et al., 2013; Xiong

and Zhu, 2016; Luzio et al., 2007). Nonetheless, the function of lysosomes is not limited to

molecular degradation. Among other roles, lysosomes are an important storage organelle, they

mediate antigen presentation, they undergo exocytosis to repair membrane damage, and are a

genuine signalling platform that senses and informs the cell of its nutrient and stress conditions

(Medina et al., 2015; Settembre and Ballabio, 2014; Sancak et al., 2010; Delamarre et al., 2005;

Xiong and Zhu, 2016; Reddy et al., 2001; Settembre et al., 2012; Roczniak-Ferguson et al.,

2012).

Strikingly, lysosomes are now considered a highly dynamic and heterogeneous organelle.

Not only can individual cells host tens to hundreds of lysosomes, lysosomes themselves are

highly heterogeneous in their pH (Johnson et al., 2016), subcellular position (Li et al., 2016a; Pu

et al., 2015; Bagshaw et al., 2006), and morphology (Swanson et al., 1987; Saric et al., 2016;

Vyas et al., 2007). Lastly, lysosomes can adapt to their environment. For example, in

macrophages and dendritic cells, lysosomes are transformed from individual puncta into a highly

tubular network (Saric et al., 2016; Vyas et al., 2007; Swanson et al., 1987). In addition, nutrient

depletion, protein aggregation, and phagocytosis activate a family of transcription factors that

can enhance expression of lysosome genes and adjust lysosome activity (Gray et al., 2016;

Pastore et al., 2016; Sardiello et al., 2009; Tsunemi et al., 2012; Medina et al., 2011; Settembre

et al., 2012; Roczniak-Ferguson et al., 2012). This is best understood for the related transcription

factors, TFEB and TFE3, which are recruited to the surface of lysosomes and subject to

phosphorylation by various kinases including mTORC1 to modulate their nucleo-cytoplasmic

transport (Li et al., 2016b; Martina et al., 2012; Peña-Llopis et al., 2011; Martina et al., 2014,

2016; Roczniak-Ferguson et al., 2012).

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Given the above, lysosomes are highly regulated organelles. A master modulator of

lysosomes is the lipid kinase PIKfyve, which converts phosphatidylinositol-3-phosphate

[PtdIns(3)P] into phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] (Sbrissa et al., 1999).

Inactivation of PIKfyve or of its regulators, Vac14/ArPIKfyve and Fig4/Sac3, causes a variety of

physiological problems including embryonic lethality, neurodegeneration and 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 et al., 2011). At the cellular level, these defects relate to impaired autophagic flux,

altered delivery to lysosomes, dysregulation of lysosomal Ca2+ transport, and massive

enlargement of lysosomes (Ferguson et al., 2009; Dong et al., 2010; de Lartigue et al., 2009;

Ferguson et al., 2010; Kim et al., 2014; Ho et al., 2012; Mccartney et al., 2014; Kim et al., 2016).

Remarkably, it remains unclear how lysosomes enlarge in cells defective for PIKfyve activity. It

has been suggested that PIKfyve, or loss of the yeast ortholog, Fab1, impairs recycling from

endosomes, lysosomes or from the yeast vacuole, thus causing an influx of membrane into

lysosomes and their enlargement (Rutherford et al., 2006; Bryant et al., 1998; Dove 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 “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 mitochondria fission (Chan, 2006).

Recently, PIKfyve impairment 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 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

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lysosome swelling proceeds through lysosome coalescence, likely caused by reduced

“run”/fission events during lysosome “kiss-and-run” and/or full fusion/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). Nevertheless, 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

with 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-silenced 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-fusion of TFE3 and MITF, which are related to TFEB (Ploper and

De Robertis, 2015; Raben and Puertollano, 2016), also translocated to the nucleus upon PIKfyve

abrogation in RAW cells (Supplemental Fig. S1A,B). Overall, our data is consistent with recent

reports that PIKfyve activity robustly controls nuclear localization of TFEB and related factors

(Gayle et al., 2017; Wang et al., 2015).

TFEB is controlled by several factors including mTOR, which phosphorylates and

maintains TFEB in the cytosol (Martina et al., 2012; Roczniak-Ferguson et al., 2012; Settembre

et al., 2012). In addition, PIKfyve has been suggested to control mTORC1 in adipocytes

(Bridges et al., 2012), though others have noted that 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 examined the

phosphorylation state of S6K, a major mTORC1 target, in cells treated with apilimod.

Strikingly, we could not observe a significant difference in the phosphorylation levels of S6K

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between control and apilimod-treated cells (Fig. 2A). This suggests that mTORC1 retains its

activity in PIKfyve-inhibited RAW macrophages. Alternatively, we questioned if mTOR might

control TFEB by regulating PIKfyve activity instead. To test this, we exposed cells to torin1, an

inhibitor of mTOR. First, we showed that apilimod reduced PtdIns(3,5)P2 by >70% with a

concomitant increase in its precursor phosphatidylinositol-3-phosphate (Fig. 2B). In

comparison, we did not observe a decrease in the levels of both phosphatidylinositol-3-phosphate

or PtdIns(3,5)P2 in torin1-treated cells relative to control cells; in fact, there was a statistically

significant increase in PtdIns(3,5)P2 levels in torin1-treated cells (Fig. 2B).

Since TFEB is regulated by phosphorylation at various sites (Dephoure et al., 2008;

Mayya et al., 2009), we decided to assess whether PIKfyve activity might govern TFEB through

phosphorylation. To achieve this, we examined changes in TFEB phosphorylation using Phos-

tag gels, which can resolve multiple phospho-isoforms of a target protein. Notably, apilimod

treatment caused a change in the phosphorylation pattern of TFEB when separated by a Phos-tag

gel that was comparable to torin1-treated cells (Fig. 2C). Importantly, lysates from tfeb-/- RAW

cells lacked most bands separated 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

phosphatase, collapsed all most bands 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 select lysosomal

genes including LAMP1, MCOLN1, cathepsin D, and the V-ATPase H and D subunits using

qRT-PCR. As expected, we observed augmented mRNA levels for these genes after 3 h

incubation with apilimod (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 cathepsin D in apilimod-treated RAW macrophages were similar to control

cells after 3 or 6 h of PIKfyve inhibition, though the small increase in LAMP1 protein levels at 6

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h apilimod was statistically higher than 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 cells deleted for TFEB and related proteins

Next, we tested whether expression of TFEB and/or TFE3 is dispensable for lysosome swelling

during PIKfyve inhibition. To do this, RAW macrophages deleted for TFEB, TFE3 or both

(Pastore et al., 2016) were treated with vehicle or apilimod for 1 h and manually assessed for the

average diameter and number of swollen vacuoles using differential interference contrast (DIC)

optics. As suspected, RAW cells carrying deletions for TFEB and/or TFE3 were not impaired in

apilimod-induced vacuolation, with vacuole number and size comparable to wild-type RAW

cells (Supplemental Fig. S2A).

We then followed manual assessment with a more robust volumetric analysis to quantify

the size and number of endo/lysosomes by decorating their luminal volume with fluid-phase

pinocytic tracers like fluorescent-dextrans or Lucifer yellow. Notably, our labelling method

likely decorates a collection of late endosomes, lysosomes and endolysosomes, a term that

describes hybrid endosome-lysosome organelles (Huotari and Helenius, 2011). Thus, we use the

term endo/lysosome to reflect the heterogeneous mixture of endosomes and lysosomes likely

labelled here and to differentiate from endolysosomes. First, we showed that fluorescent-dextran

co-localized to LAMP1-positive structures similarly well between all RAW strains, confirming

that deletion of TFEB and/or TFE3 did not disrupt trafficking of the label to endo/lysosomes

(Supplemental Fig. S2B). Second, we defined parameters such as thresholding and minimum

particle size to estimate endo/lysosome number and volume (see Methods and Supplemental

information for optimization testing; Supplemental Fig. S3). Using this approach, we conclude

that the average number and volume of individual endo/lysosomes were similar between

apilimod-treated wild-type and tfeb-/-, tfe3-/- and tfeb-/- tfe3-/- RAW macrophages (Fig. 4A),

consistent with the manual analysis (Supplemental Fig. S2A). Nevertheless, tfeb-/- tfe3-/- RAW

macrophages retain expression of the related MITF protein, which becomes nuclear upon

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addition of apilimod (Supplemental Fig. S1); this raised the possibility that MITF may be

sufficiently redundant with TFEB and TFE3 to promote lysosome expansion during PIKfyve

inhibition. To test this, we opted to compare HeLa cells to counterpart HeLa cells deleted for all

three proteins, previously described in (Nezich et al., 2015). Despite deletion of genes encoding

TFEB, TFE3 and MITF, endo/lysosome enlargement caused by apilimod was indistinguishable

between wild-type and mutant HeLa strains (Supplemental Fig. S4). Finally, we then asked

whether protein biosynthesis was necessary for lysosomes to enlarge during PIKfyve repression.

Strikingly, cycloheximide, which arrests protein biosynthesis, did not interfere with 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, a high turnover protein. As shown in Fig. 4C, resting cells exhibited

high-rate of puromycylation, which detects ribosome-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 and 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, while total lysosome volume per cell was

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not significantly altered (Supplemental 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; Supplemental Fig. S4). Interestingly,

replacing apilimod-containing medium with fresh media reversed the effects on endo/lysosome

volume and number per cell (Fig. 6). Overall, our results collectively suggest that acute PIKfyve

inhibition not only enlarges endo/lysosomes, but also decreases their numbers without disturbing

the total endo/lysosome volume.

Imbalanced fusion-fission cycles may underpin endo/lysosome swelling in PIKfyve-

hindered cells

It is unlikely that endo/lysosome number is reduced during PIKfyve inhibition via lysosome lysis

because the total lysosome volume per cell was unchanged relative to control cells. Instead, the

reduction in endo/lysosome number coupled to their enlargement suggests that there is a shift

towards endo/lysosome homotypic fusion relative to fission in PIKfyve-abrogated cells. Hence,

we predicted that conditions that abate homotypic lysosome fusion would lessen enlargement

and maintain higher lysosome numbers in PIKfyve-inhibited cells.

