Volume 26, Issue 15, Pages 1955-1964 (August 2016) Phagocytosis Enhances Lysosomal and Bactericidal Properties by Activating the Transcription Factor TFEB  Matthew A. Gray, Christopher H. Choy, Roya M. Dayam, Erika Ospina-Escobar, Alexander Somerville, Xuan Xiao, Shawn M. Ferguson, Roberto J. Botelho  Current Biology  Volume 26, Issue 15, Pages 1955-1964 (August 2016) DOI: 10.1016/j.cub.2016.05.070 Copyright © 2016 Elsevier Ltd Terms and Conditions

Current Biology 2016 26, 1955-1964DOI: (10.1016/j.cub.2016.05.070) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Fcγ Receptor Enhances the Degradative and Bactericidal Capacity in Macrophages (A and B) Quantitation of lysosome-based proteolysis in bone-marrow-derived macrophages (A) and RAW macrophages (B). Macrophages remained resting or were stimulated with AIgG for the indicated times, followed by co-endocytosis and chase of DQ-BSA and fluorescent-dextran into lysosomes. Using microscopy or flow cytometry, the fluorescence intensities for DQ-BSA and dextran per cell were quantified and the former normalized against the latter for each cell to account for possible differences in pinocytosis caused by AIgG. (C and D) Quantitation of bactericidal activity in primary macrophages (C) and RAW macrophages (D). Macrophages were either untreated cells (control), stimulated with AIgG, or allowed to ingest IgG-opsonized beads (OB) or IgG-opsonized E. coli, followed by phagocytosis of live E. coli as described in Experimental Procedures. Macrophages were then immediately lysed or allowed to mature their phagosomes before lysis to, respectively, estimate the rate of phagocytosis (Internalized) and killing (Survived). Data are shown as a normalized mean ± SEM for three to six independent experiments, where ∗ indicates statistical significance (p < 0.05) against control conditions using Student’s t test (A and B) or ANOVA followed by Tukey’s post hoc test (C and D). Current Biology 2016 26, 1955-1964DOI: (10.1016/j.cub.2016.05.070) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Fcγ Receptor Engagement Causes TFEB Translocation into the Nucleus (A) RAW cells were transfected with TFEB-GFP and then exposed to vehicle (control), the mTOR inhibitor torin1 (TOR), aggregated IgG (AIgG), or IgG-opsonized beads (OB). Cells were then fixed and counter-stained with DAPI to identify the nucleus. Arrows denote beads. Scale bar, 10 μm. (B) Percentage of cells with nuclear localization of TFEB-GFP after the indicated post-stimulation time. (C and D) The nuclear-to-cytoplasmic fluorescence intensity ratio of endogenous TFEB in RAW (C) and primary macrophages (D). Data are shown as mean ± SEM from three independent experiments and statistically assessed as above (∗p < 0.05). See Figures S1, S2, and S3 for additional information. Current Biology 2016 26, 1955-1964DOI: (10.1016/j.cub.2016.05.070) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Fcγ Receptor Stimulation Enhances Gene and Protein Expression of Specific Lysosomal Genes (A) Relative mRNA expression of select lysosomal (left) and autophagy (right) genes measured by qRT-PCR in RAW macrophages that were untreated (control) or exposed to torin1 (TOR) or IgG-opsonized beads (OB). (B) Protein expression of ATP6V1H, CTSD, and LAMP1 in RAW macrophages treated as above and detected by western blotting (left) and quantified relative to HSP60 (right panel). The 34- and 52-kDa bands in the CTSD blot, respectively, correspond to the mature and pro-CTSD isoforms and were combined for quantification. Data are shown as mean ± SEM from seven independent experiments for CTSD and ATP6V1H and three experiments for LAMP1 and statistically tested as above (∗p < 0.05). (C) Quantification of protein expression of ATP6V1H, CTSD, and LAMP1 in RAW macrophages electroporated with non-targeting siRNA (NT siRNA) or TFEB siRNA and treated as indicated. Data are shown as mean ± SEM from four independent experiments and statistically tested as above (∗p < 0.05). See Figures S2 and S3 for additional information. Current Biology 2016 26, 1955-1964DOI: (10.1016/j.cub.2016.05.070) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 TFEB Is Necessary for