Page 1 of 55 Diabetes Melanophilin accelerates insulin granule fusion without predocking to the plasma membrane Hao Wang,1 Kouichi Mizuno,1 Noriko Takahashi,2 Eri Kobayashi,1 Jun Shirakawa,3 Yasuo Terauchi,3 Haruo Kasai,4 Katsuhide Okunishi,1 and Tetsuro Izumi1* 1Laboratory of Molecular Endocrinology and Metabolism, Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, 371- 8512, Japan, 2Department of Physiology, Kitasato University School of Medicine, Sagamihara, Kanagawa, 252-0373, Japan, 3Department of Endocrinology and Metabolism, Graduate School of Medicine, Yokohama City University, Yokohama, Kanagawa, 236-0004, Japan, 4Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, Tokyo, 113-0033, Japan A short running title: A fast mode of undocked granule exocytosis *Correspondence: [email protected] Address: 3-39-15 Showa-machi, Maebashi, Gunma 371-8512 Tel: +81-27-220-8856 The word count: 4121 The number of figures: 6 1 Diabetes Publish Ahead of Print, published online September 29, 2020 Diabetes Page 2 of 55 Abstract Direct observation of fluorescence-labeled secretory granule exocytosis in living pancreatic β cells has revealed heterogeneous prefusion behaviors: some granules dwell beneath the plasma membrane before fusion, while others fuse immediately once they are recruited to the plasma membrane. Although the former mode seems to follow sequential docking-priming-fusion steps as found in synaptic vesicle exocytosis, the latter mode, which is unique to secretory granule exocytosis, has not been explored well. Here, we show that melanophilin, one of the effectors of the monomeric GTPase Rab27 on the granule membrane, is involved in such an accelerated mode of exocytosis. Both melanophilin-mutated leaden mouse and melanophilin-downregulated human pancreatic β cells exhibit impaired glucose-stimulated insulin secretion, with a specific reduction in fusion events that bypass stable docking to the plasma membrane. Upon stimulus- 2+ induced [Ca ]i rise, melanophilin mediates this type of fusion by dissociating granules from myosin-Va and actin in the actin cortex and by associating them with a fusion-competent, open form of syntaxin-4 on the plasma membrane. These findings provide the hitherto unknown mechanism to support sustainable exocytosis by which granules are recruited from the cell interior and fuse promptly without stable predocking to the plasma membrane. 2 Page 3 of 55 Diabetes Introduction Professional secretory cells store bioactive molecules in vesicles in advance and release them in response to extracellular stimuli by promoting fusion of vesicle membranes to the plasma membrane. In such regulated exocytic pathways, secretory granules carrying proteins as cargo must be regenerated at the Golgi apparatus after releasing their contents, in contrast that synaptic vesicles containing low-molecular-weight substances can recycle within the presynaptic terminal. Therefore, newly generated granules must cross a peripheral microfilament web, referred to as the actin cortex, before approaching the plasma membrane. However, the molecular mechanism by which granules link to the F-actin network and are processed towards exocytosis remains poorly understood. This process that accumulates granules in the cell periphery is thought to form a reserve pool to sustain regulated secretion after depletion of a readily releasable pool beneath the plasma membrane. Its disturbance could impair the capacity of secretory cells to cope with external changes and stresses and cause diseases such as type 2 diabetes. We have recently shown that exophilin-8 (also known as MyRIP and Slac2-c), one of the Rab27 effectors that play versatile roles in regulated secretory pathways (1), captures granules within the actin cortex via indirect interaction with myosin-VIIa through binding to RIM-BP2, and that this exophilin-8-RIM-BP2-myosin-VIIa complex formation is critical for peripheral accumulation and efficient exocytosis of insulin granules (2). However, another motor protein on actin filaments, myosin-Va, has also been suggested to function as a carrier to capture and/or transport granules to the vicinity of the plasma membrane (3-6), although the molecular mechanism by which myosin-Va functions in granule exocytosis remains unknown. Myosin-Va does not interact with exophilin-8, but binds another Rab27 effector, melanophilin (also known 3 Diabetes Page 4 of 55 as exophilin-3 and Slac2-a), in pancreatic β cells (2). Melanophilin retains melanosomes in the periphery of skin melanocytes by directly interacting with both Rab27a on melanosomes and myosin-Va on cortical actin filaments, which makes melanosomes capable of being transferred to neighboring keratinocytes (7-10). Its functional loss leads to clustering of melanosomes near the perikaryotic regions and causes hypopigmentation in both leaden mice and human Griscelli syndrome patients (11,12). However, other overt abnormalities have not been reported. The present study demonstrates in vivo function of melanophilin in insulin granule exocytosis that bypasses stable predocking to the plasma membrane. 