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 Supplementary http://www.jimmunol.org/content/suppl/2018/10/16/jimmunol.180026 Downloaded from Material 8.DCSupplemental Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: by guest on October 2, 2021 http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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