Myoferlin-Mediated Lysosomal Exocytosis Regulates Cytotoxicity by Phagocytes

Myoferlin-Mediated Lysosomal Exocytosis Regulates Cytotoxicity by Phagocytes Yuji Miyatake, Tomoyoshi Yamano and Rikinari Hanayama This information is current as J Immunol published online 17 October 2018 of October 2, 2021. http://www.jimmunol.org/content/early/2018/10/16/jimmun ol.1800268

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 17, 2018, doi:10.4049/jimmunol.1800268 The Journal of Immunology

Myoferlin-Mediated Lysosomal Exocytosis Regulates Cytotoxicity by Phagocytes

Yuji Miyatake,*,† Tomoyoshi Yamano,*,‡ and Rikinari Hanayama*,‡,x

During inflammation, phagocytes release digestive enzymes from lysosomes to degrade harmful cells such as pathogens and tumor cells. However, the molecular mechanisms regulating this process are poorly understood. In this study, we identified myoferlin as a critical regulator of lysosomal exocytosis by mouse phagocytes. Myoferlin is a type II transmembrane protein with seven C2 domains in the cytoplasmic region. It localizes to lysosomes and mediates their fusion with the plasma membrane upon calcium stimulation. Myoferlin promotes the release of lysosomal contents, including hydrolytic enzymes, which increase cytotoxicity. These data demonstrate myoferlin’s critical role in lysosomal exocytosis by phagocytes, providing novel insights into the mechanisms of inflammation-related cellular injuries. The Journal of Immunology, 2018, 201: 000–000.

hagocytes (such as macrophages, neutrophils, and den- including neutrophil azurophil granules, platelet dense granules, Downloaded from dritic cells) internalize a variety of foreign particles, eosinophil granules, basophil and mast cell histamine granules, P bacteria, and dead cells into a subcellular compartment and CTL lytic granules. Among these cells, CTLs have been most called a phagosome, where they are digested to yield a series of extensively studied in terms of the exocytic machinery of the peptides following the fusion of the phagosome with a lysosome secretory lysosomes, which cause the release of membrane-bound (1). Phagocytosis and the subsequent digestion of enclosed par- Fas ligand, the pore forming protein perforin, and granzyme serine

ticles are essential processes for eukaryotes to protect themselves protease to induce the killing of target cells (6). In CTLs, lyso- http://www.jimmunol.org/ from bacterial and viral infections and for the maintenance of somal exocytosis is mediated by a complex formed of small homeostasis (2). Lysosomes are ﬁlled with more than 50 different GTPase Rab27A and its effector Munc 13-4 that tethers the lytic hydrolytic enzymes, which are capable of digesting most cellular granules to the plasma membrane to allow fusion (7). Mutations in components, including proteins, nucleic acids, carbohydrates, and the Munc 13-4 gene cause familial hemophagocytic lymphohis- lipids (3). It has been reported that phagocytes activated by ba- tiocytosis type 3, which causes immunodeﬁciency due to defects cillus Calmette–Gue´rin infection secrete lysosomal enzymes with in the lysosomal exocytosis of cytolytic enzymes by CTLs (8). In cytotoxic activity against tumor cells, which is called heteroly- several cell types, lysosomal exocytosis is also regulated by the sis (4). During contact with the activated phagocytes, the target Ca2+ sensor synaptotagmin-VII (Syt VII) (9). Syt VII is present on

tumor cells undergo degenerative changes, including the clumping the lysosomal membrane and regulates Ca2+-triggered exocytosis by guest on October 2, 2021 of nuclear chromatin, vacuolation, and retraction of cytoplasm, by binding to the plasma membrane, a process that is required for which is inhibited by preventing lysosomal exocytosis. However, repairing damaged plasma membranes. Mice deficient in Syt VII the molecular mechanisms that regulate lysosomal exocytosis by exhibit defective membrane repair, which results in myopathy phagocytes are largely unknown. (10). Both Munc 13-4 and Syt VII possess C2 domains that me- Several hematopoietic cells have cell-specific secretory lyso- diate Ca2+-dependent binding to phosphatidylserine (PS) in the somes, which are secreted in response to external stimuli (5), inner leaflet of the plasma membrane, which is a critical process for membrane fusion (11). Myoferlin is a type II transmembrane protein with seven C2 domains in the cytoplasmic region (12, 13). *Department of Immunology, Kanazawa University Graduate School of Medical The first C2 domain (C2A domain) binds PS in a calcium- Sciences, Ishikawa 920-8640, Japan; †Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan; ‡WPI Nano Life Science Institute (NanoLSI), Kanazawa dependent manner (14). Myoferlin was identified as a protein University, Ishikawa 920-1192, Japan; and xPrecursory Research for Embryonic expressed in the plasma membrane of myoblasts undergoing fu- Science and Technology, Japan Science and Technology Agency, Saitama sion and has been implicated in the repair of injured plasma 332-0012, Japan membranes (15, 16). Myoferlin is also known to regulate various Received for publication February 23, 2018. Accepted for publication September 16, 2018. aspects of tumor progression and cancer cell motility (17, 18). It is believed that injury-induced Ca2+ influx through membrane le- This work was supported by grants from the Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology “Chronic Inflamma- sions triggers endocytosis and the generation of endocytosed tion” (4336), the Ministry of Education, Culture, Sports, Science and Technology vesicles expressing myoferlin, which fuse with the injured mem- Grant-in-Aid for Challenging Exploratory Research (16K15231) (to R.H.), and by the World Premier International Research Center Initiative, Ministry of Education, Cul- brane to yield a membrane patch (16). This characteristic of ture, Sports, Science and Technology, Japan. myoferlin led us to hypothesize that it may be a regulator of Address correspondence and reprint requests to Dr. Rikinari Hanayama, Kanazawa calcium-dependent lysosomal exocytosis by phagocytes. University Graduate School of Medical Sciences, 13-1 Takara, Kanazawa, Ishikawa 920-8640, Japan. E-mail address: [email protected] The online version of this article contains supplemental material. Materials and Methods Abbreviations used in this article: BMDM, bone marrow–derived macrophage; Fw, Cells and plasmids forward; KO, knockout; MO, membranous organelle; P-L, phagosome–lysosome; PS, phosphatidylserine; Rv, reverse; shRNA, short hairpin RNA; Syt VII, synaptotagmin- NIH3T3 cells were obtained from RIKEN BioResource Research VII; WT, wild-type. Center, (Ibaraki, Japan), and cultured in DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with 10% FBS (Biowest). Bone marrow– Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$37.50 derived macrophages (BMDMs) were prepared as previously described

