Cell Culture, Transfection, and VSV Infection
HeLa, Cos7 cells, human fibroblasts, and mouse embryonic fibroblasts from mouse models of MLIV (S7), MPSIIIA (S7), and MSD were transfected using PolyFect Transfection Reagent (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 was generated by infection with MCOLN1 lentiviral shRNA. Neuronal progenitor cells were isolated from cortices of WT and MSD and MPSIIIA pups (P0) by using standard protocols (see Supplemental Experimental Procedures
for details). NSCs were transfected by using nucleofection (Amaxa, Lonza, Walkersville, MD). Infection of control and CF7 HeLa cells with tsO45 strain of VSV was executed as described (Polishchuk et al., 2003
Tannic Acid Treatment
To prevent fusion of exocytic organelles with PM, control and CF7 HeLa cells were incubated 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 and cells 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 temperature blocks to accumulate VSVG first in the ER and then in the Golgi. Then cells were shifted to 37°C to activate VSVG transport from the Golgi either in the presence or in the absence of 0.5% TA. One hour after the temperature shift to 37°C, the cells were fixed and processed for (IF) labeling (see below).
Surface LAMP1 Analysis
Cells (collected in phosphate-buffered saline [PBS] or grown on coverslips) were incubated with anti-rat LAMP1-1DB4 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4°C for 30 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–rat secondary 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 Aria Flow Cytometer (Becton Dickinson & Co., Mountain View, CA).
Immunofluorescence and Confocal Imaging
Transfected 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 appropriate primary 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-thick serial cryosections using appropriate primary antibodies (see Supplemental Experimental Procedures
for details). The secondary antibodies were purchased from Molecular Probes (Invitrogen).
Images were taken on Vectashield mounted coverslips or sections using a confocal microscope (LSM510; Carl Zeiss, Inc.) equipped with a Plan-Neofluar 63x immersion objective (Carl Zeiss, Inc.).
Immunoblot and PM Isolation
PM proteins were isolated using the Cell Surface Protein Isolation kit (Pierce, Thermo Fischer Scientific Inc, Rockford, IL). Immunoblots were performed using standard protocols and primary antibodies as described in Supplemental Experimental Procedures
. Proteins were quantified by the Bradford method.
Electron Microscopy and Immuno-Gold Analysis
Control 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 at room temperature. The cells were then postfixed for 2 hr in OsO4. After dehydration in graded series of ethanol, the cells were embedded in Epon 812 (FLUKA) and polymerized at 60°C for 72 hr. Thin sections were cut at the Leica EM UC6 and counterstained with uranyl acetate and lead citrate.
For immuno-gold HeLa and CF cells (both intact and TA-treated) were fixed with a mixture of 4% paraformaldehyde and 0.05% glutaraldehyde, labeled with a monoclonal antibody against LAMP1 according the gold-enhance protocol, embedded in Epon-812, and cut as described previously (Polishchuk et al., 2003
EM images were acquired from thin sections using a Philips Tecnai-12 electron microscope equipped with an ULTRA VIEW CCD digital camera (Philips, Eindhoven, The Netherlands).
Quantification of vacuolization was performed using the AnalySIS software (Soft Imaging Systems GmbH, Muenster, Germany).
Evaluation of lysosome distance from the PM was done in EM images using the iTEM software (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 of Golgi membranes) were analyzed.
Calcium Measurements by Confocal Imaging
TFEB-GFP-transfected cells were treated with FuraRed and analyzed according to the protocol described previously (Luciani et al., 2010
Measurements of Ca2+ Concentration by Ratiometric Assays
HeLa cells were infected with either control adenovirus (Ad. Null) or adenovirus expressing TFEB (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 (Molecular Devices, 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 used to calculate the cytoplasmic calcium concentration by using Fura-2AM calibration curves (see Supplemental Experimental Procedures
Flow Cytometric Calcium Flux Assay
Live imaging was performed on WT and CF7 cells using a Carl Zeiss microscope (LSM 710) equipped with a perfusion, open and closed cultivation chamber fitted with a Tempcontrol and CO2-control device (Y module; Carl Zeiss). Acquisition was performed using ZEISS LSM 710 software. The trajectory and the mean velocity of vesicles were calculated by IMAGEJ using the LSM reader and particle tracking plug-ins. For details, see Supplemental Experimental Procedures
GAGs were radiolabeled using H3
-glucosamine hydrochloride (Perkin Elmer, 37.75 Ci/mmol, Boston, MA). Quantitative GAG analysis was performed according protocols described in (de Jong et al., 1989
Alcian Blue Staining
Sections of paraffin-embedded tissues were stained with 1% Alcian blue (Sigma-Aldrich) and counterstained with Nuclear-Fast red (Sigma-Aldrich). See Supplemental Experimental Procedures
In Situ Detection of Apoptotic Cells
TUNEL was performed by using the ApopTag Peroxidase In Situ Apoptosis Detection kit (Oncor, Gaithersburg, MD).