To test this hypothesis, we disrupted the microtubule system and motor activity using

nocadozole and ciliobrevin, respectively, to impair lysosome-lysosome fusion (Rosa-Ferreira and

Munro, 2011; Deng and Storrie, 1988; Jordens et al., 2001). Indeed, cells treated with these

compounds resisted apilimod-induced swelling and retained higher endo/lysosome number

relative to apilimod-only condition (Fig. 7A-C for nocadozole, Supplemental Fig. S5A-C for

ciliobrevin). Conversely, endo/lysosome shrinkage was accelerated upon washing apilimod in

microtubule-disrupted cells (Fig. 7D-F). To complement the pharmacological approach, we

transfected cells with the p50 dynamitin subunit or dominant negative kinesin-1 to respectively

impair dynein and kinesin. We observed reduced swelling and increased endo/lysosome numbers

during apilimod exposure relative to untransfected cells (Supplemental Fig. S5D-I). Overall

these data suggest that endo/lysosomes swell in PIKfyve-inhibited cells through endo/lysosome

coalescence.

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Lysosomes undergo constant rounds of fusion and fission, including by "kiss-and-run”

(Duclos et al., 2003; Bright et al., 2005; Pryor et al., 2000). We propose a model in which

PIKfyve, likely through synthesis of PtdIns(3,5)P2, controls endo/lysosome fission.

Consequently, in PIKfyve inhibited cells, lysosomes fail to 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 of

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 two minutes (Supplemental Fig. S6, Supplemental Movies

1 and 2). In addition, lysosomes appeared to become less dynamic as well. Indeed, when we

moved to a different field-of-view within the same coverslip after the period of observation,

endo/lysosomes were engorged (Supplemental 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, Supplemental 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 Sweptfield 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, Supplemental Movie S5 and S6). When imaging untreated cells,

endo/lysosomes proved to be highly dynamic structures undertaking significant number of

collisions, splitting and “tubulation” events (Fig. 8C, Supplemental Movie S5). After adding

apilimod, endo/lysosomes underwent enlargement and became fewer, through coalescence (Fig.

8D, Supplemental 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. Though this

analysis cannot distinguish between fission and separation of overlapping but independent

particles, we reasoned that this could estimate differences in fission rates if the defect is

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sufficiently large. Indeed, while approximately 85% of endo/lysosomes “split” in control cells

over a period of 30 min, this was reduced to about 50% in apilimod-treated cells (Fig. 8E).

Altogether, we propose that PIKfyve loss-of-function causes lysosome enlargement by reducing

the rate of lysosome fission/”run” events relative to lysosome fusion/”kiss” events, leading to

lysosome coalescence.

Discussion

Disruption of PIKfyve activity causes several major physiological defects including neurological

decay, lethal embryonic development, and inflammatory disease, among others (Ikonomov et al.,

2011; Min et al., 2014; Chow et al., 2007; McCartney et al., 2014). It is unclear how these

defects arise, but they likely relate to disruption of endo/lysosome homeostasis, including

impaired hydrolytic function, reduced rates of autophagic flux, and endo/lysosome swelling

(Martin et al., 2013; de Lartigue et al., 2009; Min et al., 2014; Kim et al., 2016). Endo/lysosome

vacuolation remains the most dramatic and visible defect in PIKfyve-abrogated cells. Yet, the

mechanism by which endo/lysosomes become engorged remains poorly defined. The prevailing

model is that recycling from endo/lysosomes is impaired in PIKfyve/Fab1-defective cells, thus

causing endo/lysosome and vacuole swelling (McCartney et al., 2014; Ho et al., 2012).

Arguably, this is assumed to proceed through tubulo-vesicular transport intermediates formed

during membrane fission catalysed by coat proteins like the retromer (Bryant et al., 1998; de

Lartigue et al., 2009; Rutherford et al., 2006; Zhang et al., 2007). Here, we explored two other

possibilities that could contribute to endo/lysosome enlargement: enhanced endo/lysosome

biosynthesis and/or an imbalance in endo/lysosome fusion-fission dynamics. Collectively, our

work intimates that enhanced lysosome biosynthesis does not drive endo/lysosome swelling.

Instead, our work suggests that endo/lysosomes coalescence during PIKfyve inhibition, resulting

in concurrent enlargement of endo/lysosomes and abatement in their numbers. We provide

evidence that coalescence is due to a shift away from fission during endo/lysosome fusion-

fission cycling (Supplemental Fig. S7). Our observations are consistent with recent work by

Bissig et al. that intimate a role for PIKfyve in reforming terminal lysosomes from

endolysosomes, a term they use to define a subset of lysosomes that are acidic and proteolytic

(Bissig et al., 2017).

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PIKfyve and TFEB function

Inhibition of PIKfyve caused rapid and robust nuclear translocation of TFEB, TFE3 and MITF.

Indeed, others have recently observed that TFEB shuttles into the nucleus in PIKfyve-inhibited

cells (Gayle et al., 2017; Wang et al., 2015). Consistent with nuclear translocation, we also

observed increased mRNA levels of selected lysosomal genes including LAMP1, V-ATPase

subunits and cathepsins. This increase is in line with a recent study that looked at transcriptome-

wide changes in mRNA levels in B-cell non-Hodgkins lymphoma cells treated with apilimod

(Gayle et al., 2017). This suggests that PIKfyve regulates a negative-feedback loop to control

lysosome biogenesis through suppression of TFEB and related factors. However, acute

inhibition of PIKfyve led to either no change, or in the case of LAMP1, to a small increase in

protein levels after 6 h of PIKfyve inhibition, despite the augmented mRNA levels. The

decoupling between enhanced transcription and constant protein levels may result from

pleiotropic effects of PIKfyve inhibition. For example, PIKfyve is known to govern trafficking

of newly synthesized lysosomal genes by controlling recycling of mannose-6-phosphate

receptors (Gayle et al., 2017; Ikonomov et al., 2009a; Rutherford et al., 2006; Zhang et al.,

2007). By contrast, cells chronically impaired for PIKfyve have augmented LAMP1 levels

(Ferguson et al., 2009, 2010). However, this LAMP1 accumulation is thought to occur through

impaired autophagosome turnover rather than an increase in biosynthesis of LAMP1 (Ferguson

et al., 2009; Kim et al., 2016). This may be consistent with the small increase in LAMP1 after

prolonged inhibition of PIKfyve (Fig. 3C). Overall, given that endo/lysosome enlargement was

unaffected in cells deleted for TFEB, TFE3 and/or MITF or treated with cycloheximide, we

argue that biosynthesis does not significantly contribute to lysosome enlargement in cells acutely

inhibited for PIKfyve, though we do not exclude a role in other PIKfyve-mediated functions.

PIKfyve and TFEB regulation

TFEB and TFE3 are maintained in the cytosol via phosphorylation on specific residues that form

docking sites for the 14-3-3 scaffolding protein (Roczniak-Ferguson et al., 2012; Martina et al.,

2012). We now know that TFEB and related factors are subjected to multiple kinases including

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mTORC1, PKC, PKD, GSK3β and phosphatases such as the Ca2+-dependent calcineurin (Li et

al., 2016b; Martina et al., 2014; Roczniak-Ferguson et al., 2012; Settembre et al., 2012; Najibi et

al., 2016; Ploper et al., 2015; Marchand et al., 2015; Wang et al., 2015). Yet, mTORC1

regulation of TFEB remains as the best-understood regulatory circuit. Given that PIKfyve is

needed for mTORC1 activity in adipocytes (Bridges et al., 2012), we examined the existence of a

PIKfyve-mTORC1-TFEB axis. However, S6K remained phosphorylated in apilimod-treated

cells, suggesting that mTORC1 was active. These observations are consistent with Wang et al.

and Krishna et al. who also showed that mTORC1 retains its activity in PIKfyve-abrogated cells

(Wang et al., 2015; Krishna et al., 2016). We considered the possibility that mTORC1 and

starvation controls PIKfyve activity instead, which may then modulate TFEB, perhaps by a

PtdIns(3,5)P2-dependent localization of TFEB to lysosomes, as has been shown for Tup1, a

transcription factor in yeast that controls galactose metabolism (Han and Emr, 2011). Yet, we

could not observe a significant decrease in PtdIns(3,5)P2 levels in mTOR-inhibited cells, though

we cannot exclude disruption of a specific pool of PtdIns(3,5)P2 or of PtdIns(5)P, a 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 function 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 those defective in PtdIns(3,5)P2,

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

like 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

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by unabated anterograde membrane flow and hindered recycling due to defects in forming

tubulo-vesicular intermediates (Supplemental 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 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 (Supplemental 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, likely 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” (Supplemental Fig. S7). In either case, failure to fission would

result in overt coalescence, resulting in lysosome enlargement and reduced numbers. This model

is consistent with recent work by Bissig et al. that intimate a role for PIKfyve in reforming

terminal lysosomes from endolysosomes (Bissig et al., 2017). We do not understand how

PIKfyve modulates lysosome fission dynamics. However, we speculate that mechanisms that

offset lysosome coalescence are distinct from those that drive vesiculation and recycling

(Krishna et al., 2016; Bryant et al., 1998; Rutherford et al., 2006). One interesting possibility

may relate to contact sites between endo/lysosome and the endoplasmic reticulum since these

have been shown to demarcate and assemble molecular machines that catalyse endolysosome

fission (Friedman et al., 2013; Rowland et al., 2014). Thus, it is appealing to speculate that

PIKfyve and PtdIns(3,5)P2 may mediate this process or be regulated by these contact sites to

drive endo/lysosome fission.

Overall, we examined two possible mechanisms that might have contributed to

endo/lysosome enlargement in PIKfyve-inhibited cells. While TFEB and related factors are

controlled by PIKfyve, likely establishing a negative-feedback loop for lysosome biogenesis, we

did not obtain evidence to suggest that biosynthesis contributes to lysosome swelling during

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acute PIKfyve inhibition. Instead, we observed that loss of PIKfyve activity caused concomitant

reduction in the number of and increase in the volume of endo/lysosomes, suggesting that

endo/lysosomes undergo coalescence through fusion and impaired fission. Future work should

delineate the contribution of lysosome coalescence and vesicular recycling to lysosome

enlargement.

Materials and Methods

Cell culture and transfection

RAW 264.7 macrophages strains and HeLa cell strains were cultured in Dulbecco's Modified

Eagle Medium (DMEM; Wisent, St. Bruno, QC) supplemented with 5% heat inactivated fetal

bovine serum (FBS; Wisent). Mouse embryonic fibroblast (MEF) cells were cultured in Roswell

Park Memorial Institute media (RPMI-1640: Gibco, Burlington, ON) with 15% FBS. All cells

were authenticated, cleared of contamination and were cultured at 37°C with 5% CO2. Transient

transfections were performed with FuGene HD (Promega, Madison, WI) following

manufacturer’s instructions, using a 3:1 DNA to transfection reagent ratio. The transfection

mixture was replaced with fresh media after 4-6 h and cells were employed 24 h post-

transfection. Silencing RNA oligonucleotides were electroporated into RAW cells with the Neon

Electroporation system (Life Technologies, Burlington, ON) following manufacturer’s

instructions. Briefly, two rounds of knockdown were performed using ON-TARGETplus

PIKfyve SMARTpool siRNA (L-040127-00-0005) or ON-TARGETplus non-targeting control

pool (GE Healthcare, Mississauga, ON) over 48 h.

