A TRP channel in the lysosome regulates large particle phagocytosis via focal exocytosis - PubMed (original) (raw)

. 2013 Sep 16;26(5):511-24.

doi: 10.1016/j.devcel.2013.08.003. Epub 2013 Aug 29.

Xiang Wang, Xiaoli Zhang, Andrew Goschka, Xinran Li, Xiping Cheng, Evan Gregg, Marlene Azar, Yue Zhuo, Abigail G Garrity, Qiong Gao, Susan Slaugenhaupt, Jim Pickel, Sergey N Zolov, Lois S Weisman, Guy M Lenk, Steve Titus, Marthe Bryant-Genevier, Noel Southall, Marugan Juan, Marc Ferrer, Haoxing Xu

Affiliations

A TRP channel in the lysosome regulates large particle phagocytosis via focal exocytosis

Mohammad Samie et al. Dev Cell. 2013.

Abstract

Phagocytosis of large extracellular particles such as apoptotic bodies requires delivery of the intracellular endosomal and lysosomal membranes to form plasmalemmal pseudopods. Here, we identified mucolipin TRP channel 1 (TRPML1) as the key lysosomal Ca2+ channel regulating focal exocytosis and phagosome biogenesis. Both particle ingestion and lysosomal exocytosis are inhibited by synthetic TRPML1 blockers and are defective in macrophages isolated from TRPML1 knockout mice. Furthermore, TRPML1 overexpression and TRPML1 agonists facilitate both lysosomal exocytosis and particle uptake. Using time-lapse confocal imaging and direct patch clamping of phagosomal membranes, we found that particle binding induces lysosomal PI(3,5)P2 elevation to trigger TRPML1-mediated lysosomal Ca2+ release specifically at the site of uptake, rapidly delivering TRPML1-resident lysosomal membranes to nascent phagosomes via lysosomal exocytosis. Thus phagocytic ingestion of large particles activates a phosphoinositide- and Ca2+-dependent exocytosis pathway to provide membranes necessary for pseudopod extension, leading to clearance of senescent and apoptotic cells in vivo.

Copyright © 2013 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. TRPML1 is necessary for optimal phagocytosis of large particles in bone-marrow macrophages

(A) RT-PCR analysis of wild type (WT) and TRPML1 KO (ML1 KO ) bone-marrow derived macrophages (BMMs) using a primer pair targeting the deleted region (exons 3, 4, and 5) of the TRPML1 gene (Venugopal, Browning et al. 2007). The housekeeping gene L32 served as a loading control. (B) ML-SA1 robustly activated endogenous whole-endolysosome ML1-like currents in WT, but not ML1 KO BMMs. (C) WT and ML1 KO BMMs were exposed to IgG-opsonized red blood cells (IgG-RBCs; red colored) at a ratio of 50 RBCs/BMM for time periods indicated (15, 30, 60, and 90 min). Non-ingested IgG-RBCs were lysed by briefly (1–2 min) incubating the cells in water at 4°C. Samples were then fixed and processed for confocal microscopy. (D) Average particle ingestion for WT and ML1 KO BMMs. Ingested IgG-RBCs were quantified for 150–200 BMMs per experiment, by experimenters who were blind to the genotype. (E) ML1 KO BMMs had a lower uptake index compared with WT BMMs. Uptake index was calculated based on the total number of RBCs ingested for 100 BMMs. (F) Particle-size-dependent phagocytosis defect of ML1 KO BMMs. BMMs were exposed to 3 or 6 µm IgG-coated polystyrene beads for indicated periods of time. Samples were washed extensively and briefly trypsinized to dissociate non-ingested beads attached to the cell surface or cover slips. The number of ingested particles was determined as described in (D). For all panels, unless otherwise indicated, the data represent the mean ± the standard error of the mean (SEM) from at least three independent experiments. See also Figure S1.