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800268 2 MYOFERLIN MEDIATES LYSOSOMAL EXOCYTOSIS BY PHAGOCYTES

(19). pCDNA3.1-Myoferlin HA was a gift from W. Sessa (20) (plasmid cells (2 3 104 cells) cultured in a Millicell EZ SLIDE eight-well glass no. 22443; Addgene). GFP was fused to the N terminus of myoferlin by slide (Merck Millipore), were fixed in 4% paraformaldehyde solution, amplifying the GFP locus on pCAG-GFP using the following primers: and permeabilized with ice-cold acetone. After blocking with PBS con- forward (Fw) (59-GCCACCATGGTGAGCAAGGG-39) and reverse (Rv) taining 10% goat serum (Sigma-Aldrich) and 1% BSA, the cells were (59-GATATCTTGTACAGCTCGTCCA-39). The product was cloned into stained in PBS containing 1% BSA with FITC anti-LAMP1 Ab (1D4B; pGEM-T Easy Vector (Promega), digested by NotI and EcoRV sites, and BioLegend). After staining, the cells were mounted with coverslips using subsequently inserted into the pCDNA3.1-Myoferlin HA. The D C2A VECTASHIELD mounting medium (Vector Laboratories) and were ob- mutant of myoferlin was constructed by depleting the amino acid region served by a confocal microscopy (FV10i; Olympus). from 2 to 115 loci. Each plasmid DNA was introduced into NIH3T3 cells by lipofection using FuGENE6 (Promega). Retroviral 29-mer short Flow cytometric analysis hairpin RNA (shRNA) plasmids that targeted the mouse Myoferlin coding sequence (59-GTTCCATTCAGCCACCATGTTGCAAGATG-39 for shRNA 1, For intracellular LAMP1 staining, BMDMs were incubated with an un- or 59-GACAATGACAGTGATGACGTGGAGAGCAA-39 for shRNA 2)anda conjugated LAMP1 Ab to inhibit the staining of extracellular LAMP1, then scrambled control shRNA plasmid were established using the HuSH permeabilized with FOXP3 Fix/Perm Buffer Set (BioLegend) and stained shRNA cloning plasmid pRS (OriGene). These retroviral plasmids were with FITC anti-LAMP1 Ab. For the staining of mouse peritoneal exudate introduced by lipofection using FuGENE6 into Platinum-E packaging cells, the cells were collected 2 d after i.p. injection with 1 mg pHrodo Red cells (21). Forty-eight hours after transfection, the culture supernatant E. coli BioParticles (Thermo Fisher Scientific), and then stained with FITC was used to infect NIH3T3 cells in the presence of 10 mg/ml polybrene, anti-CD11b (M1/70; BioLegend) and Alexa Fluor 647 anti–Ly-6G (1A8; thereby establishing stable transformants. BioLegend) and analyzed using a BD FACSVerse system. Mice Western blot Gene-trap 129 embryonic stem cells that targeted the Myoferlin chromo- Lysosomes were isolated from NIH3T3 cells by using a Lysosome En- richment Kit for Tissues and Cultured Cells (Thermo Fisher Scientific), somal gene (clone D135C09) were obtained from Helmholtz Zentrum Downloaded from Munchen, (Munich, Germany) and introduced into C57BL/6 embryos to according to the manufacturer’s protocol. Isolated lysosomes or whole cells produce chimeric mice. Chimeric mice with a high ES contribution were were lysed in RIPA buffer (0.1% SDS, 0.5% DOC, 150 mM NaCl, 50 mM crossed with C57BL/6 mice to produce Myoferlin+/2 mice. Myoferlin2/2 Tris (pH 7.4), 2 mM EDTA, 1% Triton X-100), which was supplemented mice were generated by crossing Myoferlin+/2 parents, and the phenotypes with a protease inhibitor mixture (Roche) and/or a phosphatase inhibitor of the Myoferlin+/+ and Myoferlin2/2 littermates were analyzed. The ge- mixture (Sigma-Aldrich). Protein concentrations were determined with a notype of the Myoferlin gene was determined by PCR with the following Pierce 660nm Protein Assay Reagent (Thermo Fisher Scientific). Western primers: wild-type (WT)–Fw (59-AGGCAACACTGCACACTCAA-39) blot analysis was performed by using Abs against myoferlin (159; Sigma- and WT-Rv (59-GCCAGACGCTGTGACAGTTA-39) for the WT allele, Aldrich), LAMP1 (1D4B; BioLegend), syntaxin6 (C34B2; Cell Signaling http://www.jimmunol.org/ and knockout (KO)-Fw (59-GAGAAGAAGAGGAGGAACCC-39) and Technology), or b-actin (AC74; Sigma-Aldrich). The Western blot signals KO-Rv (59-GTGATTGACTACCCGTCAGC-39) for the mutant allele. All were detected with ImageQuant LAS 4000 mini Biomolecular Imager (GE mice were housed in a specific pathogen-free facility, and all animal ex- Healthcare), and the intensity was quantified by ImageQuant TL Analysis periments were performed according to a protocol approved by Kanazawa Toolbox software. University, Kanazawa, Japan. Cytotoxicity assay Quantitative PCR analysis E. coli DH5a were grown at 37˚C in lysogeny broth until the mid- Total RNAwas isolated from cells using a GenElute Mammalian Total RNA exponential phase (OD.0.8–1.0). The E. coli were collected by centrifu- 3 Miniprep Kit (Sigma-Aldrich), and cDNA was synthesized with ReverTra gation at 4000 g for 10 min and inactivated with 50 ml of 70% (v/v)