Pharmacological manipulation of cells

Cells were treated with apilimod (Toronto Research Chemicals, Toronto, ON) at indicated

concentrations and times. Torin-1 (Tocris Bioscience, Minneapolis, MN) or rapamycin

(BioShop, Burlington, ON) were used as positive controls for TFEB nuclear localization and

mTORC1 inhibition. Cells were also incubated with 100 µM ciliobrevin D (EMD Millipore,

Toronto, ON) or 10 µM nocodazole (Sigma-Aldrich, Oakville, ON).

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Western blotting

Whole cell lysates were generated with 2x Laemmli sample buffer supplemented with protease

inhibitor cocktail (Sigma-Aldrich) and PhosSTOP phosphatase inhibitor (Roche, Laval, QC)

where necessary. Lysates were passed through a 27-gauge needle 6x and heated. Puromycylation

assays were carried out with the addition of 10 µg/mL puromycin for 15 minutes before cell

lysis. When protein dephosphorylation was required, cells were lysed with 1% triton-X100 in

PBS supplemented with protease inhibitor cocktail, spun at 16,000 x g for 10 minutes, and the

supernatant was subjected to lambda protein phosphatase (NEB, Whitby, ON) as per

manufacturer’s instruction. Briefly, 20 µg of protein was incubated with 50 mM HEPES, 100

mM NaCl, 2 mM DTT, 0.01% Brij 25, 1 mM MnCl2, and 200 units of phosphatase at 30°C for

60 min. The reaction as stopped with 5x Laemmli sample buffer and heated.

Most samples were subjected to SDS-PAGE through a 10% acrylamide resolving gel. To

assay for TFEB mobility shift, we used an 8% acrylamide resolving gel supplemented with 12.5

μM Phos-tag (Wako Chemicals USA, Richmond, VA) and 25 μM MnCl2. Phos-tag gels were

washed three times with 100 mM EDTA for 10 min each, followed by a wash with Transfer

buffer (25 mM Tris-HCl, 192 mM glycine, 20% methanol). Proteins were transferred to PVDF,

blocked and incubated with primary and HRP-linked secondary antibodies using Tris-buffered

saline plus 0.1% Tween-20 with either 5% skim milk or 3% BSA. Proteins were visualized using

Clarity enhanced chemiluminescence (Bio-Rad Laboratories, Mississauga, ON) with a

ChemiDoc XRS+ or ChemiDoc Touch imaging system (Bio-Rad). Quantification of proteins

were determined using Image Lab software (Bio-Rad) by normalizing against a loading control

and then normalizing against the vehicle-treated control. Rabbit monoclonal antibodies used

were against cathepsin D (1:1000, GTX62603, GeneTex Inc., Irvine, CA), HSP60 (1:1000,

12165S, Cell Signaling Technologies, Danvers, MA), and p70 S6 kinase (1:1000, 2708S, Cell

Signaling). Rabbit polyclonal antibodies were used against ATP6V1H (1:1000, GTX110778,

GeneTex), TFEB (1:2000, A303-673A, Bethyl Laboratories, Montgomery, TX), phosphoThr389-

p70 S6 kinase (1:1000, 9205S, Cell Signaling), and β-actin (1:1000, 4967S, Cell Signaling). Rat

monoclonal antibodies were used against LAMP1 (1:200, 1D4B, Developmental Studies

Hybridoma Bank, Iowa City, IO). Mouse monoclonal antibodies were used against puromycin

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(1:1000, 540411, Millipore, Burlington, MA), and p53 (1:1000, 2524S, Cell Signaling).

Secondary HRP-linked antibodies were raised in donkey (Bethyl).

Quantitative RT-PCR

Total RNA was extracted from RAW cells using the GeneJET RNA purification kit (Thermo

Fisher Scientific, Mississauga, ON). Equal quantities of mRNA were reverse transcribed with

SuperScript VILO cDNA synthesis kit (Invitrogen) following manufacturer’s guidelines. The

subsequent cDNA was diluted 1:100 and amplified for quantitative PCR using the TaqMan Fast

Advanced Master Mix (Applied Biosystems, Foster City, CA) in the presence of TaqMan assays

with a Step One Plus Real-Time PCR thermal cycler (Applied Biosystems) with Step One

software (version 2.2.2; Applied Biosystems). The TaqMan gene expression assays (Invitrogen)

for the reference gene Abt1 (Mm00803824_m1) and target genes Ctsd (Mm00515586_m1),

Atp6v1h (Mm00505548_m1), ATP6V1D (Mm00445832_m1), LAMP1 (Mm00495262_m1),

and Mcoln1 (Mm00522550_m1) were done in triplicate. Target gene expression was determined

by relative quantification (ΔΔCt method) to Abt1 and the vehicle-treated control sample.

Endo/lysosomal labelling

Endo/lysosomes in RAW cells, HeLa cells and MEFs were labelled by incubating cells with

either 200 µg/mL Alexa488- or Alexa546-conjugated dextran (ThermoFisher) or with 2.5 mg/mL

Lucifer yellow (ThermoFisher) in cell-specific complete medium for 2 h at 37 ºC in 5% CO2,

followed by washing in phosphate-buffered saline and replenishing with fresh complete medium

for 1 h before live-cell imaging. This method likely labels a spectrum of late endosomes,

lysosomes and endolysosomes, which are endosome-lysosome hybrids (Huotari and Helenius,

2011). Thus, we refer to dextran- or Lucifer yellow-labelled structures as endo/lysosomes.

Epifluorescence and spinning disc confocal microscopy

Manual quantification of lysosome size and imaging of GFP-based transfections were imaged

using a Leica DM5000B system outfitted with a DFC350FX camera and controlled by Leica

application suite (Leica Microsystems Inc., Concord, ON). TFEB-GFP transfected into PIKfyve

knockout cells and endogenous TFEB in RAW cells were imaged using an Olympus IX83

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microscope with a Hamamatsu ORCA-Flash4.0 digital camera controlled by CellSens

Dimensions software (Olympus Canada Inc., Richmond Hill, ON). Endogenous TFEB was

further deconvolved using a constrained iterative in CellSens Dimensions. All other imaging of

fixed cells was done with a Quorum DisKovery Spinning disc confocal microscope system

equipped with a Leica DMi8 microscope connected to Andor Zyla 4.2 Megapixel sCMOS

camera and controlled by Quorum Wave FX Powered by MetaMorph software (Quorum

Technologies, Guelph, ON). Live-cell imaging by spinning disc confocal microscopy was

accomplished with either an Olympus IX81 inverted microscope equipped with a Hamamatsu

C9100-13 EMCCD camera and a 60X 1.35 N.A. objective and controlled with Volocity 6.3.0

(PerkinElmer, Bolton, ON). Alternatively, we employed the Quorum DisKovery Spinning disc

confocal microscope system above but using the iXON 897 EMCCD camera. Cells were imaged

live using cell-specific complete medium in a 5% CO2 chamber at 370C. All microscopes were

equipped with standard filters appropriate to fluorophores employed in this study.

3H-myo-inositol labelling of phosphoinositides and phosphoinositide measurement by HPLC-

coupled flow scintillation

RAW cells were incubated for 24 h in inositol-free DMEM (MP Biomedical, CA) with 10

µCi/mL myo-[2-3H(N)] inositol (Perkin Elmer, MA), 10% dialyzed FBS (Gibco), 4 mM L-

glutamine (Sigma Aldrich), 1x insulin-transferrin-selenium-ethanolamine (Gibco), and 20 mM

Hepes (Gibco). Cells were then treated with torin1 or apilimod for 1 h. Reactions were arrested

in 600 µL of 4.5% perchloric acid (v/v) on ice for 15 min, scraped, and pelleted at 12,000 x g for

10 minutes. Pellets were washed with 1 mL of ice cold 0.1 M EDTA and resuspended in 50 µL

of water. Phospholipid deacylation was then done in 500 µL of methanol/40% methylamine/1-

butanol (45.7% methanol: 10.7% methylamine: 11.4% 1-butanol (v/v)) for 50 min at 53°C.

Samples were vacuum-dried and washed twice by resuspending in 300 µL of water and drying.

Dried samples were resuspended again in 450 µL of water and extracted with 300 µL of 1-

butanol/ethyl ether/ethyl formate (20:4:1), vortexed for 5 min and centrifugation at 12,000x g for

2 min. The bottom aqueous layer was collected and extracted two more times. The aqueous layer

was dried in vacuum and resuspended in 50 µL of water. Equal counts of 3H were separated by

HPLC (Agilent Technologies, Mississauga, ON) through an anion exchange 4.6 x 250-mm

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column (Phenomenex, Torrance, CA) with a flow rate of 1 mL/min and subjected to a gradient of

water (buffer A) and 1 M (NH4)2HPO4, pH 3.8 (adjusted with phosphoric acid) (buffer B) as

follows: 0% B for 5 min, 0 to 2% B for 15 minutes, 2% B for 80 minutes, 2 to 10% B for 20

minutes, 10% B for 30 minutes, 10 to 80% B for 10 minutes, 80% B for 5 minutes, 80 to 0% B

for 5 minutes. The radiolabeled eluate was detected by β-RAM 4 (LabLogic, Brandon, FL) with

a 1:2 ratio of eluate to scintillant (LabLogic) and analyzed by Laura 4 software.

Phosphoinositides were normalized against the parent phosphatidylinositol peak.

Live-cell imaging with swept-field confocal microscopy

Cells were seeded into single-glass bottom micro-well dishes (MatTek, Ashland, MA) and grown

to 70%-80% confluency. Cells were then incubated for 2 hours in 200 µL DMEM medium

supplemented with 10% FBS with a 200 µg/mL of Alexa555-conjugated dextran (ThermoFisher).

Samples were washed and incubation media was replaced with fresh complete DMEM followed

by a 90 min post-incubation period. Live cell imaging was performed with a Nikon Ti inverted

microscope equipped with a Prairie Technologies swept-field slit scanning confocal scan head

and a 100x 1.49 N.A. objective (Prairie Technologies, Sioux Falls, SD). Nikon NIS Elements

software equipped with a Photometrics Prime 95B back illuminated sCMOS camera was used

for image acquisition while high speed triggered acquisition was controlled by a National

Instruments DAQ card and Mad City Labs Piezo Z stage controller. Sample volumes were

acquired at 100 ms per z-slice at 30 s intervals over a 30 min observation period before adding

20 nM apilimod and then 2 h in the presence of apilimod.

