TFEB regulates lysosomal positioning by modulating TMEM55B expression and JIP4 recruitment to lysosomes

Abstract

Lysosomal distribution is linked to the role of lysosomes in many cellular functions, including autophagosome degradation, cholesterol homeostasis, antigen presentation, and cell invasion. Alterations in lysosomal positioning contribute to different human pathologies, such as cancer, neurodegeneration, and lysosomal storage diseases. Here we report the identification of a novel mechanism of lysosomal trafficking regulation. We found that the lysosomal transmembrane protein TMEM55B recruits JIP4 to the lysosomal surface, inducing dynein-dependent transport of lysosomes toward the microtubules minus-end. TMEM55B overexpression causes lysosomes to collapse into the cell center, whereas depletion of either TMEM55B or JIP4 results in dispersion toward the cell periphery. TMEM55B levels are transcriptionally upregulated following TFEB and TFE3 activation by starvation or cholesterol-induced lysosomal stress. TMEM55B or JIP4 depletion abolishes starvation-induced retrograde lysosomal transport and prevents autophagosome-lysosome fusion. Overall our data suggest that the TFEB/TMEM55B/JIP4 pathway coordinates lysosome movement in response to a variety of stress conditions.

Fig. 1

TMEM55B regulates lysosomal positioning. a Schematic of the predicted membrane topology of TMEM55B. b ARPE-19 cells transfected with GFP-TMEM55B for 18 h. Cells were fixed, permeabilized, and immunostained with antibodies against LAMP-1 (top) or HRS (bottom). Insets represent a 3.2- (top) and 4.5- (bottom) fold magnification of the indicated areas. c ARPE-19 cells infected with adenovirus expressing GFP-TMEM55B for 30 h. Cells were fixed, permeabilized, and immunostained with antibodies against LAMP-1 (top) or EEA1 (bottom). Arrows denote bulk of lysosomal accumulation. d HeLa cells infected with adenovirus expressing GFP-TMEM55B for 30 h. Cells were fixed permeabilized and immunostained with antibodies against LAMP-1. Arrows denote bulk of lysosomal accumulation. e, f HeLa cells treated with TMEM55B or Control siRNAs for 6 days. e Relative quantitative real-time PCR analysis of TMEM55B mRNA transcript levels (mean ± s.e.m. of the RNA fold change of indicated TMEM55B normalized to actin mRNA from three independent experiments; ^∗∗∗ P < 0.0001). f Immunoblot of lysates of cells treated with TMEM55B or Control siRNA. g HeLa cells treated with TMEM55B or Control siRNA were immunostained with antibody against LAMP-1. h Quantification of lysosome distribution, percentage of total fluorescence signal detected at 0–5 μm, 5–15 μm, or >15 μm from nuclear rim. Quantified results are presented as mean ± s.e.m. using two-tailed t-test ^∗ P < 0.05, ^∗∗ P < 0.005 were considered significant, n ≥ 30 from three or more independent experiments. Scale bars, 20 μm

Fig. 2

Microtubules and Dynein are required for TMEM55B-induced lysosome clustering. a ARPE-19 cells were transfected with 3×HA-TMEM55B and treated with 10 μM nocodazole for 2 h at 37 °C and then shifted to ice for 30 min in the presence of nocodazole. For nocodazole washouts, cells were washed and incubated in nocodazole-free culture medium for the indicated times. Cells were fixed, permeabilized, and immunostained with antibodies against LAMP-1 and HA. b ARPE-19 cells co-transfected with 3xHA-TMEM55B and GFP-P50 Dynamitin. Cells were fixed, permeabilized, and immunostained with antibodies against LAMP-1. Insets represent a three-fold magnification of the indicated area. Arrows indicate TMEM55B-induced lysosomal clustering. Scale bars, 20 μm

Fig. 3

Recruitment of TMEM55B CD initiates retrograde transport of tagged membranes towards the cell center. ARPE-19 cells transfected with a LAMP-1-CFP-FRB and RFP-FKBP-TMEM55B CD, b Melanoregulin (Mreg 1-42)-CFP-FRB and RFP-FKBP-TMEM55B CD, or c AKAP1-FRB-GFP and RFP-FKBP-TMEM55B CD. Cells were incubated in 200 nM Rapamycin for the indicated times. Cells were fixed, permeabilized, and immunostained with antibodies against CD63 (a, b) or LAMP-1 (c). Arrows indicate TMEM55B CD-induced lysosomal clustering. Scale bars, 10 μm (a, c) or 20 μm (b)