Ace (TOYOBO, Osaka, Japan), according to the manufacturer’s instruc- ethanol for 30 min. The E. coli were centrifuged, washed and suspended in by guest on October 2, 2021 tions. The cDNA products were amplified with a LightCycler 96 (Roche) PBS, then followed by UV irradiation treatment for 30 min. For the cy- using SYBR Select Master Mix (Thermo Fisher Scientific). The primers totoxicity assay, the peritoneal fluid was collected with 10 ml DMEM used for real-time PCR were as follows: Myoferlin-Fw (59-TGCTCATC- containing 1% FBS at 2 d after i.p. injection of the killed E. coli. After CTGTTGCTGTTC-39), Myoferlin-Rv (59-GTTCTTCATTGCTTGCGTGA-39); centrifugation at 1500 rpm for 5 min, the supernatant was condensed 10 b-actin–Fw (59-TGTGATGGTGGGAATGGGTCAG-39), b-actin–Rv times using an Amicon Pro Purification System and was used as a culture 3 4 (59-TTTGATGTCACGCACGATTTCC-39). The data were analyzed using medium for NIH3T3 cells (2 10 cells in 24-well plates). After 2 d, the the DCt (threshold cycle) method and normalized against levels of b-actin survival of attached NIH3T3 cells was examined using WST-1 Cell Pro- RNA expression in each sample. liferation Reagent (Roche), according to the manufacturer’s protocol, with an iMark Microplate Absorbance Reader (Bio-Rad Laboratories). For CFU 3 Secretion of lysosomal enzymes fibroblast assay, NIH3T3 cells (13 10 cells) were cultured with the mouse peritoneal fluids for 2 d, and the colonies were counted. The lysosomal enzymes secreted by Ca2+ stimulation were quantified as described previously (22). In brief, the NIH3T3 transformants or BMDMs (1 3 105 cells in 24-well plates) were cultured in PBS con- Results taining NAADP (100 mM; R&D Systems), ML-SA1 (1 mM; Wako Pure Myoferlin is a C2 domain–containing protein localized to Chemical Industries, Osaka, Japan), or ionomycin (100 nM; Wako Pure lysosomes of phagocytes Chemical Industries) for the indicated periods. The cultured PBS was incubated for 15 min at 37˚C with 4 mM 4-methyl-umbellyferyl-N- To examine whether various phagocytes express myoferlin, we acetyl-b-D-glucosaminide (Sigma-Aldrich) in 20 mM sodium citrate examined the expression profiles of myoferlin by quantitative phosphate buffer (pH 4.5). A 1:1 mixture of 2 M Na2CO3 and 1 M glycine was added to the reaction solution to terminate the reaction. The fluorescence PCR. Myoferlin was highly expressed in macrophages, such as produced by 4-methyl-umbellyferyl-N-acetyl-b-D-glucosaminide when reac- thioglycollate-elicited peritoneal macrophages, BMDMs and ted with the b-hexosaminidase (also known as N-acetyl-b-D-glucosaminidase) bone marrow-derived dendritic cells (BMDCs), macrophage cell secreted in the cultured PBS was measured using a Synergy HT multi- lines (J774.1, RAW 264, and BAM3), neutrophils, and NIH3T3 detection microplate reader (BioTek Instruments) at an excitation wavelength cells but not in T or B cell lines (WR19L or BaF3 cells) (Fig. 1A). of 360 nm and an emission wavelength of 460 nm. To quantify the lysosomal enzymes secreted in vivo, the peritoneal fluid was collected with 10 ml PBS As NIH3T3 cells are the most susceptible to transfection among at 2 d after i.p. injection of 70 mg Escherichia coli. After centrifugation at these cells, we first examined the subcellular localization of 1500 rpm for 5 min, the activity of b-hexosaminidase was measured in myoferlin by expressing GFP-myoferlin fusion protein in NIH3T3 the supernatants as described above. cells and found that myoferlin was colocalized with various lyso- Immunocytochemical analysis somal markers, including LysoTracker, LAMP1, or CD63 (Fig. 1B, Supplemental Fig. 1A). Western blot analysis confirmed that both NIH3T3 cells (1 3 105 cells) that expressed GFP-myoferlin were cultured in 3.5-cm glass-bottom dishes, incubated with LysoTracker Red DND-99 myoferlin and LAMP1 were enriched in isolated lysosomal frac- (50 nM; Thermo Fisher Scientific), or coexpressed either with RFP- tions, whereas neither syntaxin6 (a Golgi marker) nor b-actin LAMP1 or RFP-CD63 fusion protein. For intracellular staining, NIH3T3 (a cytosol marker) was enriched (Fig. 1C). The Journal of Immunology 3 Downloaded from