Immunofluorescence

Following treatments, cells were fixed with 4% paraformaldehyde (v/v) for 15 min. Cells were

blocked and permeabilized with 0.2% (v/v) Triton-X and 2% (v/v) BSA for 10 min, then

subjected to rabbit polyclonal antibody against TFEB (1:250; Bethyl) and Dylight-conjugated

donkey polyclonal antibody against rabbit (1:500; Bethyl). Nuclei were counter stained with 0.4

μg/mL of DAPI. For LAMP1 specific immunostaining, cells were fixed and permeabilized with

100% ice cold methanol for 5 minutes, followed by a 2% BSA block, incubation with Dylight-

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conjugated rat monoclonal antibody against LAMP1 (1:200, Clone 1D4B; Developmental

Studies Hybridoma Bank) and polyclonal donkey anti-rat IgG antibodies (1:500; Bethyl).

Imaging analysis and volumetrics

To quantify the proportion of cells with nuclear TFEB-GFP (including TFE3-GFP or MiTF-

GFP), RAW cells were visually scored as cytosolic if the cytosol had equal or greater intensity

than the nucleus. Alternatively, the nuclear:cytosolic ratio of endogenous TFEB was measured as

the ratio of the mean intensity in the nucleus over the mean intensity in the cytosol using ImageJ.

To manually quantify vacuole diameter in DIC images, vacuoles were first defined as having a

diameter greater than 1.5 µm and then the diameter was measuring using line plot in ImageJ.

To quantify average endo/lysosome volume and number per cells, we employed

volumetric and particle detection tools in Volocity (Volocity 6.3.0) or Icy BioImage Analysis.

Z-stack images were imported into the software and subject to signal thresholding. First, we

tried thresholding at 1.5, 2x, and 2.5x of average cytosolic fluorescence background. This was

then subject to particle detection by defining particles as those with a volume > 0.3 µm3, which

helped eliminate noise-derived particles. Regions of interest where then drawn around each cell

for analysis and particles split using watershed function. Importantly, while increasing

thresholding levels reduced absolute number of particles, particle size and total particle volume,

i) the reduction occurred at similar levels between control and apilimod-treated cells and ii) the

relative trends between control and apilimod-treated cells remained the same (Supplemental Fig.

S3A, B). Hence, we employed a threshold of 2x the average cytosol fluorescence intensity for

remaining analysis. Analysis of endo/lysosome splitting frequency was performed using Imaris

(Bitplane, Concord, MA) using its ImarisTrackLineage module, where endo/lysosome splitting

was defined as the frequency of events producing two particles from a single apparent particle.

Image adjustments such as enhancing contrast was done with ImageJ or Adobe Photoshop

(Adobe Systems, San Jose, CA) without altering the relative signals within images. Figures were

assembled with Adobe Illustrator (Adobe Systems).

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Statistical analysis

All experiments were repeated at least three independent times. The respective figure legends

indicate the total number of experiments and number of samples/cells assessed. The mean and

measure of variation (standard deviation) or error (standard of error of the mean) are indicated.

All experimental conditions were statistically analysed with either am unpaired Student’s t-test

when comparing two parameters only or with a one-way ANOVA test when comparing multiple

conditions in non-normalized controls. ANOVA tests were coupled to Tukey’s post-hoc test to

analyse pairwise conditions. For multiple comparisons with normalized controls, Student’s t-

tests were utilized with Bonferroni correction. Here, we accept p<0.05 as indication of statistical

significance.

Acknowledgements

Plasmid constructs expressing the kinesin-1 dominant negative and of the p50 dynamitin subunit

were obtained from Addgene (Cambridge, MA). Plasmids 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 kind gift from Dr. Richard Youle at the National Institutes of

Health.

Authors contributions

CHC and GS contributed equally to this work, designing and performing experiments, analysing

data and co-write the manuscript. MAG, RMD, ZAO performed and analysed experiments. CW

and SCW performed imaging with sweptfield microscopy. GL extracted mouse embryonic

fibroblasts. RP provided reagents and intellectual contributions to the manuscript. RJB acquired

funding, designed experiments and co-wrote the manuscript.

Competing Interests

The authors declare no competing interests.

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Funding

The research performed in the lab of RJB was funded by a Discovery Grant from the Natural

Sciences and Engineering Research Council, by the Early Researcher Award program by the

Government of Ontario, by a Tier II Canada Research Chair Award, and by contributions from

Ryerson University. Research by CHC was made possible through support from an Ontario

Graduate Scholarship and Queen Elizabeth II Graduate Scholarship. GL was funded through a

National Institutes of Health Grant GM24872.

Abbreviations

DIC: Differential interference contrast; Dimethyl sulfoxide: DMSO; Lucifer yellow: LY; mouse

embryonic fibroblasts: MEFs; phosphoinositides: PtdInsP; phosphatidylinositol-3-phosphate:

PtdIns(3)P; phosphatidylinositol-3,5-bisphosphate: PtdIns(3,5)P2; standard deviation: STD;

standard error of the mean: SEM

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References

Bagshaw, R.D., J.W. Callahan, and D.J. Mahuran. 2006. The Arf-family protein, Arl8b, is

involved in the spatial distribution of lysosomes. Biochem. Biophys. Res. Commun.

344:1186–1191. doi:10.1016/j.bbrc.2006.03.221.

Bissig, C., I. Hurbain, G. Raposo, and G. van Niel. 2017. PIKfyve activity regulates reformation

of terminal storage lysosomes from endolysosomes. Traffic. doi:10.1111/tra.12525.

Bridges, D., J.-T. Ma, S. Park, K. Inoki, L.S. Weisman, and A.R. Saltiel. 2012.

Phosphatidylinositol 3,5-bisphosphate plays a role in the activation and subcellular

localization of mechanistic target of rapamycin 1. Mol. Biol. Cell. 23:2955–62.

doi:10.1091/mbc.E11-12-1034.

Bright, N.A., M.J. Gratian, and J.P. Luzio. 2005. Endocytic delivery to lysosomes mediated by

concurrent fusion and kissing events in living cells. Curr. Biol. 15:360–365.

doi:10.1016/j.cub.2005.01.049.

Bryant, N.J., R.C. Piper, L.S. Weisman, and T.H. Stevens. 1998. Retrograde traffic out of the

yeast vacuole to the TGN occurs via the prevacuolar/endosomal compartment. J. Cell Biol.

142:651–663. doi:10.1083/jcb.142.3.651.

Cai, X., Y. Xu, A.K. Cheung, R.C. Tomlinson, A. Alcázar-Román, L. Murphy, A. Billich, B.

Zhang, Y. Feng, M. Klumpp, J.-M. Rondeau, A.N. Fazal, C.J. Wilson, V. Myer, G. Joberty,

T. Bouwmeester, M.A. Labow, P.M. Finan, J.A. Porter, H.L. Ploegh, D. Baird, P. De

Camilli, J.A. Tallarico, and Q. Huang. 2013. PIKfyve, a class III PI kinase, is the target of

the small molecular IL-12/IL-23 inhibitor apilimod and a player in Toll-like receptor

signaling. Chem. Biol. 20:912–21. doi:10.1016/j.chembiol.2013.05.010.

Chow, C.Y., Y. Zhang, J.J. Dowling, N. Jin, M. Adamska, K. Shiga, K. Szigeti, M.E. Shy, J. Li,

X. Zhang, J.R. Lupski, L.S. Weisman, and M.H. Meisler. 2007. Mutation of FIG4 causes

neurodegeneration in the pale tremor mouse and patients with CMT4J. Nature. 448:68–72.

doi:10.1038/nature05876.

Delamarre, L., M. Pack, H. Chang, I. Mellman, and E.S. Trombetta. 2005. Differential lysosomal

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

proteolysis in antigen-presenting cells determines antigen fate. Science. 307:1630–4.

doi:10.1126/science.1108003.

Deng, Y.P., and B. Storrie. 1988. Animal cell lysosomes rapidly exchange membrane proteins.

Proc. Natl. Acad. Sci. U. S. A. 85:3860–4.

Dong, X., D. Shen, X. Wang, T. Dawson, X. Li, Q. Zhang, X. Cheng, Y. Zhang, L.S. Weisman,

M. Delling, and H. Xu. 2010. PI(3,5)P(2) controls membrane trafficking by direct activation

of mucolipin Ca(2+) release channels in the endolysosome. Nat. Commun. 1:38.

doi:10.1038/ncomms1037.

Dove, S.K., R.C. Piper, R.K. McEwen, J.W. Yu, M.C. King, D.C. Hughes, J. Thuring, A.B.

Holmes, F.T. Cooke, R.H. Michell, P.J. Parker, and M.A. Lemmon. 2004. Svp1p defines a

family of phosphatidylinositol 3,5-bisphosphate effectors. EMBO J. 23:1922–1933.

doi:10.1038/sj.emboj.7600203.

Duclos, S., R. Corsini, and M. Desjardins. 2003. Remodeling of endosomes during lysosome

biogenesis involves “kiss and run” fusion events regulated by rab5. J. Cell Sci. 116:907–

918. doi:10.1242/jcs.00259.

Duclos, S., R. Diez, J. Garin, B. Papadopoulou, A. Descoteaux, H. Stenmark, and M. Desjardins.

2000. Rab5 regulates the kiss and run fusion between phagosomes and endosomes and the

acquisition of phagosome leishmanicidal properties in RAW 264.7 macrophages. J. Cell

Sci. 113:3531–3541.

Ferguson, C.J., G.M. Lenk, and M.H. Meisler. 2009. Defective autophagy in neurons and

astrocytes from mice deficient in PI(3,5)P2. Hum. Mol. Genet. 18:4868–4878.

doi:10.1093/hmg/ddp460.

Ferguson, C.J., G.M. Lenk, and M.H. Meisler. 2010. PtdIns(3,5)P2 and autophagy in mouse

models of neurodegeneration. Autophagy. 6:170–171. doi:10.4161/auto.6.1.10626.

Friedman, J.R., J.R. DiBenedetto, M. West, A.A. Rowland, and G.K. Voeltz. 2013. Endoplasmic

reticulum-endosome contact increases as endosomes traffic and mature. Mol. Biol. Cell.

24:1030–1040. doi:10.1091/mbc.E12-10-0733.

Gayle, S., S. Landrette, N. Beeharry, C. Conrad, M. Hernandez, P. Beckett, S.M. Ferguson, T.