Fig. 4

TMEM55B interacts with Dynein adapter JIP4. a Immunoblot of RFP pull-down from lysates of ARPE-19 cells co-transfected with LAMP-1-CFP-FRB and RFP-FKBP-TMEM55B CD and treated with 200 nM Rapamycin (Rap) (+) or vehicle (−) for 2 h. b Immunoblot of GFP pull-down from lysates of ARPE-19 cells transfected with GFP-TMEM55B FL (full length), GFP-TMEM55B CD (cytosolic domain), GFP-TMEM55A FL, or GFP-TMEM55A CD. c Immunoblot of GFP pull-down from lysates of ARPE-19 cells transfected with GFP-LAMP-1 or GFP. d Immunoblot of GFP pull-down from lysates of ARPE-19 cells transfected with GFP-TMEM55B FL or GFP-TMEM55B CD. e Immunoblot of GFP pull-down from lysates of ARPE-19 cells co-transfected with GFP-LAMP-1 and 3xHA-TMEM55B. f ARPE-19 cells were infected with adenovirus expressing GFP (top) or GFP-TMEM55B (bottom) for 24 h. Cells were fixed, permeabilized, and immunostained with antibodies against JIP4 and LAMP-1. Insets represent a 2.8-fold magnification of the indicated areas. Scale bars, 20 μm

Fig. 5

TMEM55B/JIP4 drive retrograde lysosomal trafficking independent of RILP. a HeLa cells treated with JIP4 or Control siRNA were immunostained with antibody against LAMP-1. b Quantification of lysosome distribution shown as the percentage of total fluorescence signal detected at 0–5 μm, 5–15 μm, or >15 μm from nuclear rim. Quantified results are presented as mean ± s.e.m. using two-tailed t-test ^∗ P < 0.05, ^∗∗ P < 0.005 were considered significant, n ≥ 30. c Immunoblot of HeLa cells depleted of JIP4 or Control RNAi. d HeLa cells treated with JIP4 or Control siRNA were infected with adenovirus expressing GFP-TMEM55B for 24 h. Cells were fixed, permeabilized, and immunostained with antibodies against LAMP-1. Arrows denote bulk of lysosomal accumulation. e Quantification of lysosome distribution, percentage of total fluorescence signal detected at 0–5 μm or >15 μm from nuclear rim. Quantified results are presented as mean ± s.e.m. using two-tailed t-test ^∗ P < 0.05, ^∗∗ P < 0.005 were considered significant, n ≥ 10. f HeLa cells depleted of TMEM55B, JIP4, or expressing control RNAis were transfected with GFP-RILP for 24 h. Cells were fixed, permeabilized, and immunostained with antibodies against LAMP-1. Arrows denote bulk of lysosomal accumulation. Scale bars, 20 μm

Fig. 6

TFE3 and TFEB increase TMEM55B expression to cluster lysosomes during starvation. a–c ARPE-19 cells were infected with adenovirus expressing TFE3-S321A-Myc, TFEB-S211A-FLAG, or null virus for 36 h. a Relative quantitative real-time PCR analysis of TMEM55B mRNA transcript levels (mean ± s.e.m. of the RNA fold change of indicated TMEM55B normalized to GAPDH mRNA, n = 4). b Representative immunoblot of lysates from TFE3-S321A-Myc and TFEB-S211A-FLAG expressing cells. c Quantification of TMEM55B protein levels. d Control MEFs untreated or starved in EBSS for 4 h. Cells were fixed, permeabilized, and immunostained with antibodies against TFE3. e–g Relative quantitative real-time PCR analysis of e MCOLN1, f TMEM55B and g TMEM55A mRNA transcript levels from null- or TFE3/TFEB knockout MEFs, untreated or starved in EBSS for 4 h (mean ± s.e.m. of the RNA fold change of indicated TMEM55B normalized to GAPDH) n = 6 from three independent experiments. h HeLa cells stably expressing control or TFEB shRNAs were starved in EBSS for 4 h. Cells were fixed, permeabilized, and immunostained with antibodies against LAMP-1. i HeLa cells expressing control, TMEM55B, and JIP4 RNAis were starved in EBSS for 4 h. Cells were fixed, permeabilized, and immunostained with antibodies against LAMP-1. j Quantification of lysosome distribution, percentage of total fluorescence signal detected >15 μm from nuclear rim. Quantified results are presented as mean ± s.e.m. using two-tailed t-test ^∗ P < 0.05, ^∗∗ P < 0.005 were considered significant, n ≥ 30. k Immunoblot of lysates from HeLa cells treated with siControl or siJIP4 siRNA, then starved in EBSS for 4 h, or starved in EBSS for 4 h with 2 h 100 nM bafilomycin. Note that all the samples were run in the same gel but are presented in two panels due to space limitations. l quantification of LC3_II/Actin ratios from siControl or siJIP4-treated HeLa cells starved in EBSS 4 h with 100 nM bafilomycin for 2 h. Quantified results are fold increase of LC3_II/Actin from siControl after starvation and bafilomycin treatment and data are presented as mean ± s.e.m. using two-tailed t-test **P < 0.005, n = 3. m HeLa cells treated with siControl or siJIP4 siRNA and starved with EBSS for 4 h and 100 nM bafilomycin for 2 h. Cells were fixed, permeabilized, and immunostained with antibodies against LAMP-1, LC3, and DAPI. Scale bars, 20 μm