FIGURE 1. Localization of myoferlin on lysosomes. (A) Quantitative PCR analyses were performed to compare Myoferlin mRNA levels in the cells indicated. The graph represents the gene expression levels relative to b-actin. THIO (2) and THIO (+) stand for resident and thioglycollate-elicited peritoneal macrophages, respectively. (B) NIH3T3 cells expressing GFP-myoferlin fusion protein (green) were stained with LysoTracker (red) or were http://www.jimmunol.org/ coexpressed either with RFP-LAMP1 or RFP-CD63 fusion protein (red). Next, the cells were observed by confocal microscopy. The staining proﬁles are merged in the third panel. Scale bar, 10 mm. Scatter plots with a Pearson correlation analysis, which indicated linear correlations between myoferlin (x-axis) and lysosomal markers (y-axis), are shown in the fourth panel. (C) Equivalent amounts of proteins extracted from whole-cell lysate (WCL) or isolated lysosomes of NIH3T3 cells were subjected to SDS-PAGE, and Western blot analysis was performed using Abs against indicated proteins.

Accumulation of lysosomes in myoferlin knockdown cells membranous materials compared with the control cells (control 5. 6 6 To clarify the functions of myoferlin in lysosomes, we established 9 1.8 per cell versus myoferlin-knockdown 28.6 5.5 per cell; n = 20; p , 0.02, Student t test) (Fig. 2C), which is consistent with

NIH3T3 transformants expressing shRNA against myoferlin by guest on October 2, 2021 (Supplemental Fig. 1B). LAMP1 staining revealed that the a previous report showing that autolysosomes accumulate in myoferlin knockdown cells contained significantly more lysosomes myoferlin-deficient cells (23). However, induction of autophagy compared with the control cells (Fig. 2A). The increase of lyso- was not altered by myoferlin deficiency (Supplemental Fig. 1C). somes was confirmed by FACS staining with LysoTracker, which showed a 2.5-fold increase in the myoferlin knockdown cells Myoferlin promotes lysosomal exocytosis by phagocytes (Fig. 2B). Electron microscopic analysis of the NIH3T3 trans- Based on these results, we considered the possibility that the formants showed that the myoferlin knockdown cells carried accumulation of lysosomes might be due to impaired lysosomal many more cytoplasmic vesicles filled with debris and additional exocytosis by myoferlin knockdown. To examine whether

FIGURE 2. Increase of lysosomes by myoferlin knockdown. (A) NIH3T3 cells that stably expressed control or myoferlin shRNA were established and stained with an Ab against LAMP1 (green), counter-stained with DAPI (blue), and observed by confocal microscopy. Scale bar, 10 mm. (B) NIH3T3 cells that expressed myoferlin shRNA (red line) or control shRNA (black line) were stained with LysoTracker, and their staining intensities were compared using ﬂow cytometry. (C) Electron microscopy analysis of NIH3T3 cells that stably expressed control shRNA or myoferlin shRNA. The arrows indicate debris accumulated in the autolysosomes. Myoferlin shRNA 1 and 2 were designed to target different coding sequences. Scale bar, 1 mm. 4 MYOFERLIN MEDIATES LYSOSOMAL EXOCYTOSIS BY PHAGOCYTES Downloaded from

FIGURE 3. Myoferlin regulates lysosomal exocytosis. (A) NIH3T3 transformants that expressed myoferlin shRNA (black circles) or control shRNA (white diamonds) were cultured in PBS containing a calcium mobilizer, NAADP (100 mM), for the times indicated, and amounts of b-hexosaminidase http://www.jimmunol.org/ secreted in the culture medium were quantified by reacting with 4-methyl-umbellyferyl-N-acetyl-b-D-glucosaminide. The experiment was performed in triplicate, and the average values are plotted with the SD. *p , 0.02, Student t test. (B) Secretion of b-hexosaminidase by NIH3T3 transformants were compared 5 min after stimulation with NAADP. Plasmid DNA encoding human MYOFERLIN (hMYOF) or its deletion mutant that lacks the C2A domain (hMYOF D C2A) was introduced into the NIH3T3 transformants that expressed mouse myoferlin shRNA by lipofection 3 d before the assay. The experiment was performed in triplicate and the average values are plotted with the SD. *p , 0.02, Student t test. n.s., not significant. (C) Intracellular expression of LAMP1 in BMDMs from Myoferlin+/+ (WT, solid line) and Myoferlin2/2 (KO, dotted line) mice were compared by flow cytometry. (D) Secretion of b-hexosaminidase by myoferlin WT (white bars) and KO (black bars) BMDMs were compared in response to indicated calcium stimuli. *p , 0.02, Student t test. (E) Cytoplasmic calcium flux in response to ionomycin (10 mM) was measured in BMDMs from myoferlin WT (solid line) or KO (dotted line) mice every 10 s by using a Calbryte 590 AM calcium indicator. (F) BMDMs from myoferlin WT (white bars) or KO (black bars) mice were

