Transcriptional Activation of Lysosomal Exocytosis PromotesCellular Clearance

Diego L. Medina1,6, Alessandro Fraldi1,6, Valentina Bouche1, Fabio Annunziata1,Gelsomina Mansueto1, Carmine Spampanato1, Claudia Puri1, Antonella Pignata1, Jose A.Martina2, Marco Sardiello3,4, Michela Palmieri4, Roman Polishchuk1, Rosa Puertollano2,and Andrea Ballabio1,3,4,5,∗1Telethon Institute of Genetics and Medicine (TIGEM), Via P. Castellino 111, 80131 Naples, Italy2Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, MD 20892, USA3Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030,USA4Jan and Dan Duncan Neurological Research Institute, Texas Children Hospital, Houston, TX77030, USA5Medical Genetics, Department of Pediatrics, Federico II University, Via Pansini 5, 80131 Naples,Italy

SummaryLysosomes are cellular organelles primarily involved in degradation and recycling processes.During lysosomal exocytosis, a Ca2+-regulated process, lysosomes are docked to the cell surfaceand fuse with the plasma membrane (PM), emptying their content outside the cell. This processhas an important role in secretion and PM repair. Here we show that the transcription factor EB(TFEB) regulates lysosomal exocytosis. TFEB increases the pool of lysosomes in the proximity ofthe PM and promotes their fusion with PM by raising intracellular Ca2+ levels through theactivation of the lysosomal Ca2+ channel MCOLN1. Induction of lysosomal exocytosis by TFEBoverexpression rescued pathologic storage and restored normal cellular morphology both in vitroand in vivo in lysosomal storage diseases (LSDs). Our data indicate that lysosomal exocytosis maydirectly modulate cellular clearance and suggest an alternative therapeutic strategy for disordersassociated with intracellular storage.

AbstractGraphical Abstract—

© 2011 ELL & Excerpta Medica.∗Corresponding author ballabio@tigem.it.6These authors contributed equally to this workThis document was posted here by permission of the publisher. At the time of deposit, it included all changes made during peerreview, copyediting, and publishing. The U.S. National Library of Medicine is responsible for all links within the document and forincorporating any publisher-supplied amendments or retractions issued subsequently. The published journal article, guaranteed to besuch by Elsevier, is available for free, on ScienceDirect.

Sponsored document fromDevelopmental Cell

Published as: Dev Cell. 2011 September 13; 21(3): 421–430.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Highlights—► TFEB-regulated transcription induces lysosomal docking to the plasmamembrane (PM) ► TFEB promotes lysosomal fusion with the PM by raising Ca2+ levels throughMCOLN1 ► TFEB can thus rescue pathological storage in lysosomal storage disease (LSD) cells► In vivo TFEB gene delivery rescues storage, inflammation, and apoptosis in LSD mice

IntroductionLysosomes are cellular organelles primarily involved in degradation and recycling processes(Kornfeld and Mellman, 1989). Lysosomes are also involved in a secretory pathway knownas lysosomal exocytosis, which requires two sequential steps. In the first step, Ca2+-independent (Jaiswal et al., 2002), lysosomes are recruited to the close proximity of the cellsurface, while in the second step the pool of predocked lysosomes fuse with the plasmamembrane (PM) in response to Ca2+ elevation (Andrews, 2000; Jaiswal et al., 2002; Tuckeret al., 2004). Lysosomal exocytosis plays a major role in several physiological processessuch as cellular immune response, bone resorption, and PM repair (Andrews, 2000, 2005;Bossi and Griffiths, 2005). Ca2+-dependent lysosomal exocytosis was considered to belimited to specialized secretory cells; however, recent studies indicate that this processoccurs in all cell types (Andrews, 2000; Rodríguez et al., 1999; Rodríguez et al., 1997).Although the main steps of lysosomal exocytosis have been elucidated, little is known aboutits regulation and how this process is coordinated with lysosomal biogenesis.

We recently discovered that lysosomal biogenesis and lysosomal degradative function aretranscriptionally regulated by the bHLH-leucine zipper transcription factor EB (TFEB)(Sardiello et al., 2009). TFEB activation was able to reduce the accumulation of thepathogenic protein in a cellular model of Huntington disease (Sardiello et al., 2009) andameliorated the phenotype of cells from a murine model of Parkinson disease (Dehay et al.,2010).

In this study we demonstrated that TFEB transcriptionally regulates lysosomal exocytosisboth by inducing the release of intracellular Ca2+ through its target gene MCOLN1 and byincreasing the population of lysosomes ready to fuse with the PM. Moreover, wedemonstrated that the induction of lysosomal exocytosis by TFEB promotes cellularclearance in pathological conditions such as lysosomal storage diseases (LSDs) in which thelysosomal degradative capacity of cells is compromised.

Medina et al. Page 2

Published as: Dev Cell. 2011 September 13; 21(3): 421–430.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Results and DiscussionTFEB Overexpression Induces Lysosomal Exocytosis

A typical hallmark of lysosomal exocytosis is the translocation of lysosomal membranemarkers to the PM (Reddy et al., 2001; Rodríguez et al., 1997; Yogalingam et al., 2008).TFEB overexpression in mouse embryonic fibroblasts (MEFs) and neuronal stem cells(NSCs) resulted in an increased exposure of the luminal domain of LAMP1, a lysosomalmembrane marker, on the PM (Figure 1A). Consistently, quantitative analysis by flowcytometry (FACs) showed an increase of LAMP1 staining on the PM of TFEB-overexpressing cells (Figure 1B). A direct consequence of lysosomal exocytosis is therelease of lysosomal enzymes into the cell culture medium (Rodríguez et al., 1997).Significantly higher levels of lysosomal hydrolases were detected in the medium of severalcells lines overexpressing TFEB compared with control cells (Figure 1C). The increase oflysosomal enzymes in the medium was not associated with an increase in the levels ofcytosolic lactate dehydrogenase (LDH), thus excluding that the release of lysosomalenzymes was due to cell damage (see Figure S1A available online). Together, these dataindicate that TFEB induces lysosomal exocytosis.

Western blot and FACs analyses revealed an enrichment of LAMP1 on the PM comparedwith total LAMP1 in TFEB-overexpressing cells, suggesting that the elevation of LAMP1on the PM was not a mere consequence of TFEB-mediated expansion of the lysosomalcompartment and of the consequent increase of LAMP1 protein levels (Sardiello et al.,2009), but it reflected an active movement of lysosomes toward the PM (Figures 1D and1E).