Xu, J. Rothberg, and H. Lichenstein. 2017. B-cell non-Hodgkin lymphoma: Selective

vulnerability to PIKFYVE inhibition. Autophagy. 0. doi:10.1080/15548627.2017.1304871.

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

Gray, M.A., C.H. Choy, R.M. Dayam, E. Ospina-Escobar, A. Somerville, X. Xiao, S.M.

Ferguson, and R.J. Botelho. 2016. Phagocytosis Enhances Lysosomal and Bactericidal

Properties by Activating the Transcription Factor TFEB. Curr. Biol. 26:1955–1964.

doi:10.1016/j.cub.2016.05.070.

Han, B.-K., and S.D. Emr. 2011. Phosphoinositide [PI(3,5)P2] lipid-dependent regulation of the

general transcriptional regulator Tup1. Genes Dev. 25:984–995. doi:10.1101/gad.1998611.

Ho, C.Y., T. a. Alghamdi, and R.J. Botelho. 2012. Phosphatidylinositol-3,5-bisphosphate: No

longer the poor PIP 2. Traffic. 13:1–8. doi:10.1111/j.1600-0854.2011.01246.x.

Ikonomov, O.C., J. Fligger, D. Sbrissa, R. Dondapati, K. Mlak, R. Deeb, and A. Shisheva.

2009a. Kinesin adapter JLP links PIKfyve to microtubule-based endosome-to-trans-Golgi

network traffic of furin. J. Biol. Chem. 284:3750–3761. doi:10.1074/jbc.M806539200.

Ikonomov, O.C., D. Sbrissa, K. Delvecchio, Y. Xie, J.-P.P.J.-P. Jin, D. Rappolee, and A.

Shisheva. 2011. The phosphoinositide kinase PIKfyve is vital in early embryonic

development: preimplantation lethality of PIKfyve-/- embryos but normality of PIKfyve+/-

mice. J. Biol. Chem. 286:13404–13413. doi:10.1074/jbc.M111.222364.

Ikonomov, O.C., D. Sbrissa, and A. Shisheva. 2001. Mammalian cell morphology and endocytic

membrane homeostasis require enzymatically active phosphoinositide 5-kinase PIKfyve. J.

Biol. Chem. 276:26141–7. doi:10.1074/jbc.M101722200.

Ikonomov, O.C., D. Sbrissa, and A. Shisheva. 2009b. YM201636, an inhibitor of retroviral

budding and PIKfyve-catalyzed PtdIns(3,5)P2 synthesis, halts glucose entry by insulin in

adipocytes. Biochem. Biophys. Res. Commun. 382:566–70. doi:10.1016/j.bbrc.2009.03.063.

Jin, N., C.Y. Chow, L. Liu, S.N. Zolov, R. Bronson, M. Davisson, J.L. Petersen, Y. Zhang, S.

Park, J.E. Duex, D. Goldowitz, M.H. Meisler, and L.S. Weisman. 2008. VAC14 nucleates a

protein complex essential for the acute interconversion of PI3P and PI(3,5)P(2) in yeast and

mouse. EMBO J. 27:3221–34. doi:10.1038/emboj.2008.248.

Johnson, D.E., P. Ostrowski, V. Jaumouillé, and S. Grinstein. 2016. The position of lysosomes

within the cell determines their luminal pH. J. Cell Biol. 212:677–92.

doi:10.1083/jcb.201507112.

Jordens, I., M. Fernandez-Borja, M. Marsman, S. Dusseljee, L. Janssen, J. Calafat, H. Janssen, R.

Wubbolts, and J. Neefjes. 2001. The Rab7 effector protein RILP controls lysosomal

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

transport by inducing the recruitment of dynein-dynactin motors. Curr. Biol. 11:1680–1685.

doi:10.1016/S0960-9822(01)00531-0.

Kim, G.H.E., R.M. Dayam, A. Prashar, M. Terebiznik, and R.J. Botelho. 2014. PIKfyve

inhibition interferes with phagosome and endosome maturation in macrophages. Traffic.

15:1143–63. doi:10.1111/tra.12199.

Kim, S.M., S.G. Roy, B. Chen, T.M. Nguyen, R.J. McMonigle, A.N. McCracken, Y. Zhang, S.

Kofuji, J. Hou, E. Selwan, B.T. Finicle, T.T. Nguyen, A. Ravi, M.U. Ramirez, T. Wiher,

G.G. Guenther, M. Kono, A.T. Sasaki, L.S. Weisman, E.O. Potma, B.J. Tromberg, R.A.

Edwards, S. Hanessian, and A.L. Edinger. 2016. Targeting cancer metabolism by

simultaneously disrupting parallel nutrient access pathways. J. Clin. Invest. 126:4088–4102.

doi:10.1172/JCI87148.

Krishna, S., W. Palm, Y. Lee, W. Yang, U. Bandyopadhyay, H. Xu, O. Florey, C.B. Thompson,

and M. Overholtzer. 2016. PIKfyve Regulates Vacuole Maturation and Nutrient Recovery

following Engulfment. Dev. Cell. 38:536–47. doi:10.1016/j.devcel.2016.08.001.

de Lartigue, J., H. Polson, M. Feldman, K. Shokat, S.A. Tooze, S. Urbé, and M.J. Clague. 2009.

PIKfyve regulation of endosome-linked pathways. Traffic. 10:883–893. doi:10.1111/j.1600-

0854.2009.00915.x.

Lenk, G.M., C.M. Frei, A.C. Miller, R.C. Wallen, Y.A. Mironova, R.J. Giger, and M.H. Meisler.

2016. Rescue of neurodegeneration in the Fig4 null mouse by a catalytically inactive FIG4

transgene. Hum. Mol. Genet. 25:340–347. doi:10.1093/hmg/ddv480.

Li, X., N. Rydzewski, A. Hider, X. Zhang, J. Yang, W. Wang, Q. Gao, X. Cheng, and H. Xu.

2016a. A molecular mechanism to regulate lysosome motility for lysosome positioning and

tubulation. Nat. Cell Biol. 18:404–17. doi:10.1038/ncb3324.

Li, Y., M. Xu, X. Ding, C. Yan, Z. Song, L. Chen, X. Huang, X. Wang, Y. Jian, G. Tang, C.

Tang, Y. Di, S. Mu, X. Liu, K. Liu, T. Li, Y. Wang, L. Miao, W. Guo, X. Hao, and C.

Yang. 2016b. Protein kinase C controls lysosome biogenesis independently of mTORC1.

Nat. Cell Biol. 18:1065–77. doi:10.1038/ncb3407.

Luzio, J.P., M.D.J. Parkinson, S.R. Gray, and N.A. Bright. 2009. The delivery of endocytosed

cargo to lysosomes. Biochem. Soc. Trans. 37:1019–1021. doi:10.1042/BST0371019.

Luzio, J.P., P.R. Pryor, and N.A. Bright. 2007. Lysosomes: fusion and function. Nat. Rev. Mol.

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

Cell Biol. 8:622–632. doi:10.1038/nrm2217.

Marchand, B., D. Arsenault, A. Raymond-Fleury, F.-M. Boisvert, and M.-J. Boucher. 2015.

Glycogen Synthase Kinase-3 (GSK3) Inhibition Induces Prosurvival Autophagic Signals in

Human Pancreatic Cancer Cells. J. Biol. Chem. 290:5592–605.

doi:10.1074/jbc.M114.616714.

Martin, S., C.B. Harper, L.M. May, E.J. Coulson, F.A. Meunier, and S.L. Osborne. 2013.

Inhibition of PIKfyve by YM-201636 dysregulates autophagy and leads to apoptosis-

independent neuronal cell death. PLoS One. 8:e60152. doi:10.1371/journal.pone.0060152.

Martina, J.A., Y. Chen, M. Gucek, and R. Puertollano. 2012. MTORC1 functions as a

transcriptional regulator of autophagy by preventing nuclear transport of TFEB. Autophagy.

8:903–14. doi:10.4161/auto.19653.

Martina, J.A., H.I. Diab, O.A. Brady, and R. Puertollano. 2016. TFEB and TFE3 are novel

components of the integrated stress response. EMBO J. 35:479–495.

doi:10.15252/embj.201593428.

Martina, J.A., H.I. Diab, L. Lishu, L. Jeong-A, S. Patange, N. Raben, and R. Puertollano. 2014.

The Nutrient-Responsive Transcription Factor TFE3 Promotes Autophagy, Lysosomal

Biogenesis, and Clearance of Cellular Debris. Sci. Signal. 7:ra9-ra9.

doi:10.1126/scisignal.2004754.

Mccartney, A.J., Y. Zhang, and L.S. Weisman. 2014. Phosphatidylinositol 3,5-bisphosphate: low

abundance, high significance. Bioessays. 36:52–64. doi:10.1002/bies.201300012.

Medina, D.L., A. Fraldi, V. Bouche, F. Annunziata, G. Mansueto, C. Spampanato, C. Puri, A.

Pignata, J. a. Martina, M. Sardiello, M. Palmieri, R. Polishchuk, R. Puertollano, and A.

Ballabio. 2011. Transcriptional activation of lysosomal exocytosis promotes cellular

clearance. Dev. Cell. 21:421–430. doi:10.1016/j.devcel.2011.07.016.

Medina, D.L., S. Di Paola, I. Peluso, A. Armani, D. De Stefani, R. Venditti, S. Montefusco, A.

Scotto-Rosato, C. Prezioso, A. Forrester, C. Settembre, W. Wang, Q. Gao, H. Xu, M.

Sandri, R. Rizzuto, M.A. De Matteis, and A. Ballabio. 2015. Lysosomal calcium signalling

regulates autophagy through calcineurin and TFEB. Nat. Cell Biol. 17:288–299.

doi:10.1038/ncb3114.

Min, S.H., A. Suzuki, T.J. Stalker, L. Zhao, Y. Wang, C. McKennan, M.J. Riese, J.F. Guzman,

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

S. Zhang, L. Lian, R. Joshi, R. Meng, S.H. Seeholzer, J.K. Choi, G. Koretzky, M.S. Marks,

and C.S. Abrams. 2014. Loss of PIKfyve in platelets causes a lysosomal disease leading to

inflammation and thrombosis in mice. Nat. Commun. 5:4691. doi:10.1038/ncomms5691.

Mindell, J.A. 2012. Lysosomal acidification mechanisms. Annu. Rev. Physiol. 74:69–86.

doi:10.1146/annurev-physiol-012110-142317.

Najibi, M., S.A. Labed, O. Visvikis, and J.E. Irazoqui. 2016. An Evolutionarily Conserved PLC-

PKD-TFEB Pathway for Host Defense. Cell Rep. 15:1728–1742.

doi:10.1016/j.celrep.2016.04.052.