Figure 2

Figure 2. The expression level and channel activity of ML1 regulate particle ingestion in macrophages

(A) Micromolar concentrations of ML-SA1 (0.1, 1, and 10 µM; pre-incubation for 15 min) increased particle uptake in WT, but not ML1 KO BMMs. BMMs were exposed to IgG-RBCs in the presence of ML-SA1 for 15 min. (B) Transfection of ML1-GFP, but not ML1-KK-GFP (non-conducting pore mutation (Dong, Shen et al. 2010), rescued the uptake defect in ML1 KO BMMs. BMMs were exposed to IgG-RBCs for 30 min in the presence or absence of the small molecule TRPML agonist, ML-SA1 (10 µM). (C) ML-SA1-activated whole-endolysosome TRPML-like currents in RAW 264.7 macrophage cell lines, RAW cells stably-expressing ML1-GFP (ML1 overexpression or O/E), and RAW cells stably-expressing ML1-specific RNAi (ML1 knockdown or KD) (Thompson, Schaheen et al. 2007). (D) Real-time RT-PCR analysis of ML1 RNA expression level (relative to L32) in RAW macrophages (N=3 batches of independent experiments for each cell type). (E) Particle uptake in RAW 264.7, ML1 O/E, and ML1 KD RAW macrophages. (F) Particle uptake was sensitive to intracellular Ca2+. BMMs were pre-incubated with the membrane-permeable fast Ca2+ chelator BAPTA-AM (1 µM; 15 min) prior to IgG-RBC exposure. (G) ML-SI1 inhibited large basal whole-endolysosome _I_ML1 that was seen in a subset of ML1-expressing Cos1 cells. (H) ML-SI1 (10 µM; pre-incubation for 15 min) inhibited particle ingestion in WT BMMs to a similar level of that in ML1 KO BMMs. BMMs were exposed to IgG-RBCs in the presence of ML-SI1 for 30 min. For all panels, unless otherwise indicated, the data represent the mean ± SEM from at least three independent experiments; 150–200 BMMs were analyzed for each experiment. See also Figure S2.

Figure 3

Figure 3. Particle binding to macrophages rapidly recruits ML1-GFP to the phagocytic cups and nascent phagosomes

(A) Rapid recruitment of ML1-GFP to the sites of particle ingestion (white arrow) and to expanding membrane extensions. Selected frames from time-lapse confocal microscopy of a ML1-GFP transfected RAW 264.7 macrophage that was exposed to 6 µm IgG-coated polystyrene beads. Approximately one min after the attachment of beads to the cell surface, ML1-GFP was recruited to the membrane extension surrounding the particle (Frame 1 min 15 sec; Video 1). Note that phagocytosis may have been initiated before the imaging time zero due to a technical difficulty in identifying phagocytosing macrophages and capturing phagocytosing events, which typically took 5–10 min after RBCs were added to the recording chamber. Scale bar = 5 µm. (B) ML1-GFP and Syt VII-mCherry were simultaneously recruited to nascent phagosomes. Selected frames from time-lapse confocal microscopy (Video 3) showed a RAW cell doubly-transfected with ML1-GFP and Syt VII-mCherry that was exposed to IgG-RBCs. White box indicates the site of particle ingestion; both ML1-GFP and Syt VII-mCherry were concurrently recruited to the site of phagosome formation. Scale bar = 10 µm. (C) The recruitment kinetics of different endolysosomal markers to the particle-containing phagosomes. RAW macrophage cells were transfected with various molecular endosomal and lysosomal markers. Upon particle binding, the recruitment of these markers to phagosomes was monitored using live-cell imaging over the course of 40 min. For large particles, ML1-mCherry and Lamp1-GFP were recruited to the forming phagosomes within 5 min after phagocytosis initiation; Rab7-GFP and LysoTracker were observed in the phagosomes about 20 min after phagocytosis initiation; Rab5-GFP appeared on the phagosomes 10 min after phagocytosis initiation, and then disappeared 10 –15 min later. For small particles (bottom row), ML1-GFP was recruited to the small particle-containing phagosomes 15–20 min after phagocytosis initiation. The center of the particle is indicated with an *. Scale bars = 5 µm. (D) Summary of recruitment kinetics of various endolysosomal markers to phagosomes. See also Figure S3.