To exclude the possibility that TFEB-mediated induction of LAMP1 expression resulted inan overloading of the trans-Golgi network (TGN) with a consequent abnormal sorting ofLAMP1 directly from the TGN to the PM, we performed experiments using thetemperature-sensitive VSVG-GFP protein (Figure S1B). This approach is generally used tomonitor transport through the TGN-to-PM segment of the secretory pathway (Matlin andSimons, 1984). At 20°C, both control and stable HeLa cells expressing TFEB (CF7 cells)exhibited the VSVG protein trapped in the Golgi area, while the staining for LAMP1decorated a different compartment with a typical spotty lysosome-like pattern (Figure S1B,left panel arrows). This indicates that the exposure to the low temperature did not cause theaccumulation of a detectable amount of newly synthesized LAMP1 within the Golgi, despiteoverexpression of TFEB. The temperature shift from 20°C to 37°C triggered VSVG exitfrom the Golgi toward the PM (Figure S1B, middle panel arrows), while again no VSVGoverlap with LAMP1 was detected within intracellular structures directed toward the cellsurface. Finally, blocking fusion of Golgi-derived VSVG transport carriers with the PM(Polishchuk et al., 2004; see also Experimental Procedures) by the use of 0.5% tannic acid(TA) resulted in the accumulation of VSVG transport carriers near the PM (Figure S1B,right panel arrows), as expected. However, no LAMP1 was detected within such VSVG-positive structures, suggesting that LAMP1 was not delivered to the PM from the Golgi inCF7 cells. These results clearly indicate that LAMP1 does not escape the lysosomal route inTFEB-overexpressing cells and support our conclusion that the increase of LAMP1 on thePM is due to enhanced lysosomal exocytosis.

TFEB Overexpression Enhances Lysosomal Predocking to the PM and ElevatesIntracellular Ca2+

Induction of lysosomal exocytosis involves the recruitment of lysosomes to the PM (Blottand Griffiths, 2002). TFEB overexpression resulted in an increased motility of lysosomes(Figure 2A; Movies 1 and 2). The impact of TFEB overexpression on lysosomal recruitment

Medina et al. Page 3

Published as: Dev Cell. 2011 September 13; 21(3): 421–430.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

was investigated using the immuno-EM approach. To this end, control HeLa and CF7 cellswere labeled with an antibody against LAMP1 and prepared for ultrastructural analysis, aspreviously described (Polishchuk et al., 2003). In thin sections, LAMP1 was detected atvacuolar structures that contained multiple intralumenal vesicles and/or electron-densematerial. These structures could be identified as lysosomes on the basis of theirultrastructural features (Figure 2B, arrows and arrowheads). In HeLa control cells,lysosomes were distributed randomly throughout the cells, often at a substantial distancefrom the cell surface (Figure 2B, upper left panel and arrows). To reveal the pool oflysosomes that undergo exocytosis, we incubated cells for 1 hr in the medium containing0.5% TA, which is widely used as a tool to block fusion of the different exocytic organelleswith the PM (Newman et al., 1996; Polishchuk et al., 2004). After the incubation with TA,the cells were fixed and processed for immuno-EM. Exposure of control HeLa cells to TAinduced a moderate increase in LAMP1-positive structures located very close (within200-400 nm) to the cell surface (Figure 2B, upper right panel, arrowhead and frequencyplot), indicating that only a small proportion of the lysosomes underwent exocytosis over thetreatment period. In contrast, CF7 cells exhibited numerous lysosomes in the closeproximity of the PM (Figure 2B, middle and lower left panels, arrowheads, and frequencyplot), even in the absence of TA. Block of fusion by TA further increased the pool of theLAMP1-positive structures near the cell surface, with many of them being almost attachedto the PM (Figure 2B, middle and lower right panels, arrowheads, and frequency plot). It isimportant to note that the vast majority of such LAMP1 structures in TA-treated CF7 cellsresembled lysosomes rather than Golgi-derived vesicles, in terms of both size andultrastructural features. Notably, the magnitude of such an increase in the frequency of PM-associated lysosomes upon the block of fusion was significantly higher in TFEB-overexpressing cells compared with the control HeLa line (Figure 2B, frequency plot).

We further confirmed the latter results by loading control and TFEB-overexpressing HeLacells with BSA gold (5 nm) for 30 min and chasing them in BSA gold–free medium for120 min (Figure S2). In these conditions, all of the endosomal structures that present BSAgold are late endosomes/lysosomes (Parton et al., 1989). In CF7 cells, the distance betweenthe endo/lysosomes structures and the PM was strongly reduced (mean distance HeLacontrol 2387nm versus 438nm in HeLa CF7) when the total number of endo/lysosomes (58versus 73) was increased (Figure S2). Notably, fusion of lysosomes with the PM and releaseof BSA-stained lysosomes in the extracellular space were detected in TFEB-overexpressingcells (Figure S2, lower right panel). Thus, taken together, these findings indicate that TFEBoverexpression significantly stimulates the recruitment of lysosomes to the PM, a step that isrequired for lysosomal exocytosis.

Previous studies demonstrated the requirement Ca2+ elevation for the fusion of lysosomeswith the PM but not for the recruitment of lysosomes to the cell surface (Andrews, 2000;Jaiswal et al., 2002; Rodríguez et al., 1997). Confocal imaging showed an elevation ofintracellular Ca2+ levels in TFEB-GFP-positive cells (Figure 3A), consistent with the role ofTFEB in the induction of lysosomal exocytosis. To quantify the extent of TFEB-mediatedCa2+ elevation, we measured the cytoplasmic concentration of Ca2+ by a ratiometric assay(Kao et al., 2010). We observed a very significant increase in Ca2+ concentration in TFEB-overexpressing CF7 HeLa cells compared with control cells (91.4 nM ± 1.1 and 67.5 nM ±0.8, respectively) (Figure 3B). Moreover, depletion of Ca2+ by the use of Ca2+ chelators inTFEB-overexpressing cells completely inhibited the appearance of LAMP1 on PM(Figure 3C). As expected, TFEB-mediated recruitment of lysosomes to the PM, as measuredby EM analysis of the distance of the endo-lysosomes structures to the PM in BSA gold-loaded cells, was not affected by depletion of Ca2+ in CF7 cells (Figure 3D).

Medina et al. Page 4

Published as: Dev Cell. 2011 September 13; 21(3): 421–430.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Together, these results indicate that in addition to promoting lysosomal recruitment to thePM, TFEB induces lysosomal exocytosis by enhancing Ca2+-mediated fusion of lysosomeswith the PM.