treated with ionomycin (100 nM) for the times indicated and stained for LAMP1. The expression levels of cell surface LAMP1 on the cells (n = 10) were by guest on October 2, 2021 quantified by immunofluorescence with confocal microscopy. *p , 0.02, Student t test. myoferlin promotes lysosomal exocytosis, we quantified the Myoferlin+/+ BMDMs,butitwasmuchloweronMyoferlin2/2 amount of b-hexosaminidase, which is one of the most com- BMDMs (Fig. 3F), which was consistent with the secretion of mon lysosomal enzymes, secreted by calcium stimulation. When b-hexosaminidase. These results indicate that myoferlin plays a NIH3T3 transformants were treated with NAADP, which induces crucial role in lysosomal exocytosis by phagocytes upon calcium calcium release from endolysosomes through two-pore channels stimulation. To investigate whether myoferlin also plays a role (24), b-hexosaminidase was rapidly secreted, but the secretion was in phagocytosis, we cocultured Myoferlin+/+ or Myoferlin2/2 significantly impaired by myoferlin knockdown (Fig. 3A). The se- BMDMs with FITC-labeled small or large beads, but we did not cretion of b-hexosaminidase from myoferlin knockdown cells was find any effect of myoferlin deficiency on the uptake of these restored by expressing human myoferlin protein but not by its de- beads (Supplemental Fig. 2A). During phagocytosis, myoferlin letion mutant that lacks the C2A domain, suggesting that the binding was gradually recruited to phagosomes (Supplemental Fig. 2B), of the C2A domain to PS is essential for the myoferlin activities but the phagosome–lysosome (P-L) fusion was not impaired in (Fig. 3B). We then generated Myoferlin2/2 mice to examine the Myoferlin2/2 BMDMs when acidification of phagosomes was lysosomal exocytosis by phagocytes. These mice were viable and examined with engulfed pHrodo Red E. coli BioParticles exhibited normal development. BMDMs were prepared from (Supplemental Fig. 2C). This is probably because only a small Myoferlin+/+ and Myoferlin2/2 mice, and we confirmed the ac- increase of cytoplasmic Ca2+ levels occurred during phagocy- cumulation of lysosomes in Myoferlin2/2 BMDMs by FACS tosis compared with the treatment with calcium mobilizers staining of intracellular LAMP1 (Fig. 3C). Next, we compared (data not shown), which might be insufficient to activate the the secretion of b-hexosaminidase from BMDMs upon various function of myoferlin. Accordingly, the ability of killing- calcium stimuli. As shown in Fig. 3D, Myoferlin2/2 BMDMs engulfed bacteria was not impaired in Myoferlin2/2 BMDMs secreted a significantly lower amount of b-hexosaminidase than (Supplemental Fig. 2D). These data indicate that myoferlin has Myoferlin+/+ BMDMs in response to various calcium stimuli a minimal effect on P-L fusion, suggesting that lysosomal such as NAADP, ionomycin (a calcium ionophore), and ML-SA1 exocytosis is a primary role for myoferlin. (a mucolipin1 channel agonist), although the increase of cytoplasmic Ca2+ levels were comparable (Fig. 3E). The occurrence Myoferlin promotes cytotoxicity via lysosomal exocytosis of lysosomal exocytosis can also be quantified by expression We next examined whether myoferlin promotes secretion of ly- levels of cell surface LAMP1. After ionomycin treatment, sosomal enzymes in vivo. The peritoneal injection of pHrodo Red the intensity of cell surface LAMP1 levels was increased on E. coli BioParticles induced the recruitment of CD11b+/Ly-6G+ The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 4. Impaired lysosomal secretion and cytotoxicity by myoferlin deficiency. (A) pHrodo Red E. coli BioParticles (1 mg) were injected into the peritoneal cavity of myoferlin WT or KO mice. After 2 d, the peritoneal exudate cells were collected and stained for CD11b. The CD11b+ and pHrodo+ cells were further stained for Ly-6G. (B) Confocal images of CD11b+/Ly-6G+ cells stained with Hoechst 33342 nuclear staining dye. Phase contrast images were shown in the right panels. (C) The amounts of b-hexosaminidase in the peritoneal fluid were compared in the myoferlin WT (white diamonds) and KO (black squares) mice. The fluid was collected with (day 2) or without (day 0) the i.p. injection of E. coli BioParticles (70 mg) (n = 7). *p , 0.02, Student t test. (D and E) The cytotoxicity of the peritoneal fluid was examined. Peritoneal fluid was collected by injecting DMEM containing 1% FBS i.p. 2 d after inoculation with killed E. coli (10 mg). After centrifugation, the supernatants were condensed 10 times and then used as culture media for NIH3T3 cells. The supernatants were not diluted or were diluted 3-fold or 10-fold with DMEM containing 10% FBS and used as culture media. After 2 d, the survival of attached NIH3T3 cells was examined using a WST-1 assay, and the levels were normalized against that of NIH3T3 cells cultured without peritoneal fluid. by guest on October 2, 2021 The cytotoxicity levels of peritoneal fluid from WT mice i.p. injected with (white bars) or without (black bars) killed E. coli DH5a are shown in (D), whereas those from myoferlin WT (white bars) or KO (black bars) mice are shown in (E). *p , 0.02, Student t test. (F) CFU fibroblast (CFU-F) assay was performed to estimate the number of viable NIH3T3 cells, which were cultured with peritoneal fluid for 2 d. The graph indicates total colonies per well (mean of two independent experiments). *p , 0.02, Student t test. polymorphonuclear cells [i.e., neutrophils to elicit phagocytosis of many more colonies when cultured with the peritoneal fluid the BioParticles (Fig. 4A, 4B)]. There were no differences in the obtained after E. coli injection from Myoferlin2/2 mice com- number of recruited cells in Myoferlin+/+ and Myoferlin2/2 mice. pared with that from Myoferlin+/+ mice (Fig. 4E, 4F). Overall, We then compared the activity of lysosomal enzymes in the these results demonstrate that myoferlin plays a crucial role in peritoneal fluid that was secreted by the neutrophils after the release of cytotoxic lysosomal contents from phagocytes, the bacterial infection. Two days after peritoneal injection of the which may increase the likelihood of cellular injuries in bacterial E. coli BioParticles, the increase of the lysosomal enzyme activity infections. was much greater in the peritoneal fluid of Myoferlin+/+ mice than Myoferlin2/2 mice (Fig. 4C). We then investigated whether the Discussion activity of lysosomal enzymes correlated with the cytotoxicity of In this study, we found that myoferlin is a regulator of lysosomal the peritoneal fluid. Again, we injected large numbers of killed exocytosis by phagocytes. Myoferlin belongs to the ferlin family, E. coli into the Myoferlin+/+ mouse peritoneal cavity to promote the members of which are defined by their sequence similarity to the recruitment of neutrophils to elicit phagocytosis. Two days Caenorhabditis elegans fer-1 (13). Fer-1 is localized to the later, the peritoneal fluid was collected by injecting DMEM membrane of specialized vesicles called membranous organelles containing 1% FBS and was concentrated 10 times by diafiltra- (MOs) in spermatids, where it regulates the Ca2+-dependent tion. When NIH3T3 cells were cultured with the medium for 3 d, fusion of MOs with the plasma membrane during spermiogenesis 80% of the cells were killed by the medium (Fig. 4D). This cy- (25). In fer-1 mutants, MOs do not fuse with the plasma mem- totoxic effect was also observed using the medium containing brane, thereby causing the abnormal accumulation of MOs and peritoneal fluid obtained without E. coli injection, but when the infertility. It is known that mammals possess at least six mem- media were serially diluted, the peritoneal fluid obtained after bers of the ferlin family of proteins, including dysferlin, oto- E. coli injection had greater cytotoxicity than that without E. coli ferlin, and myoferlin, which share similar domain structures, injection. This result is consistent with the activity of lysosomal with a single transmembrane domain in the C terminus and enzymes with or without E. coli injection (Fig. 4C). The cyto- multiple C2 domains in the N terminus (13). Dysferlin is toxic effect was significantly impaired, and viable cells formed the most thoroughly studied ferlin, and its mutations cause 6 MYOFERLIN MEDIATES LYSOSOMAL EXOCYTOSIS BY PHAGOCYTES limb-girdle muscular dystrophy 2B and Miyoshi myopathy (26). Disclosures Dysferlin localizes to the cytoplasmic vesicles and plays a crit- The authors have no financial conflicts of interest. ical role in muscle membrane repair by mediating the fusion of vesicles with the plasma membrane, thereby serving as a membrane patch in the disrupted regions (26). Otoferlin is highly References 1. Kinchen, J. M., and K. S. Ravichandran. 2008. Phagosome maturation: going expressed in the cochlea of the ear and has been implicated in through the acid test. Nat. Rev. Mol. Cell Biol. 9: 781–795. 2+ synaptic vesicle exocytosis, in which it acts as the major Ca 2. Haas, A. 2007. The phagosome: compartment with a license to kill. Traffic 8: sensor to trigger the fusion of the synaptic vesicles and plasma 311–330. 3. Luzio, J. P., P. R. Pryor, and N. A. Bright. 2007. Lysosomes: fusion and function. membrane (27). Thus, the common function of ferlin family Nat. Rev. Mol. Cell Biol. 8: 622–632. proteins is to mediate the fusion of cytoplasmic vesicles with the 4. Hibbs, J. B., Jr. 1974. Heterocytolysis by macrophages activated by bacillus plasma membrane in exocytosis. Myoferlin was identified as the Calmette-Gue´rin: lysosome exocytosis into tumor cells. Science 184: 468– 471. protein that shares the highest homology with dysferlin (12). 5. Blott, E. J., and G. M. Griffiths. 2002. Secretory lysosomes. Nat. Rev. Mol. Cell Despite their similarity and their common expression in skeletal Biol. 3: 122–131. muscle, myoferlin and dysferlin are thought to share few over- 6. Trapani, J. A., and M. J. Smyth. 2002. Functional significance of the perforin/ granzyme cell death pathway. Nat. Rev. Immunol. 2: 735–747. lapping functions. Previously, dysferlin was shown to regulate 7. Elstak, E. D., M. Neeft, N. T. Nehme, J. Voortman, M. Cheung, M. Goodarzifard, monocyte adhesion and phagocytosis (28, 29). However, we H. C. Gerritsen, P. M. van Bergen En Henegouwen, I. Callebaut, G. de Saint could detect dysferlin expression only in NIH3T3 cells but not in Basile, and P. van der Sluijs. 2011. The munc13-4-rab27 complex is specifically required for tethering secretory lysosomes at the plasma membrane. Blood 118: any macrophages or neutrophils, and lysosomal exocytosis was 1570–1578. intact in dysferlin-deficient BMDMs (data not shown). Given the 8. Feldmann, J., I. Callebaut, G. Raposo, S. Certain, D. Bacq, C. Dumont, N. Lambert, M. Ouacheé-Chardin, G. Chedeville, H. Tamary, et al. 2003. Downloaded from low expression levels of other ferlin family proteins in phago- Munc13-4 is essential for cytolytic granules fusion and is mutated in a form of cytes, myoferlin is likely the only ferlin protein that regulates familial hemophagocytic lymphohistiocytosis (FHL3). Cell 115: 461–473. lysosomal exocytosis by phagocytes. 9. Martinez, I., S. Chakrabarti, T. Hellevik, J. Morehead, K. Fowler, and 2+ N. W. Andrews. 2000. Synaptotagmin VII regulates Ca(2+)-dependent exocy- Myoferlin regulates lysosomal exocytosis in a Ca -dependent tosis of lysosomes in fibroblasts. J. Cell Biol. 148: 1141–1149. manner. In this study, we used a calcium mobilizer to increase 10. Chakrabarti, S., K. S. Kobayashi, R. A. Flavell, C. B. Marks, K. Miyake, cytoplasmic Ca2+ levels and thus to activate the function of D. R. Liston, K. T. Fowler, F. S. Gorelick, and N. W. Andrews. 2003. Impaired