Pastore, N., O.A. Brady, H.I. Diab, J.A. Martina, L. Sun, T. Huynh, J.-A.A. Lim, H. Zare, N.

Raben, A. Ballabio, and R. Puertollano. 2016. TFEB and TFE3 cooperate in the regulation

of the innate immune response in activated macrophages. Autophagy. 12:1240–1258.

doi:10.1080/15548627.2016.1179405.

Peña-Llopis, S., S. Vega-Rubin-de-Celis, J.C. Schwartz, N.C. Wolff, T.A.T. Tran, L. Zou, X.-J.

Xie, D.R. Corey, and J. Brugarolas. 2011. Regulation of TFEB and V-ATPases by

mTORC1. EMBO J. 30:3242–58. doi:10.1038/emboj.2011.257.

Phelan, J.P., S.H. Millson, P.J. Parker, P.W. Piper, and F.T. Cooke. 2006. Fab1p and AP-1 are

required for trafficking of endogenously ubiquitylated cargoes to the vacuole lumen in S.

cerevisiae. J. Cell Sci. 119:4225–4234. doi:10.1242/jcs.03188.

Ploper, D., and E.M. De Robertis. 2015. The MITF family of transcription factors: Role in

endolysosomal biogenesis, Wnt signaling, and oncogenesis. Pharmacol. Res. 99:36–43.

doi:10.1016/j.phrs.2015.04.006.

Ploper, D., V.F. Taelman, L. Robert, B.S. Perez, B. Titz, H.-W. Chen, T.G. Graeber, E. von

Euw, A. Ribas, and E.M. De Robertis. 2015. MITF drives endolysosomal biogenesis and

potentiates Wnt signaling in melanoma cells. Proc. Natl. Acad. Sci. U. S. A. 112:E420-9.

doi:10.1073/pnas.1424576112.

Pryor, P.R., B.M. Mullock, N.A. Bright, S.R. Gray, and J.P. Luzio. 2000. The role of

intraorganellar Ca(2+) in late endosome-lysosome heterotypic fusion and in the reformation

of lysosomes from hybrid organelles. J. Cell Biol. 149:1053–62.

Pu, J., C. Schindler, R. Jia, M. Jarnik, P. Backlund, and J.S. Bonifacino. 2015. BORC, a

Multisubunit Complex that Regulates Lysosome Positioning. Dev. Cell. 33:176–188.

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

doi:10.1016/j.devcel.2015.02.011.

Raben, N., and R. Puertollano. 2016. TFEB and TFE3: Linking Lysosomes to Cellular

Adaptation to Stress*. Annu. Rev. Cell Dev. Biol. 32:255–278. doi:10.1146/annurev-cellbio-

111315-125407.

Reddy, A., E. V Caler, and N.W. Andrews. 2001. Plasma membrane repair is mediated by

Ca(2+)-regulated exocytosis of lysosomes. Cell. 106:157–69.

Roczniak-Ferguson, A., C.S. Petit, F. Froehlich, S. Qian, J. Ky, B. Angarola, T.C. Walther, and

S.M. Ferguson. 2012. The transcription factor TFEB links mTORC1 signaling to

transcriptional control of lysosome homeostasis. Sci. Signal. 5:ra42.

doi:10.1126/scisignal.2002790.

Rosa-Ferreira, C., and S. Munro. 2011. Arl8 and SKIP Act Together to Link Lysosomes to

Kinesin-1. Dev. Cell. 21:1171–1178. doi:10.1016/j.devcel.2011.10.007.

Rowland, A.A., P.J. Chitwood, M.J. Phillips, and G.K. Voeltz. 2014. ER Contact Sites Define

the Position and Timing of Endosome Fission. Cell. 159:1027–1041.

doi:10.1016/j.cell.2014.10.023.

Rutherford, A.C., C. Traer, T. Wassmer, K. Pattni, M. V Bujny, J.G. Carlton, H. Stenmark, and

P.J. Cullen. 2006. The mammalian phosphatidylinositol 3-phosphate 5-kinase (PIKfyve)

regulates endosome-to-TGN retrograde transport. J. Cell Sci. 119:3944–57.

doi:10.1242/jcs.03153.

Sancak, Y., L. Bar-Peled, R. Zoncu, A.L. Markhard, S. Nada, and D.M. Sabatini. 2010.

Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its

activation by amino acids. Cell. 141:290–303. doi:10.1016/j.cell.2010.02.024.

Sardiello, M., M. Palmieri, A. di Ronza, D.L. Medina, M. Valenza, V.A. Gennarino, C. Di

Malta, F. Donaudy, V. Embrione, R.S. Polishchuk, S. Banfi, G. Parenti, E. Cattaneo, and A.

Ballabio. 2009. A Gene Network Regulating Lysosomal Biogenesis and Function. Science

(80-. ). 325:473–7. doi:10.1126/science.1174447.

Saric, A., V.E.B. Hipolito, J.G. Kay, J. Canton, C.N. Antonescu, and R.J. Botelho. 2016. mTOR

controls lysosome tubulation and antigen presentation in macrophages and dendritic cells.

Mol. Biol. Cell. 27:321–33. doi:10.1091/mbc.E15-05-0272.

Sbrissa, D., O.C. Ikonomov, C. Filios, K. Delvecchio, and A. Shisheva. 2012. Functional

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

dissociation between PIKfyve-synthesized PtdIns5P and PtdIns(3,5)P2 by means of the

PIKfyve inhibitor YM201636. Am. J. Physiol. Cell Physiol. 303:C436-46.

doi:10.1152/ajpcell.00105.2012.

Sbrissa, D., O.C. Ikonomov, Z. Fu, T. Ijuin, J. Gruenberg, T. Takenawa, and A. Shisheva. 2007.

Core protein machinery for mammalian phosphatidylinositol 3,5-bisphosphate synthesis and

turnover that regulates the progression of endosomal transport: Novel Sac phosphatase joins

the ArPIKfyve-PIKfyve complex. J. Biol. Chem. 282:23878–23891.

doi:10.1074/jbc.M611678200.

Sbrissa, D., O.C. Ikonomov, and A. Shisheva. 1999. PIKfyve, a mammalian ortholog of yeast

Fab1p lipid kinase, synthesizes 5-phosphoinositides. Effect of insulin. J. Biol. Chem.

274:21589–21597. doi:10.1074/jbc.274.31.21589.

Schwake, M., B. Schröder, and P. Saftig. 2013. Lysosomal Membrane Proteins and Their Central

Role in Physiology. Traffic. 14:739–748. doi:10.1111/tra.12056.

Settembre, C., and A. Ballabio. 2014. Lysosomal adaptation: how the lysosome responds to

external cues. Cold Spring Harb. Perspect. Biol. 6:a016907–a016907.

doi:10.1101/cshperspect.a016907.

Settembre, C., R. Zoncu, D.L. Medina, F. Vetrini, S.S. Erdin, S.S. Erdin, T. Huynh, M. Ferron,

G. Karsenty, M.C. Vellard, V. Facchinetti, D.M. Sabatini, and A. Ballabio. 2012. A

lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR

and TFEB. Eur. Mol. Biol. Organ. J. 31:1095–108. doi:10.1038/emboj.2012.32.

Swanson, J., E. Burke, S.C. Silverstein, A. Bushnell, and S.C. Silverstein. 1987. Tubular

lysosome morphology and distribution within macrophages depend on the integrity of

cytoplasmic microtubules. Proc. Natl. Acad. Sci. U. S. A. 84:1921–1925.

doi:10.1073/pnas.84.7.1921.

Tsunemi, T., T.D. Ashe, B.E. Morrison, K.R. Soriano, J. Au, R.A.V. Roque, E.R. Lazarowski,

V.A. Damian, E. Masliah, and A.R. La Spada. 2012. PGC-1α rescues Huntington’s disease

proteotoxicity by preventing oxidative stress and promoting TFEB function. Sci. Transl.

Med. 4:142ra97. doi:10.1126/scitranslmed.3003799.

Vyas, J.M., Y.-M. Kim, K. Artavanis-Tsakonas, J.C. Love, A.G. Van der Veen, and H.L. Ploegh.

2007. Tubulation of class II MHC compartments is microtubule dependent and involves

Jour

nal o

f Cel

l Sci

ence

• A

ccep

ted

man

uscr

ipt

multiple endolysosomal membrane proteins in primary dendritic cells. J. Immunol.

178:7199–210.

Wang, W., Q. Gao, M. Yang, X. Zhang, L. Yu, M. Lawas, X. Li, M. Bryant-Genevier, N.T.

Southall, J. Marugan, M. Ferrer, and H. Xu. 2015. Up-regulation of lysosomal TRPML1

channels is essential for lysosomal adaptation to nutrient starvation. Proc. Natl. Acad. Sci.

112:E1373–E1381. doi:10.1073/pnas.1419669112.

Xiong, J., and M.X. Zhu. 2016. Regulation of lysosomal ion homeostasis by channels and

transporters. Sci. China Life Sci. 59:777–791. doi:10.1007/s11427-016-5090-x.

Zolov, S.N., D. Bridges, Y. Zhang, W.-W. Lee, E. Riehle, R. Verma, G.M. Lenk, K. Converso-

Baran, T. Weide, R.L. Albin, A.R. Saltiel, M.H. Meisler, M.W. Russell, and L.S. Weisman.

2012. In vivo, Pikfyve generates PI(3,5)P2, which serves as both a signaling lipid and the

major precursor for PI5P. Proc. Natl. Acad. Sci. U. S. A. 109:17472–7.

doi:10.1073/pnas.1203106109.

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Figures

Figure 1: PIKfyve inhibition causes nuclear translocation of TFEB. A, B: RAW cells

expressing TFEB-GFP (A) or stained for endogenous TFEB (B) treated with vehicle or 20 nM

apilimod for 1 h. C: RAW cells silenced for PIKfyve or mock-silenced and expressing TFEB-

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GFP. D: HeLa cells stained for endogenous TFEB treated with vehicle or 20 nM apilimod for 1

h. Where shown, DAPI stains the nuclei. Nuclear translocation of TFEB was quantified either as

percent of cells containing nuclear GFP-tagged TFEB (A, C) or as the nuclear-to-cytosol

fluorescence intensity ratio for endogenous TFEB (B, D). Shown is the mean and standard error

of the mean from at least three independent experiments and based on 50-300 cells per condition

per experiment. Asterisks (*) indicate statistical difference from respective control conditions

(p<0.05) using one-way ANOVA and Tukey’s post-hoc test or with an unpaired Student’s t-test,

as appropriate. Scale bar = 5 µm.