TFEB Releases Ca2+ from Acidic Stores through the Activation of Mucolipin-1Mucolipin 1 (MCOLN1) is a lysosomal nonselective cation channel that is mutated inmucolipidosis IV (MLIV), a severe type of LSD (Bargal et al., 2000,2002; Bassi et al.,2000). Previous studies reported that lysosomal exocytosis is reduced in MLIV cells(LaPlante et al., 2006) and increased in cells expressing gain-of-function MCOLN1mutations (Dong et al., 2009). These observations suggest that MCOLN1, upon a propercellular stimulation, mediates intralysosomal Ca2+ release to trigger lysosomal exocytosis.In a separate study, we demonstrated that the gene encoding MCOLN1 is regulated byTFEB (Palmieri et al., 2011), making it an appealing candidate to mediate TFEB effects onlysosomal exocytosis. Remarkably, TFEB-mediated increase of intracellular Ca2+ wasblocked in CF7 cells and in NSCs overexpressing TFEB by transient silencing of MCOLN1with a pool of four fluorescently tagged vectors carrying specific MCOLN1-shRNAs(Figures 4A and 4B). Confocal Ca2+ imaging of HeLa cells stably depleted for MCOLN1using a single shRNA vector (HeLashMCOLN1) failed to detect an elevation of intracellularCa2+ in TFEB-overexpressing cells (Figure 4C). Consistently, Ca2+ levels were not affectedby TFEB overexpression in human MLIV cells that carry loss of function mutations ofMCOLN1 (Figure 4D). In addition, stable depletion of MCOLN1 in HeLa cells (Figure 4E)or transient depletion by a pool of shRNA vectors against MCOLN1 (Figure 4F) impairedTFEB-mediated fusion of LAMP1 with the PM, and inhibited the secretion of lysosomalenzymes into the medium (Figure 4G). Similar results were obtained by transient expressionof individual shRNAs against MCOLN1 (Figure S3A). To exclude off-target effects ofshRNA contructs, we performed a rescue experiment using a vector carrying an shRNA-resistant mutant form of MCOLN1 (MCOLN1 mut) (Vergarajauregui and Puertollano,2006). Overexpression of MCOLN1 mut in HeLashMCOLN1 significantly recovered theappearance of surface of LAMP1 after TFEB overexpression (Figure S3B).

The lysosomal localization of MCOLN1 and its channel properties suggest that the elevationof intracellular Ca2+ levels induced by TFEB overexpression is due to the release of Ca2+

from lysosomal stores through MCOLN1. To test this hypothesis, we measured cytosolicCa2+ levels in TFEB-overexpressing cells by confocal imaging time-lapse after inducingacidic Ca2+ release with Bafilomycin A, an inhibitor of the vacuolar H+-ATPase, which isresponsible for the proton gradient driving lysosomal Ca2+ uptake (Christensen et al., 2002).The results clearly showed a local increase of cytosolic Ca2+ levels in the stable HeLa-CF7clone, in the transiently transfected TFEB-GFP overexpressing HeLa cells and in HeLa cellsinfected with an adenovirus encoding MCOLN1 (Figure S3C), compared with control HeLacells. Conversely, HeLa cells stably depleted of MCOLN1 were not able to increase Ca2+

levels after TFEB-GFP overexpression (Figure S3C). Notably, both TFEB and MCOLN1overexpressing cells were less sensitive to Bafilomycin A1-dependent depletion oflysosomal Ca2+ compared with HeLa control cells (Figure S3D). Together these datastrongly suggest that MCOLN1-dependent release of Ca2+ from acidic Ca2+ stores plays amajor role in TFEB-mediated lysosomal exocytosis.

TFEB-Mediated Lysosomal Exocytosis Promotes Cellular Clearance in Cellular Models ofLSDs

LSDs are monogenic disorders characterized by the progressive accumulation of undegradedmaterial in the lysosomes due to the deficiency of specific lysosomal proteins (Suzuki, 2002;Neufeld, 1979-1980). This is often associated with intracellular trafficking abnormalitiesand with a block of the autophagic pathway, leading to a progressive multisystemic

Medina et al. Page 5

Published as: Dev Cell. 2011 September 13; 21(3): 421–430.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

phenotype that is often associated with neurodegeneration (Ballabio and Gieselmann, 2009;Fraldi et al., 2010; Futerman and van Meer, 2004; Platt and Walkley, 2004; Settembre et al.,2008). Interestingly, a previous study reported induction of lysosomal exocytosis inmetachromatic leukodystrophy, a neurodegenerative form of LSD (Klein et al., 2005). Wetested whether TFEB-induced lysosomal exocytosis could be exploited to induce cellularclearance in multiple sulfatase deficiency (MSD) and mucopolysaccharidosis type IIIA(MPS-IIIA), two different types of LSDs with a severe neurodegenerative outcome due tothe progressive accumulation of glycosaminoglycans (GAGs) and severe vacuolization inpostmitotic neurons and glial cells (Platt and Walkley, 2004; Futerman and van Meer, 2004).

Overexpression of TFEB in MEFs derived from the murine models of MSD and MPSIIIAresulted in an even more significant increase of LAMP1 on the PM (Figure S4A) and oflysosomal enzymes into the culture medium (Figure S4B), compared with that observed incontrol cells overexpressing TFEB. This indicates that LSD cells efficiently respond toTFEB-mediated induction of lysosomal exocytosis. Therefore, we evaluated the effect ofTFEB overexpression on the clearance of GAGs in glia-differentiated neuronal stem cells(NSCs) isolated from mouse models of MSD and MPSIIIA. TFEB overexpression resultedin a striking reduction of alcian blue-stained GAGs in both MSD and MPSIIIA NSC-derivedglial cells (Figure 5A). The latter result was further confirmed by pulse-and-chaseexperiments using H3-glucosamine to label GAGs, showing a significant reduction of thelevels of labeled GAGs after 48 hr of chase in both MSD and MPSIIIA NSC-derived glialcells overexpressing TFEB (Figure 5B). Finally, EM analysis revealed that TFEB-mediatedclearance of GAGs in TFEB-overexpressing MSD and MPS-IIIA cells was associated withboth significant reduction of cellular vacuolization and recovery of normal cellularmorphology (Figure 5C).

The involvement of Ca2+ in TFEB-mediated reduction of pathologic storage was confirmedby the inhibition of GAG clearance after the addition of BAPTA to TFEB overexpressingNSCs (Figure S4C). Consistent with this observation, viral-mediated overexpression ofMCOLN1 in MSD glia-differentiated NSCs reduced GAG accumulation, but to a lesserextent compared with TFEB (Figure S4D), suggesting that Ca2+ elevation is required but notsufficient to mediate TFEB effects on lysosomal exocytosis.

We extended these studies to other types of LSDs associated with the storage of differenttypes of lysosomal substrates. Neuronal ceroid lipofuscinoses (NCL) and glycogenosis arecharacterized by the accumulation of lipofuscin and glycogen, respectively (Beratis et al.,1978; Persaud-Sawin et al., 2007; Saftig, 2003). We found that TFEB overexpressionstrongly reduced lipofuscin autofluorescence in cells from a murine model of a juvenileform of NCL, known as Batten disease, due to mutations of the CLN3 gene (Figure 5D).Similarly, the accumulation of glycogen labeled with the exogenous fluorescent sugar 2-NBDG (Louzao et al., 2008) was significantly reduced upon TFEB overexpression infibroblasts from a patient affected by glycogenosis type II, Pompe disease (Figure 5E).However, TFEB overexpression in mucolipidosis IV due to mutations in the MCOLN1 genefailed to induce cellular clearance, consistent with the involvement of MCOLN1 in TFEB-mediated induction of lysosomal exocytosis and clearance (Figure S4E). These data indicatethat induction of lysosomal exocytosis promotes cellular clearance in diseases due toaccumulation of different types of lysosomal substrates. Notably, all four diseases tested(i.e., MSD, MPS-IIIA, Batten, and NCL) are due to deficiency of proteins whose activity isinvolved in crucial steps of different catabolic pathways. In addition, the cells analyzed werederived from patients and murine models carrying either null mutations or mutations thatseverely inactivate protein function. Therefore, it is unlikely that clearance of lysosomalsubstrates in these cells is due to either the enhancement of the activity of the defectiveenzymes, or to an overall induction of lysosomal catabolic processes, again suggesting that

Medina et al. Page 6

Published as: Dev Cell. 2011 September 13; 21(3): 421–430.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

clearance is mediated by exocytosis. This was confirmed by the observation of an increasein GAG secretion, measured by H3-GAG, in cell culture medium from MSD cells uponTFEB overexpression (Figure 5F).