Fcγ-Receptor-Mediated Enhancements in Degradation and Bactericidal Activities in Macrophages (A) Quantitation of DQ-BSA proteolysis in control and TFEB-silenced cells. (B) Quantification of bacterial uptake and survival in control and TFEB-silenced cells. Shown is the normalized mean ± SEM from three independent experiments. All experimental and control conditions were statistically assessed as above (∗p < 0.05). See Figures S2 and S4 for additional information. Current Biology 2016 26, 1955-1964DOI: (10.1016/j.cub.2016.05.070) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Frustrated Phagocytosis Does Not Elicit Nuclear Translocation of TFEB (A) Western blotting (left) and blot densitometry (right) showing that frustrated phagocytosis on IgG-coated surfaces elicits Syk phosphorylation, while attachment to surfaces coated with BSA do not (control). The quantification of total Syk and phospho-Syk was done against HSP60 and is illustrated as the normalized mean ± SEM from seven independent experiments. (B) Frustrated phagocytosis by RAW cells failed to induce nuclear localization of TFEB-GFP. TFEB-GFP-transfected cells were parachuted onto BSA-only (control) or anti-BSA antibody-coated coverslips (FP). After attachment, cells were fixed and stained with DAPI and Alexa-647-conjugated secondary antibodies to anti-BSA antibodies to visualize the nuclei and demonstrate the presence of opsonizing antibodies during frustrated phagocytosis. Torin1 was used as positive control to show that TFEB-GFP can enter the nucleus. Scale bar, 10 μm. (C) Quantitation of nuclear localization of TFEB-GFP. Shown is the mean percentage of cells with nuclear TFEB-GFP ± SEM from three independent experiments. Conditions were statistically assessed as above (∗p < 0.05). See Figure S1 for additional information. Current Biology 2016 26, 1955-1964DOI: (10.1016/j.cub.2016.05.070) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 MCOLN1 and Ca2+ Are Required for TFEB Nuclear Localization in Response to Phagocytosis (A) RAW cells were transfected with TFEB-GFP and then exposed to vehicle (control) or BAPTA-AM, followed by phagocytosis of IgG-opsonized beads (OB). (B) Percentage of cells with nuclear localization of TFEB-GFP after the indicated stimulus. (C) RAW cells were electroporated with MCOLN1 siRNA or non-targeting (NT) siRNA oligonucleotides, followed by transfection with TFEB-GFP. They were then exposed to vehicle (control) or allowed to phagocytose IgG-opsonized beads (OB). (D) Percentage of cells with nuclear localization of TFEB-GFP after the indicated stimulus. For (A) and (C), cells were fixed and counter-stained with DAPI to identify the nucleus. Arrows denote internalized beads. Scale bar, 10 μm. For (B) and (D), percentage mean ± SEM from three independent experiments are shown. Conditions were statistically tested as before (∗p < 0.05). See Figures S1 and S5 for additional information. Current Biology 2016 26, 1955-1964DOI: (10.1016/j.cub.2016.05.070) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 7 Phagocytosis of Non-opsonized Bacteria Augments Lysosomal-Based Degradation in a TFEB-Dependent Mechanism (A) TFEB-GFP remains cytosolic in control cells (top) but is nuclear in macrophages that internalized unopsonized E. coli. Nuclei and E. coli were, respectively, visualized with DAPI and anti-E. coli antibodies as described in Experimental Procedures. Scale bar, 10 μm. (B) Quantitation of TFEB-GFP nuclear localization in cells untreated or exposed to unopsonized E. coli. Data are shown as the percentage mean ± SEM from three independent experiments. (C) Time course of DQ-BSA proteolysis after phagocytosis of unopsonized E. coli. (D) Quantitation of DQ-BSA proteolysis in TFEB-silenced cells after phagocytosis of unopsonized E. coli relative to resting cells (control) and non-targeting siRNA oligonucleotide-treated cells (NT). For (C) and (D), data are shown as mean normalized fluorescence ± SEM from three independent experiments and was statistically analyzed as above (∗p < 0.05). See Figure S1 for additional information. Current Biology 2016 26, 1955-1964DOI: (10.1016/j.cub.2016.05.070) Copyright © 2016 Elsevier Ltd Terms and Conditions