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Figure 2: mTOR and PIKfyve function independently. A. Western blot detection of

phosphoThr389-p70S6K and total p70S6K in PIKfyve-inhibited cells treated as indicated. The

ratio of phosphor-S6K-to-total S6K was quantified from three independent blots. Shown is the

mean ratio and standard deviation normalized to DMSO-treated cells. B. Quantification of

PtdIns(3)P and PtdIns(3,5)P2 levels using 3H-myo-inositol incorporation and HPLC-coupled flow

scintillation. Results are shown as the mean and standard deviation from three independent

experiments, normalized to their PtdInsP levels and to the respective DMSO-treated controls.

For A and B, asterisks (*) indicate statistical difference from respective control conditions

(p<0.05) using multiple Student’s t-test with the Bonferroni correction. C. Western blot against

TFEB from whole cell lysates resolved by a Phos-tag gel. Shown in a representative blot from

three independent experiments showing differential migration of TFEB in lysates from control

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relative to torin1- and apilimod-treated cells. Anti-TFEB antibody specificity was confirmed by

the loss of most bands in tfeb-/- RAW whole cell lysates. In addition, most bands represent

phosphorylated isoforms of TFEB since pre-treatment of lysates with lambda protein

phosphatase led to a single fast-running band.

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Figure 3: Acute PIKfyve inhibition enhances lysosomal gene transcription but not protein

levels. A. Expression of select lysosomal genes quantified by qRT-PCR and normalized against

Abt1. Shown is the mean ± SEM from seven independent experiments. B. Western blot against

selected lysosomal proteins in RAW cells treated as indicated. Numbers on the right represent

relative molecular weight (kDa). C. Quantification of indicated proteins demonstrated in B and

shown as the mean intensity ± SEM from four independent blots. Asterisks (*) indicate

statistical difference from respective control conditions (p<0.05) using multiple Student’s t-test

with the Bonferroni correction.

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Figure 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

subjected to 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 with cycloheximide. Lysosome volume and number

were quantified. In all cases, data shown is the mean ±SEM 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-treatement obliterated 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 3

independent experiments. D. Cycloheximide reduces p53 abundance relative to β-actin.

Representative blot shown from 3 independent experiments. Numbers of the right represent

relative molecular weight (in kDa). Data shown is the mean abundance ±SEM by measuring p53

signal as a ratio against -actin. Asterisks (*) indicate statistical difference from respective

control conditions (p<0.05) using Student’s t-test. Scale bar = 5 µm.

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Figure 5: Volumetric analysis of endo/lysosomes during acute and chronic PIKfyve

suppression. A. RAW cells were pre-labelled with Lucifer yellow as before and then 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, C, D: Quantification of individual endo/lysosome volume (B), endo/lysosome number per

RAW macrophage (C) and total endo/lysosome volume (D). E. Wild-type and fig4-/- mouse

embryonic fibroblasts labelled with Lucifer yellow. Scale bar = 30 µm. F,G, 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 ±SEM from three independent experiments, with at least 15-20

cells per condition per experiment. Asterisks (*) indicate statistical difference from respective

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control conditions (p<0.05) using one-way ANOVA and Tukey’s post-hoc test for data shown in

B-D, and unpaired Student’s t-test for data displayed in E-H.

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Figure 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 media 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, C, 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 ±SEM from three independent

experiments, with at least 15-20 cells per condition per experiment. Asterisks (*) indicates

statistical difference between data indicated by lines (p<0.05) using one-way ANOVA and

Tukey’s post-hoc test.

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Figure 7: Volumetric analysis of endo/lysosome number and volume in cells disrupted for

microtubule function. A. Cells were pre-labelled with Alexa488-conjugated dextran, followed

by vehicle-alone, 10 µM nocadozole for 60 min, or co-treated with apilimod and vehicle or

nocadozole for 1 h. Fluorescence micrographs are z-projections of 45-55 z-planes acquired by

spinning disc confocal microscopy. B, C: Quantification of individual endo/lysosome volume

(B) and endo/lysosome number per RAW macrophage (C). D. Cells were pre-labelled with

Alexa488-conjugated dextran as before, followed by 20 nM apilimod for 1 h and then replaced

with fresh media or media containing nocadozole for the indicated time period. E, F: Volumetric

quantification of individual endo/lysosome volume (E) and endo/lysosome number per cell (D).

In all cases, data shown are the mean ±SEM from three independent experiments, with at least

15-20 cells per condition per experiment. Asterisks (*) indicates statistical difference between

conditions indicated by lines (p<0.05) using one-way ANOVA and Tukey’s post-hoc test. Scale

bar = 5 µm.

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Figure 8: Live-cell imaging of apilimod-induced lysosome swelling.

Cells were pre-labelled with Alexa546-conjugated dextran, followed by (A) vehicle-alone or (B)

20 nM apilimod at time 0 min. Single-plane imaging was effected by spinning disc microscopy

at 1 frame/2 min. Time in min refers to time after adding vehicle or apilimod. See supplemental

Movies 3 and 4 for full capture. Scale bar of image stills for (A) and (B) = 5 µm. Sweptfield

imaging of RAW cells exposed to (C) vehicle or (D) 20nM apilimod. See supplemental 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 bar of image stills for (C) and (D) = 3 µm.

E. Rate of "splitting" events over 30 min of vehicle or apilimod-treated cells showing a reduction

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in splitting in PIKfyve-inhibited cells, which may reflect reduced fission. Data were statistically

analysed by Student's t-test. Asterisk represents p<0.05.

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TFE3-GFP DAPI

DM

SO

Api

limod

DM

SO

Api

limod

MiTF-GFP DAPI

DMSO Torin1 Apilimod0

50

100

DMSO Torin1 Apilimod0

50

100

Per

cent

of c

ells

with

TF

E3-

GFP

in th

e nu

cleu

s (%

)P

erce

nt o

f cel

ls w

ith

MiT

F-G

FP in

the

nucl

eus

(%)

A

B

* *

* *

Supplemental Figure S1

Supplemental Figure S1: PIKfyve inhibition causes nuclear translocation of GFP-tagged

TFE3 and MITF. RAW cells expressing TFE3-GFP (A) and MITF-GFP (B) treated with

vehicle or apilimod as before. DAPI identifies the nuclei. Nuclear translocation was quantified

by scoring the percent of cells with nuclear accumulation of these proteins. Shown is the mean

percent and standard error of the mean from a minimum of 30 cells across at least three

independent experiments. Asterisks (*) indicates statistical difference between conditions

indicated by lines (p<0.05) using one-way ANOVA and Tukey’s post-hoc test. Scale bar = 5

µm..

Supplemental Information

J. Cell Sci. 131: doi:10.1242/jcs.213587: Supplementary information

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DM

SO

Api

limod

WT TFEB-/- TFE3-/-TFEB-/- TFE3-/-

Num

ber o

f vac

uole

s (>

1.5

µm)

Supplemental Figure S2

DMSO Apilimod0

2

4

6

WTTFEB-/-

TFE3-/-

TFEB-/- TFE3-/-

WT TFEB-/- TFE3-/-TFEB-/- TFE3-/-

0

1

2

3

4

Vac

uole

dia

met

er (μ

m)

WT TFEB-/- TFE3-/- TFEB-/- TFE3-/-

0.0

0.2

0.4

0.6

0.8

1.0

Pea

rson

’s c

oeffi

cien

t bet

wee

n de

xtra

n an

d LA

MP

1

A

B

Supplemental Figure S2: Manual quantification of vacuole diameter and number in wild-

type, tfeb-/-, tfe3-/- and tfeb-/- tfe3-/- double deleted cells inhibited for PIKfyve. A. Cells were

incubated with vehicle or 20 nM apilimod for 1 h and then imaged by DIC optics. Mean vacuole

number ± SEM and mean diameter ± SEM for vacuoles greater than 1.5 µm in diameter. B)

Pearson's coefficient of dextran and LAMP1 in wild-type and RAW cells deleted for TFEB

and/or TFE3. Data are from three independent experiments and from 100 cells or 300 vacuoles,

respectively. No statistical difference was detected among apilimod-treated cell types.

J. Cell Sci. 131: doi:10.1242/jcs.213587: Supplementary information

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Manual countingAutomatic counting

0

50

100

150

DMSO Apilimod

Tota

l end

olys

osom

e nu

mbe

r per

cel

l

A

0

20

40

60

80

100

Threshold value (x background)

DMSOApilimod

0

2

4

6

8

10 DMSOApilimod

1.5x 2.0x 2.5x

Tota

l end

olys

osom

e nu

mbe

r per

cel

l

1.5x 2.0x 2.5xThreshold value (x background)

Ave

rage

end

olys

osom

e vo

lum

e (μ

m3 )

1.5x 2.0x 2.5x0

200

400 DMSOApilimod

Tota

l end

olys

osom

e vo

lum

e (μ

m3 )

per c

ell

Threshold value (x background)

B

C

Supplemental Figure S3

D

Supplemental Figure S3: Comparison of endo/lysosome number, size and total

endo/lysosome volume at different intensity thresholding values. Vehicle and apilimod-

treated cells were imaged with the same parameters. A-C. Images were then subject to

thresholding levels set at 1.5x, 2x and 2.5x that of cytosolic fluorescence intensity before

volumetric analysis. Quantification of endo/lysosome number (A), endo/lysosome volume (B)

and total endo/lysosome volume per cell (C). While the absolute values reduced with increasing

thresholding, the relative trends between control and apilimod-treated cells remained similar

across all thresholding measures. D. Number of endo/lysosomes counted manually and

automatically at 2x thresholding and using a minimum particle size of 0.3 µm3. In all cases,

shown is the mean ± STD from 27 different cells.