TFEB Delivery Rescues Tissue Pathology in a Mouse Model of MSDWe tested whether in vivo overexpression of TFEB in a mouse model of MSD, a severe typeof LSD, had similar effects on cellular clearance. To this end, we injected systemically anadeno-associated type 2/9 virus (AAV2/9) that carries TFEB-3xflag into adult MSD mice.One month after injection, several tissues were collected to monitor transduction efficiencyand GAG storage. AAV-mediated TFEB delivery resulted in efficient TFEB transduction(Figure S5) and significant reduction of GAG staining in liver and skeletal muscles, asdetected by alcian blue staining (Figure 6A) and GAG quantification (Figure 6B).Subsequently, we investigated whether TFEB-mediated clearance of GAGs resulted in thereduction of the pathologic hallmarks of MSD, such as macrophage infiltration andapoptosis (Settembre et al., 2007). We found a striking reduction of CD68-positive cells inAAV-TFEB injected MSD mice compared with untreated mice (Figure 6C). Mostimportantly, we also observed a significant reduction of TUNEL-positive cells (Figure 6D).These results indicate that TFEB activation of lysosomal exocytosis reduced both primaryaccumulation of GAGs and secondary pathological processes associated with LSDs such asinflammation and cell death.

ConclusionsThis study demonstrates that lysosomal exocytosis is transcriptionally regulated by TFEB, amaster gene for lysosomal biogenesis and autophagy (Sardiello et al., 2009; Settembre et al.,2011). TFEB modulates lysosomal exocytosis by triggering intracellular Ca2+ elevationthrough the cation-channel MCOLN1. Cells may use this mechanism to coordinatelysosomal catabolic and secretory functions in response to specific stimuli. Our data alsoindicate that lysosomal exocytosis can be exploited to promote cellular clearance inlysosomal storage diseases, suggesting an alternative strategy to treat disorders due tointracellular storage, such as LSDs and common neurodegenerative diseases. Severalapproaches, such as gene delivery, pharmacological induction of TFEB, and target geneactivation, could be exploited to promote cellular clearance in target tissues. These strategieswill have to be tested by long-term in vivo studies in animal models to verify the therapeuticpotential of this discovery.

Experimental ProceduresCell Culture, Transfection, and VSV Infection

HeLa, Cos7 cells, human fibroblasts, and mouse embryonic fibroblasts from mouse modelsof MLIV (S7), MPSIIIA (S7), and MSD were transfected using PolyFect TransfectionReagent (QIAGEN, Hilden, Germany) or lipofectamine 2000 Reagent (Invitrogen, Carlsbad,CA), according to the manufacturer's protocols. TFEB-3xFLAG HeLa stable cell lines (CF7)was previously described (Sardiello et al., 2009). The shMCOLN1 HeLa cell line wasgenerated by infection with MCOLN1 lentiviral shRNA. Neuronal progenitor cells wereisolated from cortices of WT and MSD and MPSIIIA pups (P0) by using standard protocols(see Supplemental Experimental Procedures for details). NSCs were transfected by usingnucleofection (Amaxa, Lonza, Walkersville, MD). Infection of control and CF7 HeLa cellswith tsO45 strain of VSV was executed as described (Polishchuk et al., 2003).

Medina et al. Page 7

Published as: Dev Cell. 2011 September 13; 21(3): 421–430.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Tannic Acid TreatmentTo prevent fusion of exocytic organelles with PM, control and CF7 HeLa cells wereincubated for 1 hr at 37°C in 20 mM HEPES-buffered DMEM supplemented with 0.5%tannic acid (TA). At the end of the incubation, TA-containing medium was removed andcells were fixed and processed for immuno-EM (see below). For immunofluorescense (IF)experiments VSV-infected cells were exposed subsequently to 40°C and 20°C temperatureblocks to accumulate VSVG first in the ER and then in the Golgi. Then cells were shifted to37°C to activate VSVG transport from the Golgi either in the presence or in the absence of0.5% TA. One hour after the temperature shift to 37°C, the cells were fixed and processedfor (IF) labeling (see below).

Surface LAMP1 AnalysisCells (collected in phosphate-buffered saline [PBS] or grown on coverslips) were incubatedwith anti-rat LAMP1-1DB4 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4°C for30 min. Cells were washed in PBS and fixed in 2% paraformaldehyde (PFA). Anti-LAMP1-1D4B–treated cells were further incubated with Alexa-594 conjugated anti–ratsecondary antibodies (Molecular Probes, Invitrogen) for 30 min at room temperature.Finally, cells were analyzed on a confocal microscope (LSM510; Carl Zeiss, Inc.,Thornwood, NY) equipped with Plan-Neofluar 63x immersion objective or on a FACs AriaFlow Cytometer (Becton Dickinson & Co., Mountain View, CA).

Immunofluorescence and Confocal ImagingTransfected cells were grown on glass coverslips fixed with 4% paraformaldehyde (PFA,Sigma, St. Louis, MO), quenched with 50 mM NH4Cl, and permeabilized in blocking buffer(0.05% saponin/0.2% BSA in PBS/Ca/Mg). Coverslips were then incubated with appropriateprimary antibodies described in Supplemental Experimental Procedures.

Tissues were fixed with 4% PFA and then subjected to a sucrose gradient (from 10 to 30%),before OCT embedding. Immunofluorescence analyses were performed on 10-μm-thickserial cryosections using appropriate primary antibodies (see Supplemental ExperimentalProcedures for details). The secondary antibodies were purchased from Molecular Probes(Invitrogen).

Images were taken on Vectashield mounted coverslips or sections using a confocalmicroscope (LSM510; Carl Zeiss, Inc.) equipped with a Plan-Neofluar 63x immersionobjective (Carl Zeiss, Inc.).

Immunoblot and PM IsolationPM proteins were isolated using the Cell Surface Protein Isolation kit (Pierce, ThermoFischer Scientific Inc, Rockford, IL). Immunoblots were performed using standard protocolsand primary antibodies as described in Supplemental Experimental Procedures. Proteinswere quantified by the Bradford method.

Electron Microscopy and Immuno-Gold AnalysisControl and TFEB-overexpressing cells (both intact and TA-treated) were washed in PBS,and fixed in 0.05% glutaraldehyde dissolved in 0.2 M HEPES buffer (pH 7.4) for 30 min atroom temperature. The cells were then postfixed for 2 hr in OsO4. After dehydration ingraded series of ethanol, the cells were embedded in Epon 812 (FLUKA) and polymerized at60°C for 72 hr. Thin sections were cut at the Leica EM UC6 and counterstained with uranylacetate and lead citrate.