membrane resealing and autoimmune myositis in synaptotagmin VII-deficient http://www.jimmunol.org/ myoferlin. In physiological settings, various immune or inflam- mice. J. Cell Biol. 162: 543–549. matory responses elevate Ca2+ levels in phagocytes (30). For 11. Martens, S., and H. T. McMahon. 2011. C2 domains and membrane fusion. Curr. example, it is well known that signals from cell surface receptors Top. Membr. 68: 141–159. 12. Davis, D. B., A. J. Delmonte, C. T. Ly, and E. M. McNally. 2000. Myoferlin, a such as Fc receptors activate the phospholipase C g and inositol candidate gene and potential modifier of muscular dystrophy. Hum. Mol. Genet. 1,4,5-triphosphate signaling pathways, thereby causing the re- 9: 217–226. 2+ 2+ 13. Posey, A. D., Jr., A. Demonbreun, and E. M. McNally. 2011. Ferlin proteins in lease of Ca from the endoplasmic reticulum (31). Such Ca myoblast fusion and muscle growth. Curr. Top. Dev. Biol. 96: 203–230. elevation may trigger myoferlin-mediated lysosomal exocytosis 14. Davis, D. B., K. R. Doherty, A. J. Delmonte, and E. M. McNally. 2002. Calcium- in response to bacterial infection. In contrast, myoferlin did not sensitive phospholipid binding properties of normal and mutant ferlin C2 domains. J. Biol. Chem. 277: 22883–22888. appear to control phagocytosis or phagosome maturation al-