Fig. 7

TFEB/3 and SREBF2 co-operate to regulate TMEM55B levels in response to changes in cholesterol levels. a, b HeLa cells were treated with drugs to deplete cellular cholesterol for 120 h, treated with 10 μM U18666A for 18 h, or left untreated. a Relative quantitative real-time PCR analysis of TMEM55B mRNA transcript levels (mean ± s.e.m. of the RNA fold change of indicated TMEM55B normalized to GAPDH mRNA, using two-tailed t-test **P < 0.005 were considered significant) n = 3. b Representative immunoblot of lysates from cholesterol depleted cells. c Control MEFs were untreated or incubated in 10 μM U18666A for 18 h. Cells were fixed, permeabilized, and immunostained with antibodies against TFE3. d Relative quantitative real-time PCR analysis of TMEM55B mRNA transcript levels from null- or TFE3/TFEB knockout MEFs, untreated or starved in EBSS for 4 h (mean ± s.e.m. of the RNA fold change of indicated TMEM55B normalized to GAPDH, using two-tailed t-test *P < 0.05 were considered significant) n = 6 from three independent experiments. e HeLa cells depleted of TMEM55B, JIP4, or control RNAi were starved in EBSS for 4 h and then immunostained with antibody against LAMP-1. f Control MEFs treated with control and SREBF2 siRNAs and TFEB/TFE3 KO MEFs treated with SREBF2 siRNAs were incubated with 10 μM U18666A for 18 h and the TMEM55B mRNA transcript levels were quantified by qRT-PCR (mean ± s.e.m. of the RNA fold change normalized to TMEM55B levels in siControl-treated cells, using two-tailed t-test *P < 0.05, **P < 0.005 were considered significant) n = 6 from three independent experiments. g Immunoblot of lysates from primary skin fibroblasts from Niemann-Pick C (NPC1) patient 1, NPC1 patient 2, and genetic matched control patient. h NPC1 patient fibroblasts depleted of JIP4 with RNAi. Cells were fixed, permeabilized, and immunostained with antibodies against LAMP-1, JIP4, and DAPI. i Model of TMEM55B transcriptional regulation. Nutrient deprivation inactivates mTOR to allow TFEB/3 translocation to the nucleus to activate transcription of TMEM55B and induce lysosome retrograde trafficking. Decreased sterol levels at the ER stimulates SREBF2 to translocate to the nucleus to activate transcription of TMEM55B and induces lysosome retrograde trafficking. TMEM55B recruits scaffold JIP4 that binds dynein–dynactin motor complex. Scale bars, 20 μm

Fig. 8

JIP4-dependent retrograde lysosomal transport in response to acute oxidative stress. a, b MEFs were incubated with 20 µM curcumin for the indicated times. Cells were fixed, permeabilized, and immunostained with antibodies against LAMP-1 (a) or JIP4 and p150^Glued (b). c Quantification of cells with JIP4 recruitment to lysosomes in DMSO or curcumin treated MEFs. DMSO n = 745, curcumin n = 805 from three independent experiments. Error bars denote s.e.m. P value calculated using two-tailed t-test ****P < 0.0001. d MEFs treated with control or JIP4 siRNAs and incubated with curcumin for 4 h. Cells were fixed, permeabilized, and immunostained with antibody against LAMP-1. e Immunoblot of U2OS expressing GFP-TMEM55B cells untreated or treated with 150 µM NaAsO₂. f Immunoblot of lysates from MEFs treated with curcumin for the indicated times. g MEFs were pre-incubated with MAPK inhibitors, followed by combination of inhibitors and curcumin. Cells were fixed, permeabilized, and immunostained with antibody against LAMP-1. h Quantification of cells with JIP4 recruitment to lysosomes in curcumin/MAPK inhibitor-treated MEFs. Control n = 568, curcumin n = 583, curcumin + p38 inhibitor (20 µM) n = 598, curcumin + JNK inhibitor (25 µM) n = 590, curcumin + ERK inhibitor (20 µM) n = 575 from two independent experiments. Error bars denote s.e.m. P value calculated using one-way ANOVA ***P < 0.001. Scale bars, 20 μm