Medina et al. Page 8

Published as: Dev Cell. 2011 September 13; 21(3): 421–430.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

For immuno-gold HeLa and CF cells (both intact and TA-treated) were fixed with a mixtureof 4% paraformaldehyde and 0.05% glutaraldehyde, labeled with a monoclonal antibodyagainst LAMP1 according the gold-enhance protocol, embedded in Epon-812, and cut asdescribed previously (Polishchuk et al., 2003).

EM images were acquired from thin sections using a Philips Tecnai-12 electron microscopeequipped with an ULTRA VIEW CCD digital camera (Philips, Eindhoven, TheNetherlands).

Quantification of vacuolization was performed using the AnalySIS software (Soft ImagingSystems GmbH, Muenster, Germany).

Evaluation of lysosome distance from the PM was done in EM images using the iTEMsoftware (Soft Imaging Systems GmbH).

Selection of cells for quantification was based on their suitability for stereologic analysis,i.e., only cells sectioned through their central region (detected on the basis of the presence ofGolgi membranes) were analyzed.

BSA-GoldElectron microscopy and BSA immunogold were performed on HeLa control cells andoverexpressing TFEB (CF7) according to the protocol described previously (Puri, 2009).See Supplemental Experimental Procedures for details.

Calcium Measurements by Confocal ImagingTFEB-GFP-transfected cells were treated with FuraRed and analyzed according to theprotocol described previously (Luciani et al., 2010).

Measurements of Ca2+ Concentration by Ratiometric AssaysHeLa cells were infected with either control adenovirus (Ad. Null) or adenovirus expressingTFEB (Ad. TFEB-FLAG). After 24 hr, cells were loaded with 20 μM Fura-2AM(Invitrogen) for 1 hr at 37°C. After washing, pseudocolor ratiometric images were acquired.The acquisition and analysis of the images were done using MetaFluor software (MolecularDevices, Sunnyvale, CA). Fura-2AM ratios (F340/F380) from cells infected with Ad. Null(n = 2584; where n represent the number of cells) or Ad. TFEB-FLAG (n = 2238) were usedto calculate the cytoplasmic calcium concentration by using Fura-2AM calibration curves(see Supplemental Experimental Procedures for details).

Flow Cytometric Calcium Flux AssayThe assay was performed following the protocol described by Schepers et al. (2009) withsome modifications as described in the Supplemental Experimental Procedures.

Enzymatic ActivitiesEnzyme activities were measured using the appropriate fluorimetric or colorimetricsubstrates as described in Supplemental Experimental Procedures.

Live ImagingLive imaging was performed on WT and CF7 cells using a Carl Zeiss microscope (LSM710) equipped with a perfusion, open and closed cultivation chamber fitted with aTempcontrol and CO2-control device (Y module; Carl Zeiss). Acquisition was performedusing ZEISS LSM 710 software. The trajectory and the mean velocity of vesicles were

Medina et al. Page 9

Published as: Dev Cell. 2011 September 13; 21(3): 421–430.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

calculated by IMAGEJ using the LSM reader and particle tracking plug-ins. For details, seeSupplemental Experimental Procedures.

GAG AnalysisGAGs were radiolabeled using H3-glucosamine hydrochloride (Perkin Elmer, 37.75 Ci/mmol, Boston, MA). Quantitative GAG analysis was performed according protocolsdescribed in (de Jong et al., 1989).

Alcian Blue StainingSections of paraffin-embedded tissues were stained with 1% Alcian blue (Sigma-Aldrich)and counterstained with Nuclear-Fast red (Sigma-Aldrich). See Supplemental ExperimentalProcedures for details.

In Situ Detection of Apoptotic CellsTUNEL was performed by using the ApopTag Peroxidase In Situ Apoptosis Detection kit(Oncor, Gaithersburg, MD).

ReferencesAndrews N.W. Regulated secretion of conventional lysosomes. Trends Cell Biol.. 2000; 10:316–321.

[PubMed: 10884683]Andrews N.W. Membrane repair and immunological danger. EMBO Rep.. 2005; 6:826–830.

[PubMed: 16138093]Ballabio A. Gieselmann V. Lysosomal disorders: from storage to cellular damage. Biochim. Biophys.

Acta. 2009; 1793:684–696. [PubMed: 19111581]Bargal R. Avidan N. Ben-Asher E. Olender Z. Zeigler M. Frumkin A. Raas-Rothschild A. Glusman G.

Lancet D. Bach G. Identification of the gene causing mucolipidosis type IV. Nat. Genet.. 2000;26:118–123. [PubMed: 10973263]

Bargal R. Goebel H.H. Latta E. Bach G. Mucolipidosis IV: novel mutation and diverse ultrastructuralspectrum in the skin. Neuropediatrics. 2002; 33:199–202. [PubMed: 12368990]

Bassi M.T. Manzoni M. Monti E. Pizzo M.T. Ballabio A. Borsani G. Cloning of the gene encoding anovel integral membrane protein, mucolipidin-and identification of the two major founder mutationscausing mucolipidosis type IV. Am. J. Hum. Genet.. 2000; 67:1110–1120. [PubMed: 11013137]

Beratis N.G. LaBadie G.U. Hirschhorn K. Characterization of the molecular defect in infantile andadult acid alpha-glucosidase deficiency fibroblasts. J. Clin. Invest.. 1978; 62:1264–1274. [PubMed:34626]

Blott E.J. Griffiths G.M. Secretory lysosomes. Nat. Rev. Mol. Cell Biol.. 2002; 3:122–131. [PubMed:11836514]

Bossi G. Griffiths G.M. CTL secretory lysosomes: biogenesis and secretion of a harmful organelle.Semin. Immunol.. 2005; 17:87–94. [PubMed: 15582491]

Christensen K.A. Myers J.T. Swanson J.A. pH-dependent regulation of lysosomal calcium inmacrophages. J. Cell Sci.. 2002; 115:599–607. [PubMed: 11861766]

de Jong J.G. Wevers R.A. Laarakkers C. Poorthuis B.J. Dimethylmethylene blue-basedspectrophotometry of glycosaminoglycans in untreated urine: a rapid screening procedure formucopolysaccharidoses. Clin. Chem.. 1989; 35:1472–1477. [PubMed: 2503262]

Dehay B. Bové J. Rodríguez-Muela N. Perier C. Recasens A. Boya P. Vila M. Pathogenic lysosomaldepletion in Parkinson's disease. J. Neurosci.. 2010; 30:12535–12544. [PubMed: 20844148]

Suzuki, K. Lysosomal diseases. In: Graham, D.I.; Lantos, P.L., editors. Greenfield's Neuropathology.Arnold Hodder Publication; London: 2002. p. 653-735.