4 Page 5 of 55 Diabetes Research Designs and Methods Mice and phenotypic characterization Leaden (C57J/L) and C57BR/cdJ mice were purchased from the Jackson Laboratory. To minimize potential effects of spontaneous mutations occurring after separation of these inbred strains, C57L/J were crossed with C57BR/cdJ and resultant heterozygous mice were intercrossed to generate Mlphln/Mlphln mice, which were used in the current study. Animal experiments were performed according to the rules and regulations of the Animal Care and Experimentation Committees of Gunma University and the University of Tokyo. Only male mice and their tissues and cells were phenotypically characterized in this study. Blood glucose levels were determined by a glucose oxidase method using Glutest Pro GT-1660 (Sanwa Kagaku Kenkyujyo). Insulin was measured by an AlphaLISA insulin kit (PerkinElmer). Pancreatic islet isolation, perifusion secretion assays, and morphometric electron microscopic analysis of granule distribution were performed as described previously (13,14). Antibodies and Immunoprocedures The sources of antibodies and their concentrations used are listed in Supplementary Table 1. Cells lysate proteins separated by gel electrophoresis were transferred onto an Immobilon-P membrane (Millipore), and were visualized by means of enhanced chemiluminescence (GE Healthcare Biosciences). Immunoprecipitation was performed at 4ºC by incubation with primary antibody overnight followed by the addition of protein G-agarose beads (GE Healthcare Bioscience) for 1 h, or by direct incubation with anti-HA affinity matrix beads (Roche Diagnostics) or anti-FLAG affinity gel (Sigma-Aldrich) for 1 h. For immunofluorescence, primary β cells were fixed by 4% paraformaldehyde for 30 min at room temperature, and were 5 Diabetes Page 6 of 55 rehydrated with PBS for 5 min followed by PBS plus 0.1% Triton X-100 for 30 min. The cells incubated with primary antibody overnight at 4ºC followed by Alexa Fluor 488- or 568- conjugated secondary antibody for 1 h at room temperature were observed by a confocal laser scanning microscope. Each image is representative of at least three independent experiments. DNA and RNA manipulation Mouse melanophilin and syntaxin-4 cDNAs were derived from MIN6 cells. Point and deletion mutants were generated using a standard PCR-based mutagenesis strategy, and were verified by DNA sequencing. The sequences of the primers used were listed in Supplementary Table 2. These cDNAs were subcloned into pcDNA3-HA, pcDNA3-FLAG (Invitrogen), pmCherry-C1, pEGFP-C1 (Clontech), pMAL-cR1 (New England Biolabs), pGEX4T-1 (GE Healthcare Bioscience), or pCAG with a One-STrEP-Flag (OSF) tag as described previously (2,13). Neuropeptide Y (NPY)-mCherry cDNA was generated by subcloning a mCherry cDNA into the pNPY-Venus-N1 vector. To generate recombinant adenoviruses, they were inserted into pENTR- 3C (Invitrogen) and were transferred into pAd/CMV by LR Clonase recombination (Invitrogen). To express exogenous protein, HEK293A cells were transfected with the plasmids using Lipofectamine 2000 reagent (Invitrogen), whereas MIN6 cells were infected with adenoviruses. Total internal reflection fluorescence (TIRF) microscopy Human islets (Supplementary Human Islet Checklist) were provided by the Alberta Diabetes Institute IsletCore of the University of Alberta under full ethical clearance (Yokohama City University Ethics Board, B171100025 and Human Tissue MTA from the University of Alberta, UA17-DSA-64). Mouse and human islets were dissociated into monolayer cells by incubation 6 Page 7 of 55 Diabetes with trypsin-EDTA solution, and were cultured on poly-L-lysine-coated 35-mm glass base dishes for 2 days. The cells were infected with adenovirus encoding preproinsulin-enhanced green fluorescent protein (Insulin-EGFP) or NPY-mCherry, and was further cultured for 2 days. TIRF microscopy (the penetration depth of the evanescent field: 100 nm) was performed as described previously (15,16). The cells were preincubated for 30 min in 2.8 mM glucose-containing Krebs- Ringer bicarbonate (KRB) buffer at 37ºC, and were exposed to 25 mM glucose stimulation for 20 min. Images were acquired at 103 ms intervals. Fusion events with a flash were manually selected and assigned to one of three types: residents, which are