Figure 4

Figure 4. Whole-phagosome recording of ML1 channels in the isolated nascent phagosomes

(A) Cartoon illustrations of whole-phagosome recording procedures. Upon RBC uptake (left panel), patch electrodes are used to slice open the phagocytosing macrophage within 5 min of particle binding and ingestion. After RBC-containing phagosomes are isolated, a patch pipette is used to achieve the whole-phagosome configuration. (B) An illustration of whole-phagosome configuration. Cells were transfected with Lamp-1-GFP or ML1-GFP and then exposed to IgG-coated beads on ice for 20 min; after phagocytosis was induced by transferring the cells to 37°C for 5 min, the newly formed phagosomes were isolated for electrophysiology. (C) ML-SA1- or PI(3,5)P2-activated endogenous whole-phagosome TRPML-like currents in RAW 246.7 cells. (D) Whole-phagosome _I_ML1 in WT, but not ML1 KO BMMs. ML-SA1 (25 µM) - activated _I_ML1 was inhibited by ML-SI1 (50 µM) in WT phagosomes (left panel). No ML-SA1- activated _I_ML1 was seen in ML1 KO phagosomes, in which PI(3,5)P2 (1 µM) robustly activated _I_TPC.

Figure 5

Figure 5. Particle binding induces ML1-dependent lysosomal Ca2+ release and lysosomal exocytosis in macrophages

(A) ML-SA1 (10 µM for 30 min) treatment resulted in localization of Lamp1 (red) on the plasma membrane in non-permeabilized WT, but not ML1 KO BMMs. Lamp1 surface expression was detected using an antibody recognizing a luminal epitope (1DB4). After the cells were permeabilized, total (cell surface + intracellular; green) Lamp1 proteins were detected by using the same antibody. Scale bar = 5 µm. (B) IgG-RBC binding triggered a localized Ca2+ increase at the site of uptake, shown with selected image frames of a RAW cell doubly-transfected with GCaMP3-ML1 and Syt VII-mCherry. Upon IgG-RBC binding, GCaMP3-ML1 fluorescence increased rapidly (frame 0 min 45 sec) near the site of uptake and around the Syt VII-mCherry-positive compartments in the membrane extension, and then decreased after phagosome formation (frame 6 min 15 sec). Lower panel shows the time-dependent changes of GCaMP3-ML1 fluorescence upon particle binding, at the site of uptake (phagocytic region) and in the cell body (whole-cell region). White arrow indicates the phagocytic cup. Scale bar = 10 µm. (C) RBC-ingestion-induced lysosomal acid phosphatase (AP) release in WT BMMs was reduced by treatment of BAPTA-AM (500 nM, pre-incubation for 15 min) or ML-SI1 (10 µM, pre-incubation for 10 min). (D) Whole-cell ML1-like currents in WT BMMs that were exposed to IgG-RBCs for 10 min. Dynasore (Dyn, 100 µM) was used to block Dynamin-dependent endocytosis to facilitate the detection of whole-cell _I_ML1. No significant whole-cell _I_ML1 was detected in ML1 KO BMMs (IgG-RBCs for 10 min). (E) Summary of whole-cell _I_ML1 under different experimental conditions. For all panels, unless otherwise indicated, the data represent the mean ± SEM from at least three independent experiments. See also Figure S5.