Dong X.P. Wang X. Shen D. Chen S. Liu M. Wang Y. Mills E. Cheng X. Delling M. Xu H. Activatingmutations of the TRPML1 channel revealed by proline-scanning mutagenesis. J. Biol. Chem..2009; 284:32040–32052. [PubMed: 19638346]

Medina et al. Page 10

Published as: Dev Cell. 2011 September 13; 21(3): 421–430.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Fraldi A. Annunziata F. Lombardi A. Kaiser H.J. Medina D.L. Spampanato C. Fedele A.O. PolishchukR. Sorrentino N.C. Simons K. Ballabio A. Lysosomal fusion and SNARE function are impaired bycholesterol accumulation in lysosomal storage disorders. EMBO J.. 2010; 29:3607–3620.[PubMed: 20871593]

Futerman A.H. van Meer G. The cell biology of lysosomal storage disorders. Nat. Rev. Mol. CellBiol.. 2004; 5:554–565. [PubMed: 15232573]

Jaiswal J.K. Andrews N.W. Simon S.M. Membrane proximal lysosomes are the major vesiclesresponsible for calcium-dependent exocytosis in nonsecretory cells. J. Cell Biol.. 2002; 159:625–635. [PubMed: 12438417]

Kao J.P. Li G. Auston D.A. Practical aspects of measuring intracellular calcium signals withfluorescent indicators. Methods Cell Biol.. 2010; 99:113–152. [PubMed: 21035685]

Klein D. Büssow H. Fewou S.N. Gieselmann V. Exocytosis of storage material in a lysosomaldisorder. Biochem. Biophys. Res. Commun.. 2005; 327:663–667. [PubMed: 15649398]

Kornfeld S. Mellman I. The biogenesis of lysosomes. Annu. Rev. Cell Biol.. 1989; 5:483–525.[PubMed: 2557062]

LaPlante J.M. Sun M. Falardeau J. Dai D. Brown E.M. Slaugenhaupt S.A. Vassilev P.M. Lysosomalexocytosis is impaired in mucolipidosis type IV. Mol. Genet. Metab.. 2006; 89:339–348.[PubMed: 16914343]

Louzao M.C. Espiña B. Vieytes M.R. Vega F.V. Rubiolo J.A. Baba O. Terashima T. Botana L.M.“Fluorescent glycogen” formation with sensibility for in vivo and in vitro detection. Glycoconj. J..2008; 25:503–510. [PubMed: 17973187]

Luciani A. Villella V.R. Esposito S. Brunetti-Pierri N. Medina D. Settembre C. Gavina M. Pulze L.Giardino I. Pettoello-Mantovani M. Defective CFTR induces aggresome formation and lunginflammation in cystic fibrosis through ROS-mediated autophagy inhibition. Nat. Cell Biol.. 2010;12:863–875. [PubMed: 20711182]

Matlin K.S. Simons K. Sorting of an apical plasma membrane glycoprotein occurs before it reaches thecell surface in cultured epithelial cells. J. Cell Biol.. 1984; 99:2131–2139. [PubMed: 6501415]

Neufeld E.F. Lessons from genetic disorders of lysosomes. Harvey Lect.. 1979-1980; 75:41–60.[PubMed: 122311]

Newman T.M. Tian M. Gomperts B.D. Ultrastructural characterization of tannic acid-arresteddegranulation of permeabilized guinea pig eosinophils stimulated with GTP-gamma-S. Eur. J. CellBiol.. 1996; 70:209–220. [PubMed: 8832205]

Palmieri M. Impey S. Kang H. di Ronza A. Pelz C. Sardiello M. Ballabio A. Characterization of theCLEAR network reveals an integrated control of cellular clearance pathways. Hum. Mol. Genet..2011 in press. Published online July 13, 2011.

Parton R.G. Prydz K. Bomsel M. Simons K. Griffiths G. Meeting of the apical and basolateralendocytic pathways of the Madin-Darby canine kidney cell in late endosomes. J. Cell Biol.. 1989;109:3259–3272. [PubMed: 2557351]

Persaud-Sawin D.A. Mousallem T. Wang C. Zucker A. Kominami E. Boustany R.M. Neuronal ceroidlipofuscinosis: a common pathway? Pediatr. Res.. 2007; 61:146–152. [PubMed: 17237713]

Platt, F.M.; Walkley, S.U. Oxford University Press; New York: 2004. Lysosomal Disorders of theBrain.

Polishchuk E.V. Di Pentima A. Luini A. Polishchuk R.S. Mechanism of constitutive export from thegolgi: bulk flow via the formation, protrusion, and en bloc cleavage of large trans-golgi networktubular domains. Mol. Biol. Cell. 2003; 14:4470–4485. [PubMed: 12937271]

Polishchuk R. Di Pentima A. Lippincott-Schwartz J. Delivery of raft-associated, GPI-anchoredproteins to the apical surface of polarized MDCK cells by a transcytotic pathway. Nat. Cell Biol..2004; 6:297–307. [PubMed: 15048124]

Puri C. Loss of myosin VI no insert isoform (NoI) induces a defect in clathrin-mediated endocytosisand leads to caveolar endocytosis of transferrin receptor. J. Biol. Chem.. 2009; 284:34998–35014.[PubMed: 19840950]

Reddy A. Caler E.V. Andrews N.W. Plasma membrane repair is mediated by Ca(2+)-regulatedexocytosis of lysosomes. Cell. 2001; 106:157–169. [PubMed: 11511344]

Medina et al. Page 11

Published as: Dev Cell. 2011 September 13; 21(3): 421–430.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Rodríguez A. Martinez I. Chung A. Berlot C.H. Andrews N.W. cAMP regulates Ca2+-dependentexocytosis of lysosomes and lysosome-mediated cell invasion by trypanosomes. J. Biol. Chem..1999; 274:16754–16759. [PubMed: 10358016]

Rodríguez A. Webster P. Ortego J. Andrews N.W. Lysosomes behave as Ca2+-regulated exocyticvesicles in fibroblasts and epithelial cells. J. Cell Biol.. 1997; 137:93–104. [PubMed: 9105039]

Saftig, P. Springer; New York: 2003. Medical Intelligence Unit: Lysosomes.Sardiello M. Palmieri M. di Ronza A. Medina D.L. Valenza M. Gennarino V.A. Di Malta C. Donaudy

F. Embrione V. Polishchuk R.S. A gene network regulating lysosomal biogenesis and function.Science. 2009; 325:473–477. [PubMed: 19556463]

Schepers E. Glorieux G. Dhondt A. Leybaert L. Vanholder R. Flow cytometric calcium flux assay:evaluation of cytoplasmic calcium kinetics in whole blood leukocytes. J. Immunol. Methods.2009; 348:74–82. [PubMed: 19616551]

Settembre C. Annunziata I. Spampanato C. Zarcone D. Cobellis G. Nusco E. Zito E. Tacchetti C.Cosma M.P. Ballabio A. Systemic inflammation and neurodegeneration in a mouse model ofmultiple sulfatase deficiency. Proc. Natl. Acad. Sci. USA. 2007; 104:4506–4511. [PubMed:17360554]

Settembre C. Fraldi A. Jahreiss L. Spampanato C. Venturi C. Medina D. de Pablo R. Tacchetti C.Rubinsztein D.C. Ballabio A. A block of autophagy in lysosomal storage disorders. Hum. Mol.Genet.. 2008; 17:119–129. [PubMed: 17913701]