15. Doherty, K. R., A. Cave, D. B. Davis, A. J. Delmonte, A. Posey, J. U. Earley, by guest on October 2, 2021 though it was gradually recruited to phagosomes. Because we M. Hadhazy, and E. M. McNally. 2005. Normal myoblast fusion requires detected only a small increase of cytoplasmic Ca2+ levels during myoferlin. Development 132: 5565–5575. 16. Bernatchez, P. N., A. Sharma, P. Kodaman, and W. C. Sessa. 2009. Myoferlin is phagocytosis and P-L fusion occurs calcium independently (32), critical for endocytosis in endothelial cells. Am. J. Physiol. Cell Physiol. 297: myoferlin, a Ca2+-dependent protein, plays only a minimal role C484–C492. 17. Volakis, L. I., R. Li, W. E. Ackerman, IV, C. Mihai, M. Bechel, T. L. Summerfield, in these processes. However, as we observed the accumulation of C. S. Ahn, H. M. Powell, R. Zielinski, T. J. Rosol, et al. 2014. Loss of myoferlin autolysosomes filled with debris in the myoferlin knockdown redirects breast cancer cell motility towards collective migration. PLoS One cells, it is speculated that myoferlin on phagolysosomes might 9: e86110. 18. Blackstone, B. N., R. Li, W. E. Ackerman, IV, S. N. Ghadiali, H. M. Powell, and promote expelling of indigestible debris generated inside phag- D. A. Kniss. 2015. Myoferlin depletion elevates focal adhesion kinase and olysosomes by exocytosis (33, 34). It has also been shown that paxillin phosphorylation and enhances cell-matrix adhesion in breast cancer cells. Am. J. Physiol. Cell Physiol. 308: C642–C649. phagocytes have a potent cytotoxic capacity that is induced by 19. Ishidome, T., T. Yoshida, and R. Hanayama. 2017. Induction of live cell mAbs against target cells (e.g., tumor cells and pathogens). This phagocytosis by a specific combination of inflammatory stimuli. EBioMedicine is known as Ab-dependent cell-mediated cytotoxicity (35). The 22: 89–99. 20. Bernatchez, P. N., L. Acevedo, C. Fernandez-Hernando, T. Murata, C. Chalouni, mAbs activate Fc receptors to trigger the signaling pathway that J. Kim, H. Erdjument-Bromage, V. Shah, J. P. Gratton, E. M. McNally, et al. 2+ induces Ca -dependent lysosomal exocytosis. Neutrophils have 2007. Myoferlin regulates vascular endothelial growth factor receptor-2 stability a particularly strong Ab-dependent cell-mediated cytotoxic ac- and function. J. Biol. Chem. 282: 30745–30753. 21. Morita, S., T. Kojima, and T. Kitamura. 2000. Plat-E: an efficient and tivity, during which they secrete large amounts of cytotoxic stable system for transient packaging of retroviruses. Gene Ther. 7: 1063– molecules, including hydrolytic enzymes, oxidative metabolites, 1066. and host defense peptides such as defensins (35). It would be 22. Sato, M., S. Yoshimura, R. Hirai, A. Goto, M. Kunii, N. Atik, T. Sato, K. Sato, R. Harada, J. Shimada, et al. 2011. The role of VAMP7/TI-VAMP in cell polarity useful to study whether myoferlin regulates the release of these and lysosomal exocytosis in vivo. Traffic 12: 1383–1393. molecules from neutrophils. In any case, our demonstration that 23. Demonbreun, A. R., A. D. Posey, K. Heretis, K. A. Swaggart, J. U. Earley, P. Pytel, and E. M. McNally. 2010. Myoferlin is required for insulin-like growth myoferlin deficiency decreased the cytotoxicity by phagocytes is factor response and muscle growth. FASEB J. 24: 1284–1295. clinically important and may facilitate the development of novel 24. Morgan, A. J., F. M. Platt, E. Lloyd-Evans, and A. Galione. 2011. Molecular therapeutic approaches based on myoferlin-mediated lysosomal mechanisms of endolysosomal Ca2+ signalling in health and disease. Biochem. J. 439: 349–374. exocytosis. 25. Washington, N. L., and S. Ward. 2006. FER-1 regulates Ca2+ -mediated membrane fusion during C. elegans spermatogenesis. J. Cell Sci. 119: 2552– 2562. Acknowledgments 26. Han, R., and K. P. Campbell. 2007. Dysferlin and muscle membrane repair. Curr. We thank H. Ohgi and J. Song for technical assistance in the initial stage of Opin. Cell Biol. 19: 409–416. this project and Y. Okayasu for secretarial assistance. We also thank 27. Roux, I., S. Safieddine, R. Nouvian, M. Grati, M. C. Simmler, A. Bahloul, I. Perfettini, M. Le Gall, P. Rostaing, G. Hamard, et al. 2006. Otoferlin, defective H. Omori for electron microscopy analysis and Y. Esaki and S. Nishioka in a human deafness form, is essential for exocytosis at the auditory ribbon for generating the myoferlin-deficient mice. synapse. Cell 127: 277–289. The Journal of Immunology 7

28. de Morreé, A., B. Flix, I. Bagaric, J. Wang, M. van den Boogaard, L. Grand 32. Zimmerli, S., M. Majeed, M. Gustavsson, O. Stendahl, D. A. Sanan, and Moursel, R. R. Frants, I. Illa, E. Gallardo, R. Toes, and S. M. van der Maarel. J. D. Ernst. 1996. Phagosome-lysosome fusion is a calcium-independent event in 2013. Dysferlin regulates cell adhesion in human monocytes. J. Biol. Chem. 288: macrophages. J. Cell Biol. 132: 49–61. 14147–14157. 33. Medina, D. L., A. Fraldi, V. Bouche, F. Annunziata, G. Mansueto, C. Spampanato, 29. Nagaraju, K., R. Rawat, E. Veszelovszky, R. Thapliyal, A. Kesari, S. Sparks, C. Puri, A. Pignata, J. A. Martina, M. Sardiello, et al. 2011. Transcriptional acti- N. Raben, P. Plotz, and E. P. Hoffman. 2008. Dysferlin deficiency enhances vation of lysosomal exocytosis promotes cellular clearance. Dev. Cell 21: 421–430. monocyte phagocytosis: a model for the inflammatory onset of limb-girdle 34. Xu, J., K. A. Toops, F. Diaz, J. M. Carvajal-Gonzalez, D. Gravotta, muscular dystrophy 2B. Am. J. Pathol. 172: 774–785. F. Mazzoni, R. Schreiner, E. Rodriguez-Boulan, and A. Lakkaraju. 2012. 30. Nunes, P., and N. Demaurex. 2010. The role of calcium signaling in phago- Mechanism of polarized lysosome exocytosis in epithelial cells. J. Cell Sci. cytosis. J. Leukoc. Biol. 88: 57–68. 125: 5937–5943. 31. Sańchez-Mejorada, G., and C. Rosales. 1998. Signal transduction by immuno- 35. Braster, R., T. O’Toole, and M. van Egmond. 2014. Myeloid cells as effector globulin Fc receptors. J. Leukoc. Biol. 63: 521–533. cells for monoclonal antibody therapy of cancer. Methods 65: 28–37. Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021