Figure 6

Figure 6. ML1-mediated facilitation of particle ingestion is dependent on lysosomal exocytosis

(A) ML-SA1 (10 µM) failed to increase particle ingestion in ML1 O/E RAW cells transfected with a Syt VII dominant-negative (DN) construct. Cells were pre-incubated with DMSO or ML-SA1 for 15 min before they were exposed to IgG-RBCs for 30 min. (B) ML-SA1 (10 µM) failed to increase particle ingestion in ML1 O/E RAW cells transfected with a VAMP7 dominant-negative (VAMP7-DN) construct. (C) A microtubule-disrupting agent colchicine (10 µM) inhibited ML-SA1-enhanced particle ingestion in WT BMMs. (D) FIG4 KO macrophages were defective in IgG-RBC ingestion. WT and FIG4 KO BMMs were exposed to IgG-RBCs for time periods indicated. (E, F) YM0201636 (1 µM for 2 hrs) treatment inhibited uptake of 6 µm beads, but not 3 µm beads in WT BMMs. (G) Upon particle binding, ML1-2N-GFP and Syt VII-mCherry accumulated at the site of uptake within minutes (see Video 4). Note that phagocytosis initiation may have occurred before the imaging time zero due to a technical difficulty in identifying macrophages undergoing phagocytosis. (H) HPLC analysis of the levels of PI(3,5)P2 and PI(4,5)P2 during phagosome formation. Myo-[2-3H] inositol-labeled RAW cells were exposed to IgG-coated beads (6 µm) for 0, 1, 2, 5, and 10 min at 37°C. At each time point, cells were centrifuged at 4°C and prepared for HPLC analysis. Data were from three independent experiments. (I) Particle binding activates lysosomal ML1 to initiate Ca2+-dependent lysosomal exocytosis. Upon particle (IgG-RBC) binding, the activity of PIKfyve/Fab1 (not shown) is stimulated at the site of particle uptake. The subsequent increase of PI(3,5)P2 level can then activate lysosomal ML1 to induce lysosomal Ca2+ release. Juxtaorganellar Ca2+ can then bind the Ca2+ sensor Syt VII to trigger lysosome-plasma membrane fusion, which provides substantial amount of lysosomal membranes for the pseudopod extension around IgG-RBCs. As a result, lysosomal membrane proteins, such as ML1 and Syt-VII, are inserted into the plasma membrane. Pseudopod extension continues until the IgG-RBCs are completely internalized. Phagocytic cups are then closed, and nascent phagosomes are formed. Newly formed phagosomes then undergo a series of membrane fusion and fission processes, collectively called phagosome maturation, to become late phagosomes. Lysosomes are also delivered to the late phagosomes through late phagosome maturation (phagosome-lysosome fusion), which is required for the degradation of phagocytic materials. ML1 may also plays a role in phagosome-lysosome fusion, however this requires further investigation. For all panels, unless otherwise indicated, the data represent the mean ± SEM from at least three independent experiments; 150–200 macrophages were analyzed for each experiment. For all panels, scale bars = 10 µm. See also Figure S6.

Figure 7

Figure 7. ML1 deficiency results in defective clearance of senescent red blood cells in the spleen and microglia activation in the brain

(A) Accumulation of RBCs in the red (RP) and white (WP) pulps of a ML1 KO spleen revealed by H&E staining. (B) Perl’s (Prussian blue) staining for ferric iron in the splenic red pulp (RP) from 4-month-old mice. Scale bar = 200 µm. (C) ICP-MS analysis of the splenic iron content using digested whole spleens from WT and ML1 KO mice. Data were presented as ratios of different ions. (D) Propidium iodide (PI, 20 µg/ml) -labeled cells in the cerebral cortex and hippocampus of WT, ML1 KO, and FIG4 KO mouse brain sections. (E) Iba1 was up-regulated in the whole-brain lysates of ML1 and FIG4 KO mice. (F) Iba1-positive cells were up-regulated in the sections of the brain cortex of ML1 KO mice. For all panels, unless otherwise indicated, the data represent the mean ± SEM from at least three independent experiments. See also Figure S7.

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