Settembre C. Di Malta C. Polito V.A. Garcia Arencibia M. Vetrini F. Erdin S. Erdin S.U. Huynh T.Medina D. Colella P. TFEB links autophagy to lysosomal biogenesis. Science. 2011; 332:1429–1433. [PubMed: 21617040]

Tucker W.C. Weber T. Chapman E.R. Reconstitution of Ca2+-regulated membrane fusion bysynaptotagmin and SNAREs. Science. 2004; 304:435–438. [PubMed: 15044754]

Vergarajauregui S. Puertollano R. Two di-leucine motifs regulate trafficking of mucolipin-1 tolysosomes. Traffic. 2006; 7:337–353. [PubMed: 16497227]

Yogalingam G. Bonten E.J. van de Vlekkert D. Hu H. Moshiach S. Connell S.A. d'Azzo A.Neuraminidase 1 is a negative regulator of lysosomal exocytosis. Dev. Cell. 2008; 15:74–86.[PubMed: 18606142]

Supplemental InformationRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsWe thank P. Barba, J. Cancino, P. Colella, F. Donaudy, L. Pisapia, and A. Luciani for technical assistance. Wethank E. Neufeld, B. Davidson, N. Andrews, C. Tam, and P. De Camilli for helpful discussions. We would like toacknowledge Elena Polishchuk and Anastasia Egorova for help in execution of microscopy experiments as well asTelethon Electron Microscopy Core Facility (IBP, CNR, Naples) and Integrated Microscopy Facility (IGB, CNR,Naples) for EM support. We also thank B. Lelouvier and the NHLBI Light Microscopy Core Facility for their helpwith the calcium experiments. We also thank G. Diez-Roux, G. Parenti, A. Luini, and A. De Matteis for commentson the manuscript. We acknowledge the support of the Italian Telethon Foundation (D.L.M., A.F., V.B., F.A.,G.M., C.S., A.P., and A.B); the European Research Council Advanced Investigator grant number # 250154 (A.B);the European Commission under the FP7 EUCLYD project (Grant No. HEALTH-2007-A-201678); the BeyondBatten Disease Foundation (M.S and A.B); MPS Society (D.L.M, A.F.and A.B.). J.M. and R.P. are supported bythe Intramural Research Program of the NIH, National Heart, Lung, and Blood Institute (NHLBI). In addition, wewould like to thank the TIGEM AAV vector core for virus production.

Medina et al. Page 12

Published as: Dev Cell. 2011 September 13; 21(3): 421–430.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Figure 1.TFEB Overexpression Induces Lysosomal Exocytosis(A) Confocal microscopy images showing the exposure of LAMP1 on the PM in bothnonpermeabilized NSC cells and MEFs transfected with either a bicystronic plasmidexpressing TFEB-GFP or with an empty vector. LAMP1 was detected using an antibodyagainst its luminal portion (LAMP1-1DB4). TFEB-transfected cells were localized by theexpression of GFP; nontransfected cells are indicated by asterisks.(B) Quantitative analysis by flow cytometry of LAMP1 levels on the PM in both NSCs andMEFs that express either a bi-cystronic TFEB-GFP plasmid or GFP. Bars represent the foldincrease of LAMP1 fluorescence on PM in TFEB-transfected versus GFP-transfected(control) cells.(C) TFEB overexpression increases the release of lysosomal enzymes in the culture mediumof MEFs, NSCs, HeLa, and COS-7 cells. Activities of lysosomal enzymes acid phosphatase,β-galactosidase, and β-hexosaminidase were determined in the culture medium and in cells

Medina et al. Page 13

Published as: Dev Cell. 2011 September 13; 21(3): 421–430.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

transfected with either an empty vector or with a TFEB-expression vector. The figure showspercentages of enzyme activities released compared with total activities.(D) Representative immunoblots showing LAMP1 levels in both total lysates and enrichedPM extracts from MEFs transfected with either TFEB or with an empty vector. Results werenormalized using an antibody against actin and the PM protein β1-integrin, respectively. Thehistogram shows the quantification of LAMP1 detected by the immunoblot.(E) Total and surface LAMP1 levels after TFEB overexpression were analyzed by flowcytometry analyses using the LAMP1-1DB4 and the LAMP1-L1418 antibodies that are ableto detect PM- and intracellular-LAMP1, respectively. Data represent mean ± SEM; ∗p <0.05 (B, C, D, and E). Scale bar represents 10 μm (A).

Medina et al. Page 14

Published as: Dev Cell. 2011 September 13; 21(3): 421–430.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Figure 2.TFEB Enhances Trafficking and PM Proximity of Lysosomes(A) TFEB enhances the movement of lysosomes to PM. Merged time-lapse frames ofcontrol and stable TFEB-overexpressing HeLa cells (HeLa-CF7) transfected with a LAMP1-GFP vector showing the trajectories of lysosomes. The mean velocity was calculated usingIMAGEJ software (see Experimental Procedures for details).(B) Immuno-EM reveals increase in lysosomal exocytosis upon overexpression of TFEB.Control and stable TFEB-overexpressing HeLa cells (HeLa-CF7) were fixed directly or60 min after addition of 0.5% TA to the medium (see Experimental Procedures). Then cellswere labeled with antibodies against LAMP1 and prepared for immuno-EM (seeSupplemental Experimental Procedures). In control HeLa cells, lysosomes decorated byLAMP1 are often detected at a substantial distance from the cell surface (arrows), whiletreatment with TA only slightly increases the frequency of lysosomes that are close to thePM (arrowheads). TFEB-overexpressing cells (CF7) contain numerous lysosomes located

Medina et al. Page 15

Published as: Dev Cell. 2011 September 13; 21(3): 421–430.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

near the cell surface (arrowheads), although several LAMP1-positive structures could bedetected toward the cell interior as well (arrow). TA further induced accumulation oflysosomes in subsurface area of TFEB-overexpressing cells with many lysosomes almosttouching the PM (arrows). The distance between lysosomes and PM was estimated in thinsections as described in Experimental Procedures in control and TFEB-overexpressing cellsincubated with or without TA (100 lysosomes were counted for each condition). Thenumber of lysosomes, as a function of their distance from the cell surface, was counted over200 nm intervals (100 lysosomes were counted for each experimental condition). Scale barsrepresent 10 μm (A) and 350 nm (B). Data represent mean ± SEM; ∗p < 0.05 (A).