J. Cell Sci. 131: doi:10.1242/jcs.213587: Supplementary information

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Wild

-Typ

e +

vehi

cle

TFE

B-/-TF

E3-/-

MIT

F-/-

+ v

ehic

leW

ild-T

ype

+ A

pilim

odTF

EB-/-

TFE

3-/-M

ITF-/-

+

Api

limod

A B

0

200

400

600

800

Tot

al e

ndol

ysos

ome

volu

me

(μm

3 ) pe

r cel

lW

ild-T

ype

+ ve

hicle

TFEB

-/- TFE

3-/- M

ITF

-/-

+ v

ehicl

eW

ild-T

ype

+ Ap

ilimod

TFEB

-/- TFE

3-/- M

ITF

-/-

+ A

pilim

od

Wild

-Typ

e +

vehi

cleTF

EB-/- T

FE3-

/- MIT

F-/-

+ v

ehicl

eW

ild-T

ype

+ Ap

ilimod

TFEB

-/- TFE

3-/- M

ITF

-/-

+ A

pilim

od

C

0

2

4

6

8

Ave

rage

end

olys

osom

e

vol

ume

(μm

3 ) W

ild-T

ype

+ ve

hicle

TFEB

-/- TFE

3-/- M

ITF

-/-

+ v

ehicl

eW

ild-T

ype

+ Ap

ilimod

TFEB

-/- TFE

3-/- M

ITF

-/-

+ A

pilim

od

*

0

100

200

300

400

Tota

l end

olys

osom

e

num

ber p

er c

ell

Wild

-Typ

e +

vehi

cleTF

EB-/- T

FE3-

/- MIT

F-/-

+ v

ehicl

eW

ild-T

ype

+ Ap

ilimod

TFEB

-/- TFE

3-/- M

ITF

-/-

+ A

pilim

od

D

*

*

*

Supplemental Figure S4: Volumetric analysis of endo/lysosomes during acute PIKfyve

suppression in HeLa cells. A. “Wild-type” HeLa cells and HeLa cells deleted for MITF, TFEB

and TFE3 were pre-labelled with Alexa546-conjugated dextran and treated with vehicle-only or

50 nM apilimod for 1 h. Quantification of endo/lysosome volume (B), number (C) and total

endo/lysosome volume per cell (D). Shown is the mean ± SEM from 3 independent experiments

and 15-20 cells per condition per experiment. Asterisk (*) indicates statistical difference relative

to respective controls. No difference was observed between wild-type and mutant HeLa cells for

changes during apilimod treatment. Scale bar = 10 µm.

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OSM

DD ni ver boili

CD niverboili

CAp

ilimod

DIC Dextran Alexa-546 Dynamitin-GFP

DICDextran

Alexa-488 Kif5BDN-RFP

0

5

10

15

Ciliobrevin DApilimod

--

+-

-+

++

0

50

100

150

Ciliobrevin DApilimod

--

+-

-+

++

emososylodne latoTllec rep reb

mun

A B

C

D

**

**

0

1

2

3

4

5

Kif5BDN-RFPApilimod

--

+-

-+

++

0

50

100

150

emososylodne latoTllec rep reb

mun

Kif5BDN-RFPApilimod

--

+-

-+

++

E

F

**

**G

H

0

1

2

3

4

5

Dynamitin-GFPApilimod

--

+-

-+

++

I

0

50

100

150

200

Dynamitin-GFPApilimod

--

+-

-+

++

emososylodne latoTllec rep reb

mun

**

*

OSM

Ddo

mili pA

DM

SOAp

ilimod

Supplemental Figure S5

Aver

age

endo

lyso

som

e vo

lum

e (µ

m3 )

Aver

age

endo

lyso

som

e vo

lum

e (µ

m3 )

Aver

age

endo

lyso

som

e vo

lum

e (µ

m3 )

J. Cell Sci. 131: doi:10.1242/jcs.213587: Supplementary information

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Supplemental Figure S5: Volumetric analysis of endo/lysosomes during acute PIKfyve

suppression in RAW cells disrupted for microtubule-motor proteins. A. Cells were pre-

labelled with Alexa488-conjugated dextran followed by DMSO alone or 1 h of 20 nM apilimod.

This was then followed by vehicle or 100 µM ciliobrevin D for an additional 2.5 h in presence

and absence of 20 nM apilimod 1 h. Scale bar = 5 µm. B, C. Volumetric quantification of

endo/lysosome volume (B) and number (C) for A. D. Cells were transfected with dominant-

negative kinesin (Kif5BDN-RFP) to block kinesin. Cells were then pre-labelled with Alexa488-

conjugated dextran followed by vehicle-alone or apilimod 20 nM treatment for 30 min to induce

vacuolation. Scale bar = 10 µm. E, F. Volumetric quantification of endo/lysosome volume (E)

and number (F) for D. G. Cells were transfected with p50-dynamitin-GFP (Dynamitin-GFP) to

block dynein. Cells were then pre-labelled with Alexa546-conjugated dextran followed by

vehicle-alone or apilimod 20 nM treatment for 30 min to induce vacuolation. Scale bar = 5 µm.

H, I. Volumetric quantification of endo/lysosome volume (H) and number (I) for G. In all cases,

data shown are the normalized mean ±SEM from three independent experiments, with at least

15-20 cells per condition per experiment. Asterisks (*) indicates statistical difference from

respective control conditions.

J. Cell Sci. 131: doi:10.1242/jcs.213587: Supplementary information

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Duration of DMSO treatment (min)

0 5 10 15 20 25 30Alternate field

of view

Duration of Apilimod treatment (min)

0 5 10 15 20 25 30Alternate field

of view

Supplemental Figure S6

Supplemental Figure S6: Live-cell imaging precludes lysosome enlargement. RAW

macrophages were pre-labelled with Alexa546-conjugated dextran as before. Cells were then

imaged live by spinning disc confocal microscopy after the addition of vehicle or 20 nM

apilimod every 10 s for 30 min with 6 z-slices imaged per time point. A. Time-sequence of

vehicle-treated cells over 30 min. Frames are from Supplemental Movie 1. An alternate field of

view from the same coverslip immediately post imaging shows lysosome signal at the end of 30

min of imaging. B. Time sequence of apilimod-treated cells showing no vacuolation over 30

min from Supplemental Movie 2. An alternate field of view from the same coverslip

immediately post imaging shows lysosome enlargement elsewhere. Time shown in min. Scale

bar = 5 µm.

J. Cell Sci. 131: doi:10.1242/jcs.213587: Supplementary information

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PIKfyve-dependent�ssion

PIKfyve

Membranein�ux

MembraneRecycling

MembraneRecycling

PIKfyve

PIKfyve-dependentPrediction during PIKfyve inhibition:Total endolysosome volume increasesIndividual endolysosomes enlargeEndolysosome number constant

Recycling from endo/lysosomes

Coalescence of endo/lysosomesLysosome fusion/”kiss”

Lysosome �ssion/”run”

Prediction during PIKfyve inhibition:Total endolysosome volume constantIndividual endolysosomes enlargeEndolysosome number drops

Supplemental Figure 7

J. Cell Sci. 131: doi:10.1242/jcs.213587: Supplementary information

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Supplemental Figure S7: Models of endo/lysosome enlargement during PIKfyve inhibition.

PIKfyve inhibition causes endo/lysosome enlargement. This may occur because of i) a defect in

membrane recycling from endo/lysosomes and/or ii) due to endo/lysosome coalescence. A

defect in membrane recycling should lead to an increase in total lysosome volume as membrane

influx is no longer balanced by an efflux pathway. In this model, individual lysosomes should

swell but their numbers remain similar. In comparison, endo/lysosomes undergo constant rounds

of homotypic and heterotypic fusion and splitting. This may occur through full endo/lysosome

merger, followed by splitting, or during partial fusion (kiss) and separation (run). Our work

suggests that endo/lysosome enlargement proceeds through a failure to separate/run causing

endo/lysosomes to become coalescence and enlarge. In this scenario, total lysosome volume

should remain constant but the total numbers of endo/lysosomes should drop.

J. Cell Sci. 131: doi:10.1242/jcs.213587: Supplementary information

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Supplemental Movie 1: Endo/lysosome dynamics of DMSO-treated cells at high-frame rate

imaging using spinning disc confocal microscopy. RAW macrophages were pre-labelled with

Alexa546-conjugated dextran, followed by live-cell imaging using spinning disc confocal

microscopy after the addition of DMSO. Imaging was done by acquiring 6 planes along the z-

axis every 10 s for 30 min. Refer to Supplemental Figure S6 for select stills. Scale and time are

indicated.

J. Cell Sci. 131: doi:10.1242/jcs.213587: Supplementary information

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Supplemental Movie 2: Endo/lysosome dynamics of apilimod-treated cells at high-frame

rate imaging using spinning disc confocal microscopy. RAW macrophages were pre-labelled

with Alexa546-conjugated dextran as before, followed by live-cell imaging using spinning disc

confocal microscopy after the addition of 20 nM apilimod. Imaging was done by acquiring 6

planes along the z-axis once every 10 s for 30 min after apilimod addition. Refer to

Supplemental Figure S6 for select stills. Vacuolation was not observed in the field under

observation. Scale and time are indicated.

J. Cell Sci. 131: doi:10.1242/jcs.213587: Supplementary information

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Supplemental Movie 3: Endo/lysosome dynamics of DMSO-treated cells at low-frame rate

imaging using spinning disc confocal microscopy. RAW macrophages were pre-labelled with

Alexa546-conjugated dextran as before, followed by live-cell imaging using spinning disc

confocal microscopy after the addition of DMSO. Imaging was done at a single-plane once

every 2 min for 60 min. Refer to Figure 8A for select stills. Scale and time are indicated.

J. Cell Sci. 131: doi:10.1242/jcs.213587: Supplementary information

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Supplemental Movie 4: Endo/lysosome dynamics of apilimod-treated cells at low-frame

rate imaging using spinning disc confocal microscopy. RAW macrophages were pre-labelled

with Alexa546-conjugated dextran as before, followed by live-cell imaging using spinning disc

confocal microscopy after the addition of 20 nM apilimod. Imaging was done at a single-plane

once every 2 min for 60 min. Refer to Figure 8B for select stills. Vacuolation occurs during

lower light exposure frequency. Scale and time are indicated.

J. Cell Sci. 131: doi:10.1242/jcs.213587: Supplementary information

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Supplemental Movie 5: Endo/lysosome dynamics of DMSO-treated cells at high-frame rate

imaging using sweptfield confocal microscopy. RAW macrophages were pre-labelled with

Alexa555-conjugated dextran, followed by live-cell imaging using sweptfield confocal

microscopy after the addition of DMSO. Imaging was done by acquiring 10 planes along the z-

axis every 30 s for 30 min. The movie depicts a projection of all z-planes. Refer to Figure 8C for

select stills. Scale and time are indicated.

J. Cell Sci. 131: doi:10.1242/jcs.213587: Supplementary information

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Supplemental Movie 6: Endo/lysosome dynamics of apilimod-treated cells at high-frame

rate imaging using sweptfield confocal microscopy. RAW macrophages were pre-labelled

with Alexa555-conjugated dextran, followed by live-cell imaging using sweptfield confocal

microscopy after the addition of 20 nM apilimod. Imaging was done by acquiring 10 planes

along the z-axis every 30 s for 2 h. The movie depicts a projection of all z-planes. Refer to

Figure 8D for select stills. Scale and time are indicated.

J. Cell Sci. 131: doi:10.1242/jcs.213587: Supplementary information

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