Medina et al. Page 16

Published as: Dev Cell. 2011 September 13; 21(3): 421–430.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Figure 3.TFEB-Mediated Induction of Lysosomal Exocytosis Is Associated with Intracellular Ca2+

Release(A) Analysis of intracellular Ca2+ by confocal microscopy of HeLa cells transfected with abi-cystronic TFEB-GFP construct. Data are displayed as the percentage of cells with Ca2+

response compared with the nontransfected cells.(B) TFEB overexpression elevates cytosolic Ca2+ levels. HeLa cells were infected witheither control adenovirus (Ad. Null) or adenovirus expressing TFEB (Ad. TFEB-FLAG) andloaded with Fura-2AM. Cytosolic Ca2+ concentration was calculated as described inSupplemental Experimental Procedures.(C) TFEB-mediated lysosomal exocytosis is Ca2+-dependent. MEFs and HeLa cellstransfected with either TFEB or an empty vector were treated with either the Ca2+ ionophoreionomycin (10 micromolar) or the membrane-permeable Ca2+ chelator BAPTA-AM(30 micromolar), or both. Mean values of cell surface LAMP1 fluorescence were quantifiedby flow cytometry.(D) Ca2+ depletion in HeLa-overexpressing TFEB (HeLa CF7) does not affectendolysosomes' distance to the PM. HeLa q < CF7 loaded with BSA gold, following thesame protocol described in (B), were treated with the Ca2+ chelator BAPTA-AM(30 micromolar) for 30 min before the fixation. Morphometric analysis was performed tovisualize the distance of endo-lysosomes to the PM. Scale bar represents 10 μm (A). Datarepresent mean ± SEM; ∗p < 0.05 (A, B, and C).

Medina et al. Page 17

Published as: Dev Cell. 2011 September 13; 21(3): 421–430.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Figure 4.TFEB Elevates Intracellular Ca2+ Levels through the Activation of MCOLN1(A) Flow cytometric Ca2+ flux assay of stable TFEB-transfected HeLa (CF7) and controlcells transfected with a vector containing a scramble shRNA or a specific shRNA againstMCOLN1. Data represent the mean values of the Fluo3/FuraRed ± SD ratio.(B) Flow-cytometry Ca2+ flux assay in NSCs transfected with a control vector (CTRL), avector containing a scramble shRNA plus TFEB plasmid, and a vector containing a specificshRNA against MCOLN1 plus TFEB plasmid. Ca2+ was determined in resting conditionand after stimulation with ionomycin (10 micromolar).(C and D) Analyses of intracellular Ca2+ by confocal microscopy of (C) a stable HeLa cloneexpressing specific shRNA against MCOLN1 (HeLa shMCOLN1) and (D) human MLIVfibroblasts transfected with a bi-cystronic TFEB-GFP construct. Data are displayed as thepercentage of cells with Ca2+ response compared with nontransfected cells.

Medina et al. Page 18

Published as: Dev Cell. 2011 September 13; 21(3): 421–430.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

(E) Flow-cytometry analysis of LAMP1 on the PM of HeLashMCOLN1 cells transfected withTFEB.(F) Silencing of MCOLN1 reduces TFEB-mediated translocation of LAMP1 to the PM.Representative confocal microscopy images of nonpermeabilized wild-type MEFstransfected with either a bicystronic TFEB-GFP (green in the figure) or with a bi-cystronicTFEB-GFP vector. Cells were cotransfected with a vector containing a specific shRNAagainst MCOLN1 tagged with RFP (gray in the figure), and stained for luminal LAMP1(LAMP1-1DB4; red in the figure).(G) Secretion of lysosomal β-galactosidase in HeLashMCOLN1 cells. The displayed valuesrepresent secretion efficiency of the indicated enzyme, which was calculated as the % ofenzymatic activity in the medium with respect to the total activity (medium and cellularpellet). Data represent mean ± SEM; ∗p < 0.05 (A–E and G). Scale bars represent 10 μm (Cand F) and 25 μm (D).

Medina et al. Page 19

Published as: Dev Cell. 2011 September 13; 21(3): 421–430.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Figure 5.TFEB Overexpression Reduces Storage in LSD Cells(A) NSCs isolated from the cerebral cortex of MSD and MPSIIIA mice were nucleofectedwith either a TFEB plasmid or with an empty plasmid and cultured on coverslips. Alcianblue was then used to stain the GAGs.(B) Wild-type, MSD, and MPSIIIA NSCs were nucleofected either with a TFEB plasmid orwith an empty plasmid. After 16 hr, cells were pulsed with H3-glucosamine indifferentiation medium and chased for the indicated time-points. Cell extracts obtained atdifferent time points were quantified to determine the levels of labeled GAGs.(C) Glia-differentiated MSD and MPS-IIIA NSCs were nucleofected with either TFEB orwith an empty vector, fixed on glutaraldehyde, and processed for standard electronmicroscopy. The number of vacuoles per cell was counted from 20 different cells anddisplayed as mean ± SEM.(D) TFEB promotes clearance of lipofuscin in fibroblasts from a patient with Batten disease.Cells were transfected with a vector carrying TFEB-Ruby (continuous red staining). After

Medina et al. Page 20

Published as: Dev Cell. 2011 September 13; 21(3): 421–430.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

24 hr, cells were examined by live imaging confocal analysis. Cells with increased TFEB(i.e., cells with red signal in the picture and outlined by dashed white lines in the middlepanel) display highly reduced levels of lipofuscin (punctate green signal) and a normalcellular morphology compared with nontransfected cells (i.e., cells with intense greenstaining).(E) Human Pompe disease fibroblasts were transfected with either TFEB or with an emptyvector, loaded with the fluorescent sugar 2-NBDG, and analyzed by epifluorescencemicroscopy.(F) Secretion efficiency of radioactive GAGs was measured in the culture medium of NSCsfrom MSD mice. Cells were nucleofected with either TFEB or an empty vector after pulse-chase incorporation of H3-glucosamine. Scale bars represent 100 μm (A), 10 μm (C, MSDcells), 500 nm (C, MPSIIIA cells), and 10 μm (D). Data represent mean ± SEM; ∗p < 0.05(B, C, and F).

Medina et al. Page 21

Published as: Dev Cell. 2011 September 13; 21(3): 421–430.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Figure 6.TFEB Overexpression Reduces Storage and Tissue Pathology in a Mouse Model of MSD(A) Alcian blue staining of GAG content in skeletal muscle and liver from mice injectedsystemically with either an AAV2/9-CMV-GFP or with an AAV2/9-CMV-TFEB3xflagviral vector.(B) Quantitative analysis of GAG content in skeletal muscle and liver of MSD mice injectedwith either an AAV2/9-CMV-GFP or with an AAV2/9-CMV-TFEB3xflag viral vector.GAG content was displayed as mg of GAGs/mg of tissue extract. At least four mice pergroup were analyzed for each tissue examined (∗p < 0.05). Data are mean ± SEM.(C) TFEB reduces inflammation in the liver of MSD mice. Macrophages and macrophage-related inflammatory cells were detected in liver sections from mice injected systemicallywith either an AAV2/9-CMV-GFP or with an AAV2/9-CMV-TFEB3xflag viral vector byimmunofluorescence analysis using an antibody against CD68.(D) Reduction of TUNEL-positive cells (arrows) in 4-month-old MSD mice injected with anAAV2/9-CMV-TFEB3xflag viral vector compared with age-matched MSD noninjectedmice. At least four mice per group were analyzed for each tissue examined). Data aremean ± SEM; ∗p < 0.05 (B and D). Scale bars represent 100 μm (A, C, and D).

Medina et al. Page 22

Published as: Dev Cell. 2011 September 13; 21(3): 421–430.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent