The FASEB Journal article fj.14-261529. Published online February 12, 2015.

The FASEB Journal • Research Communication

Transient Ca2+ depletion from the endoplasmic reticulum is critical for skeletal myoblast differentiation Keiko Nakanishi,*,1 Kisa Kakiguchi,†,2 Shigenobu Yonemura,†,2 Akihiko Nakano,*,‡,3 and Nobuhiro Morishima*,1,4 *Molecular Membrane Biology Laboratory, RIKEN Advanced Science Institute, Wako, Saitama, Japan; † Electron Microscope Laboratory, RIKEN Center for Developmental Biology, Kobe, Hyogo, Japan; and ‡Graduate School of Science, The University of Tokyo, Bunkyo, Tokyo, Japan Endoplasmic reticulum (ER) stress is a cellular condition in which unfolded proteins accumulate in the ER because of various but specific causes. Physiologic ER stress occurs transiently during myoblast differentiation, and although its cause remains unknown, it plays a critical role in myofiber formation. To examine the mechanism underlying ER stress, we monitored ER morphology during differentiation of murine myoblasts. Novel ER-derived structures transiently appeared prior to myoblast fusion both in vitro and in vivo. Electron microscopy studies revealed that these structures consisted of pseudoconcentric ER cisternae with narrow lumens. Similar structures specifically formed by pharmacologically induced ER Ca2+ depletion, and inhibition of ER Ca2+ efflux channels in differentiating myoblasts considerably suppressed ER-specific deformation and ER stress signaling. Thus, we named the novel structures stress-activated response to Ca2+ depletion (SARC) bodies. Prior to SARC body formation, stromal interaction molecule 1 (STIM1), an ER Ca2+ sensor protein, formed ER Ca2+ depletion-specific clusters. Furthermore, myoblast differentiation manifested by myoblast fusion did not proceed under the same conditions as inhibition of ER Ca2+ depletion. Altogether, these observations suggest that ER Ca2 + depletion is a prerequisite for myoblast fusion, causing both physiologic ER stress signaling and SARC body formation.—Nakanishi, K., Kakiguchi, K., Yonemura, S., Nakano, A., Morishima, N. Transient Ca2+ depletion from the endoplasmic reticulum is critical for skeletal myoblast differentiation. FASEB J. 29, 000–000 (2015). www.fasebj.org ABSTRACT

Key Words: ER deformation • ER stress • myoblast fusion STIM1

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FOR DECADES, SKELETAL muscle cells have been used as model systems for studying regulatory mechanisms of cell differentiation. Cell culture studies and genetic analyses have revealed a framework of genetic controls that Abbreviations: 2-APB, 2-aminoethoxydiphenyl borate; ATF6, activation transcription factor 6; BFA, brefeldin A; CHOP, CCAAT/enhancer binding protein-homologous protein; CLEM, correlative light-electron microscopy; CPA, cyclopiazonic acid; DM, differentiation medium; ER, endoplasmic reticulum; GFP, green fluorescent protein; IP3R, (continued on next page)

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induce muscle-specific gene expression. The MyoD family of muscle-specific transcription factors plays a central role in muscle-specific gene expression and is induced in muscle precursor cells (myoblasts) by the action of morphogenic molecules (1). In addition to induction of muscle-specific proteins, skeletal myoblast differentiation is accompanied by multiple rounds of myoblast fusion that ultimately generate a myofiber syncytium. Myoblast fusion is controlled by additional regulatory mechanisms that require ER stress (2). ER stress was originally defined as accumulation of unfolded proteins in the ER (3). However, ER stress is also caused by nonprotein factors, such as oxidative stress, glucose shortage, or ER Ca2+ depletion. ER stress modifies gene expression and post-translational modifications, thereby altering cell physiology (4, 5). Primarily, ER stress activates a cytoprotective signaling cascade known as the unfolded protein response or ER stress signaling, which manages ER stress. We previously demonstrated that ER stress occurs prior to cell fusion in differentiating myoblasts under physiologic conditions (2). Although ER stress activates caspases and induces apoptosis in a subpopulation of differentiating myoblasts, it is also important for differentiation because blocking ER stress signaling inhibits synthesis of muscle-specific proteins, cell fusion, and apoptosis. Hence, ER stress signaling in differentiating myoblasts does not necessarily play a defensive role, but can also act as a driving force of differentiation. A recent study suggested that apoptotic myoblasts stimulate specific cell surface receptors in live myoblasts leading to enhanced fusion of surviving cells (6). Therefore, ER stress contributes to successful management of myogenesis by activating ER stress signaling in live myoblasts and also by generating apoptotic cells as “helpers.” 1 Current affiliation: Lipid Biology Laboratory, Chief Scientist Laboratories, RIKEN, Wako, Saitama, Japan. 2 Current affiliation: Ultrastructural Research Team, RIKEN Center for Life Science Technologies, Kobe, Hyogo, Japan. 3 Current affiliation: Live Cell Molecular Imaging Research Team, RIKEN Center for Advanced Photonics, Wako, Saitama, Japan. 4 Correspondence: Lipid Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. E-mail: morishim@ riken.jp doi: 10.1096/fj.14-261529 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

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Figure 1. ER deformation at the stage of myoblast fusion. Data shown are from 1 experiment, representative of 6 independent experiments performed with different batches of cells. A) ER in proliferating C2C12 cells stained with ER-Tracker Blue-White DPX. Scale bar, 20 mm. B) Globular structures (arrows) appear on the ER after culturing cells in DM. Scale bar, 20 mm. C) Globular structures at the cell alignment stage. Scale bar, 20 mm. (D) Globular structures are smaller in multinucleated cells. They are indicated by arrows in a mononucleated cell for comparison. Scale bar, 50 mm. Insets in B, C, and D are higher magnification photomicrographs of the respective boxed areas. E) Fused cells containing 10 nuclei. Scale bar, 50 mm.

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By enhancing ER stress signaling, we previously succeeded in increasing efficiency of apoptosis and contracting myofiber formation (7). Myoblast fusion was increased by pharmacological ER stressors acting through different mechanisms, including inhibition of sarcoplasmic reticulum (SR)/ER-specific Ca2+-ATPase (SERCA), suggesting that ER stress signaling drives myoblast differentiation irrespective of the cause of ER stress. Because the cause of physiologic ER stress in differentiating myoblasts remains elusive, here we examined the mechanism underlying ER stress signaling during myoblast differentiation by monitoring ER morphology during cell fusion. We identified novel ER-derived structures that may be markers of ER Ca2+ depletion in differentiating myoblasts. Moreover, these structures and the behavior of ER Ca2+ sensor proteins suggest that Ca2+ depletion evokes ER stress in differentiating myoblasts. MATERIALS AND METHODS Cell culture and transfection C2C12 cells (RIKEN Cell Bank, Tsukuba, Ibaraki, Japan) were grown in DMEM (Life Technologies, Rockville, MD, USA) supplemented with 20% fetal bovine serum (Life Technologies) at 37°C in 5% CO2. Cell differentiation was induced by adding 2% horse serum (Life Technologies) and 1 mg/ml insulin (SigmaAldrich, St. Louis, MO, USA) to the medium. Cell transfections were performed using FuGENE HD Transfection Reagent (Promega, Madison, WI, USA) according to the manufacturer’s protocol.

(continued from previous page) inositol 1,4,5-trisphosphate receptor; PBS, phosphate buffered saline; SARC, stress-activated response to Ca2+ depletion; SERCA, sarcoplasmic/endoplasmic reticulum calcium ATPase; SR, sarcoplasmic reticulum; STIM1, stromal interaction molecule 1; TG, thapsigargin; TUN, tunicamycin

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Reagents ER-Tracker Blue-White DPX, ER-Tracker Green, and BODIPY TR-X thapsigargin were from Life Technologies and were used according to the manufacturer’s protocols. Thapsigargin (TG), cyclopiazonic acid (CPA), 2-aminoethoxydiphenyl borate (2-APB), dantrolene, brefeldin A (BFA), and DAPI were purchased from Sigma-Aldrich, and tunicamycin (TUN) and A23187 from Merck Millipore (Darmstadt, Germany). Inhibitors were used at the following concentrations: TG, 10 nM; CPA, 40 mM; TUN, 1 or 2 mg/ml; BFA, 0.8 mg/ml; A23187, 10 mm; 2-APB, 100 mM; and dantrolene, 20 mM. Plasmid DNAs Sec12 and STIM1 cDNAs were obtained from the Genome Exploration Research Group, Genomic Science Center of RIKEN (8). The other cDNAs were PCR amplified from mouse pancreas or kidney cDNA pools (Zyagen, San Diego, CA, USA). Green fluorescent protein (GFP)-tagged STIM1 cDNA was created by inserting the AcGFP coding sequence (Takara Bio, Otsu, Shiga, Japan), tagged with SacII recognition sequences at both ends, into amino acid 39 of STIM1 (9) in pcDNA 3.1 (2) (Life Technologies). Six bases (CCGCGG) had been inserted at this position using the Quickchange II Site-Directed Mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA). Other cDNAs were cloned into the vectors pEGFPC1, pEGFPN3, and pAcGFPN3 (Takara Bio). To detect inositol 1,4,5-trisphosphate receptor (IP3R), cDNA encoding the C-terminal channel domain of IP3R (amino acids 2261–2749) (10) was fused to EGFPC1 (Takara Bio) by the linker sequence TCCGGA, and the resulting cDNA was transfected into C2C12 cells. Live cell imaging C2C12 cells were stained with ER-Tracker Blue-White DPX and observed under a microscope (Model IX-70; Olympus, Shinjuku, Tokyo, Japan) equipped with an ORCA-cooled charge-coupled

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device camera (Hamamatsu Photonics, Hamamatsu, Shizuoka, Japan) and a 340 LCPlanF1 lens (0.60 NA). For time-lapse imaging, cells were grown in glass bottom dishes (MatTek, Ashland, MA, USA), and time-lapse images were acquired using an FV1000D confocal microscope (Olympus). ER in C2C12 cells was labeled with transiently expressed GFP-tagged Sec12 protein. All images were acquired at 360 magnification using a PLAPON 60XO oil objective lens (1.42 NA). Images were processed using FV10-ASW software (Olympus) and edited with MetaMorph (Molecular Devices, Sunnyvale, CA, USA). Representative data from 9 independent experiments are presented in Supplementary Movie 1. Immunocytochemistry C2C12 cells were grown on 8-chamber slides (Thermo Fisher Scientific, Waltham, MA, USA). Cells were fixed in 4% paraformaldehyde/phosphate buffered saline (PBS) at room temperature for 10 minutes and permeabilized in 0.1% Triton X-100 at room temperature for 20 minutes. Fixed and permeabilized cells were blocked in PBS containing 3% BSA (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and incubated at 4°C overnight with the primary antibodies, antiCCAAT/enhancer binding protein-homologous protein (CHOP) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-myosin (MY-32; Sigma-Aldrich). Immunoreactivity was detected using a combination of biotin-conjugated secondary antibody (Vector Laboratories, Burlingame, CA, USA) and Alexaconjugated streptavidin (Life Technologies). Cell specimens were embedded inProLongGoldmountingmedium(LifeTechnologies). ER-Tracker Green staining was observed before specimens were embedded in mounting medium. CHOP immunostaining was performed separately from ER-Tracker staining. Immunohistochemistry Frozen sections of unfixed mouse embryos were purchased from Genostaff (Bunkyo, Tokyo, Japan). Sections were fixed in 4% paraformaldehyde/PBS at 4°C for 10 minutes and then blocked in PBS containing 2% normal donkey serum (Jackson ImmunoResearch Laboratories). Fixed sections were incubated at 4°C overnight with an anti-myosin antibody (MY-32) in 1% BSA/PBS. Immunoreactivity was detected using a biotin-conjugated antimouse IgG antibody (Jackson ImmunoResearch Laboratories) and Avidin-Alexa 594 (Life Technologies). ER was visualized using ER-Tracker Blue-White DPX staining.

Figure 2. ER deformation in myoblast cells treated with a SR/ ER-specific Ca2+ pump inhibitor. A) Globular structures (arrows) are detected in C2C12 cells after TG treatment for 4 hours. Left, ER-Tracker Green staining; right, CHOP immunostaining; UT, untreated. Scale bars, 20 mm. B) ER membranes are absent from central portions of globular structures. Scale bar, 4 mm. C) Transient appearance of globular structures in TG-treated cells stained with ER-Tracker Blue-White DPX. Scale bars, 20 mm. Data shown are from 1 experiment representative of 5 (A), 5 (B), or 13 (C) independent experiments performed with different batches of cells.

Light microscopy for stained specimens Immunostained images of tissue sections were acquired using an FV1000D confocal microscope (Olympus) and UPlanSApo objective lens (0.4 NA) at 310 magnification, or an UPLSAPO 100XO oil objective lens (1.4 NA) at 3100 magnification. Images were processed using FV10-ASW software (Olympus). Selected images were pseudocolored for presentation using ImageJ software (National Institute of Health, Bethesda, MD, USA). CHOP immunostaining was observed using a BX51 fluorescent microscope and UPlanApo lens (0.70 NA, Olympus) at 320 magnification. 5GFP-tagged STIM1 was observed using a LSM700 confocal microscope (Zeiss, Oberkochen, Germany) and C-APOCHOROMAT water objective lens (1.2 NA) at 363 magnification. For 3-dimensional reconstruction of SARC bodies, ER was stained using ER-Tracker Green and observed with a CV1000 confocal microscope (Yokogawa Electric Corporation, Musashino, Tokyo, Japan). Three-dimensional images were reconstructed from Z-stacked images using ImageJ software.

ER CA2+ DEPLETION DURING MYOBLAST DIFFERENTIATION

Correlative light electron microscopy on thin sections C2C12 cells were grown in cell culture dishes on coverslips with imprinted relocation grids (Matsunami Glass, Kishiwada, Osaka, Japan), and stained with ER-Tracker Blue-White DPX to detect SARC body-containing cells. Cells were fixed in 2% formaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) at room temperature for 2 hours. Secondary fixation was performed using the same buffer containing 1% osmium tetroxide at 0°C for 1 hour. Cells were dehydrated in a graded series of ethyl alcohol and propylene oxide and then embedded in Poly/Bed 812 (Polysciences, Warrington, PA, USA). Ultrathin sections of embedded cells were prepared using an ultramicrotome, stained with 0.5% aqueous uranyl acetate, and observed using a JEM 1010 transmission electron microscope (JEOL, Akishima, Tokyo, Japan) at an accelerating voltage of 100 kV.

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RESULTS ER-derived globular structures form during myoblast differentiation and in ER Ca2+-depleted myoblasts For live cell imaging of ER membranes, we labeled the ER in myoblast C2C12 cells using an ER-Tracker stain (Fig. 1A). C2C12 cultures were placed in differentiation medium (DM) for terminal differentiation of myoblasts, as reflected by cell alignment, elongation, and fusion (2). Previously unidentified globular structures (1–4 mm in diameter) were observed in these myoblasts, which associated with the ER prior to (Fig. 1B) or upon (Fig. 1C) cell alignment. Globular structures were detectable from day 3, and by day 5, up to 20 globular structures were detected in a single cell. Eventually, these globular structures were present in the majority of differentiating cells and persisted for several days during cell fusion. Subsequently, the structures shrank in fused cells (Fig. 1D) and completely disappeared in cells with approximately 10 nuclei (Fig. 1E), indicating they are transient structures. Appearance of these globular structures in myoblasts was coincident with CHOP induction and caspase-12 activation, both markers of ER stress (2). The time course of these events suggests that ER stress caused formation of the novel structures. To determine if pharmacological ER stress can induce these globular structures, we treated proliferating C2C12 cells with representative ER stressors that act through different mechanisms. TG (11), an inhibitor of SERCA, blocks SERCA-mediated Ca2+ transfer into the ER (12), leading to ER Ca2+ depletion (13). We used TG at a relatively low concentration (10 nM), which was approximately a hundredth of that used for efficient induction of apoptosis (14), to ensure that ER stress signaling was elicited without extensive cellular apoptosis. TG treatment induced formation of globular structures similar to those found in differentiating cells (Fig. 2A). Most, if not all, of the structures were ring-shaped (Fig. 2B) and appeared at 2–36 hours after adding TG (Fig. 2C). However, the number and size of these structures decreased gradually from approximately 8 hours (Fig. 2C), as observed using time-lapse imaging (Supplementary Movie 1). This sequence of events was similar to that in differentiating myoblasts. Similarities between TG- and DMinduced structures were confirmed by electron microscopy (see below). ER stress signaling was activated by low-dose TG treatment, as shown by induction of the transcription factor

CHOP (Fig. 2A) (15). CPA, another SERCA inhibitor (16), also induced CHOP and similar globular structures (Supplemental Fig. 1A). However, TUN (17), an inhibitor of N-linked glycosylation, elicited ER stress signaling without inducing globular structures (Supplemental Fig. 1B). Overall, these results suggest that formation of globular structures and ER stress signaling are independent of each other. BFA, an inhibitor of GTP exchange factors in the Golgi apparatus (18), induced CHOP and small vesicle-like structures (Supplemental Fig. 1C). However, these structures exhibited a micromorphology that was distinct from those induced by TG or CPA (see below), suggesting that formation of globular structures is dependent on a specific type of ER stress, specifically that caused by ER Ca2+ depletion. Thus, we tentatively named these globular structures as SARC bodies. To determine if ER Ca2+ depletion occurs in differentiating myoblasts, we examined the behavior of GFP-STIM1 fusion proteins expressed in C2C12 cells. Maintaining ER Ca2+ concentrations at normal levels ensures uniform GFPSTIM1 localization on the ER in various cell types (e.g., PC12, Jurkat, and HeLa). However, ER Ca2+ depletion causes cluster formation of GFP-STIM1 at contact sites between the ER and plasma membrane (e.g., see references 9, 19, 20). Clustered STIM1 proteins can activate ORAI1 Ca2+ ion channels on the plasma membrane, inducing Ca2+ influx from extracellular spaces into the ER (21). Consistent with previous observations, GFP-STIM1 expressed at relatively low levels in C2C12 cells showed a typical reticular pattern (Fig. 3A). In a control experiment, GFP-STIM1 clusters appeared within 15 minutes after treating transfectants with 10 nM TG (Fig. 3B), confirming that GFP-STIM1 redistributes in response to ER Ca2+ depletion. Appearance of GFP-STIM1 clusters was followed by SARC body formation, which occurred after ;2 hours of TG treatment (Fig. 2C). At higher expression levels in untreated cells, GFP-STIM1 proteins formed many amorphous sheets, suggesting abnormal localization and/ or aggregation of the expressed proteins (data not shown). We excluded these GFP-STIM1 sheets from further analysis because redistribution of GFP-STIM1 did not appear to occur after TG treatment (data not shown). Under differentiation conditions, GFP-STIM1 redistribution was observed at day 1, with dots of transient GFP-STIM1 signal (Fig. 3C), suggesting that ER Ca2+ depletion occurs before redistribution during myoblast differentiation. This observation supports the proposition

Figure 3. STIM1 clustering occurs in differentiating myoblasts. A) STIM1 was detected by overexpression of GFP-tagged proteins in C2C12 cells. A representative image from 7 independent experiments. B) GFP-STIM1 cluster formation in C2C12 cells treated with 10 nM TG for 50minutes.Arepresentativeimage obtained with data shown in (A). C) Mixed pattern of GFP-STIM1 localization in differentiating C2C12 cells. Transfectants were incubated in DM for 23 hours. Scale bars, 20 mm. A representative photograph from 2 independent experiments.

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that transient ER Ca2+ depletion in differentiating myoblasts causes ER stress and activation of the unfolded protein response. Unlike redistribution in TG-treated transfectants, both GFP-STIM1 dots and reticular patterns were observed in single cells, implying less marked ER Ca2+ depletion than in TG-treated cells. SARC body formation is dependent on Ca2+ release from ER Ca2+ channels TG does not directly induce Ca2+ depletion from the ER but inhibits ER Ca2+ uptake of SERCA, leading to Ca2+ leakage through various ER channels (13). To confirm dependence of SARC body formation on decreased ER

Ca2+ concentrations, we treated myoblast cells with TG in the presence of ER Ca2+ channel inhibitors. Myoblasts express the IP3R and ryanodine receptor, with the former being the major Ca2+ channel in the ER of undifferentiated myoblasts (22). Both 2-APB, an IP3R inhibitor (23), and dantrolene, a ryanodine receptor inhibitor (24), considerably suppressed SARC body formation, with only 1 or 2 SARC bodies detected in approximately 10 and 30% of cells, respectively (Fig. 4A). The inhibitory effect of dantrolene was lower than that of 2-APB, reflecting differing expression levels of the corresponding receptors. Combined 2-APB and dantrolene treatment resulted in near complete suppression of SARC bodies (Fig. 4A), indicating that SARC body formation is dependent on Ca2+ depletion from the ER, but independent of decreased Ca2+ influx into the ER per se.

Figure 4. ER Ca2+ depletion causes SARC body formation. A) Inhibition of Ca2+ release from the ER suppressed formation of SARC bodies induced by TG. C2C12 cells were treated with TG (4 hours), with or without ER-specific Ca2+ channel inhibitors. SARC bodies formed when inhibitors were used independently (arrows). Scale bars, 20 mm. The experiment shown is representative of 3 independent replicates. B) A23187 treatment (9 hours) induced SARC body formation (left) and CHOP activation (right). Scale bars, 20 mm. Similar images were obtained in 2 independent experiments. C) Combination of 2-APB and dantrolene counteracted A23187-induced SARC body formation. Scale bars, 20 mm. Similar images were obtained in 2 independent experiments. D) Visualization of endogenous Ca2+-regulating proteins. (Left) C2C12 cells were treated with 100 nM BODIPY TR-X TG for 5 hours, and then cells counterstained with ER-Tracker Blue-White DPX before observation by fluorescent microscopy. Note that fluorescent dye alone induced SARC body formation. (Right) The IP3R channel domain (amino acids 2261–2749) fused to EGFP was expressed in C2C12 cells. At 24 hours post-transfection, SARC bodies were induced by 10 nM TG treatment for 4 hours and detected using ER-Tracker Blue-White DPX staining. Scale bars, 20 mm. Data shown are from 1 experiment, representative of 4 (TR-X TG) independent experiments performed with different batches of cells. Images of GFPIP3R in C2C12 cells were obtained from a single experiment.

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Ca2+ release from the ER is accompanied by increased cytosolic Ca2+ (11), which is used by Ca2+ binding proteins such as calmodulin to transmit important physiologic signals. To exclude the possibility that increased cytosolic Ca2+ may contribute to SARC body formation, we treated myoblasts with the calcium ionophore A23187, which chelates Ca2+ ions and causes influx through the plasma membrane (25). Cellular Ca2+ uptake results in Ca2+ depletion from the ER (e.g., see reference 26), although the mechanism of ER Ca2+ release has not yet been revealed (e.g., see reference 27), as in the case of ionomycin, another calcium ionophore (28). Therefore, A23187 induces both an increase in cytosolic Ca2+ and decrease in ER Ca2+ levels. After treatment with A23187, SARC bodies and CHOP expression were detected in myoblasts (Fig. 4B). However, although blockade of ER Ca2+ release with 2-APB and dantrolene had minimal effect on A23187-mediated influx of extracellular Ca2+, it prevented induction of SARC body formation (Fig. 4C). Thus, these results indicate that SARC body formation is dependent on ER Ca2+ depletion and not on increased cytosolic Ca2+. To further examine the mechanisms underlying SARC body formation, we examined SERCA and IP3R distribution in the ER. Using a fluorescent analog of TG, SERCA proteins were homogeneously distributed throughout the ER including SARC bodies (Fig. 4D). To investigate IP3R localization, we overexpressed GFP-tagged IP3R because commercially available anti-IP3R antibodies did not work for immunocytochemistry in C2C12 cells (data not shown). However, neither N- nor C-terminal-tagged IP3R localized to the ER, which showed a cytoplasmic distribution (data not shown) inconsistent with previous studies by other groups (e.g., see reference 10). A GFP-tagged channel domain (10) expressed at relatively low levels shows a reticular pattern, although GFP-tagged proteins tend to form aggregates when expressed at higher levels (10). In TGtreated cells, GFP-tagged protein was predominantly distributed in SARC bodies compared with other ER regions (Fig. 4D), suggesting that SARC body formation may depend on differences in local concentrations of ER Ca2+ channels that leak luminal Ca2+.

Figure 5. Structural features of SARC bodies determined by thin section electron microscopy. A) SARC bodies induced in DM (day 3). Scale bar, 1 mm. B) SARC bodies in C2C12 cells treated with either TG (5.5 hours) or CPA (4 hours). Scale bars, 1 mm. (C) Cytoplasmic components trapped in a SARC body. mt, mitochondrion; *, lipid droplet. Arrow indicates open rings. Scale bars, 0.5 mm. D) SARC bodies are characterized by a lack of ribosomes and often surrounded by rough ER (arrows). A SARC body induced by TG for 4 hours. Scale bar, 1 mm. Enlarged images of the boxed areas (upper, SARC body; lower, rough ER) are shown on the right. Ribosomes were detected on rough ER (lower). E) Ribosomes trapped inside SARC bodies in C2C12 cells cultured in DM (day 3). Scale bar, 0.4 mm. Data shown are from 1 experiment, representative of 4 (DM), 2 (TG), and 1 (CPA) independent experiments performed with different batches of cells.

SARC bodies are composed of rough ER-derived sheets in a pseudoconcentric shape To examine SARC body structure in detail, we used correlative light-electron microscopy (CLEM) in cells stained with ER-Tracker. CLEM revealed that SARC bodies in differentiating myoblasts were comprised of 4–10 pseudoconcentric, almost equally spaced rings per SARC body (Fig. 5A). Similar ring structures were detected in cells after TG or CPA treatment (Fig. 5B), but with hollow regions in the center that sometimes contained cytoplasmic particles (Fig. 5C, left). In pharmacologically induced SARC bodies, open ring structures were often found at later stages, possibly reflecting an initial stage of disappearance (Fig. 5C, right). SARC bodies were surrounded by rough ER but characterized by a lack of ribosomes (Fig. 5D), suggesting they formed from a rough ER subdomain. Furthermore, SARC body walls appeared to be formed from sheets (Fig. 5A–D), which are commonly found in rough ER (29). In 6

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differentiating myoblasts, ribosome-like dense dots were often detected between the inner cisternae of SARC bodies (Fig. 5E), suggesting that rough ER subdomains may have been transformed into SARC bodies during differentiation. Rings in SARC bodies were not interconnected, and their pseudoconcentric structures were not coiled. Serial sections through a SARC body induced in DM revealed that each ring was independent (Fig. 6A), with the number of pseudoconcentric circles highest at the quasi-equatorial plane, suggesting that these membranes were stacked like the layers of an onion. Alternatively, the inner cisternae may have formed pseudoconcentric spheres isolated from other regions of the ER. Three-dimensional image reconstructions of SARC bodies showed either cup-shaped or spherical cisternae in sagittal planes generated by virtual sectioning of the SARC body (Fig. 6B), therefore both possibilities are supported (Fig. 6C).

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Figure 6. Membrane structure of multilayered SARC bodies. A) Serial sectioning of a SARC body induced in DM (day 3). Photomicrographs of selected sections are arranged in the sectioning sequence. Scale bars, 1 mm. Data shown are from 1 experiment, representative of 2 independent experiments performed with different batches of cells. B) Three-dimensional image reconstruction of SARC bodies. Two representative sagittal plane images of SARC bodies with line drawings of their cisternae are shown. The 2 photomicrographs used for reconstruction are shown to the left of each sagittal view. SARC bodies in boxed areas were selected for image reconstruction. Insets (upper right) show higher magnifications of boxed areas and optical section positions. Scale bars, 10 mm. Data shown are from 1 experiment, representative of 3 independent experiments performed with different batches of cells. C) Possible SARC body geometries.

ER Ca2+ depletion specifically causes narrowing of the ER lumen Under differentiation conditions, SARC bodies had narrower lumens (approximately 20 nm) than rough ER (Fig. 7A). However, the cisternae of SARC bodies induced by TG (for 5.5 hours) had no apparent luminal spaces (Fig. 7A). Notably, proteins typically abundant in the ER lumen (binding protein, calnexin, calreticulin, and Grp94) were not detected by immunostaining in SARC bodies (data not shown), possibly because of the limited luminal space. At later time points (25.5 hours), SARC bodies were smaller yet the luminal space was restored to approximately 20 nm and similar to differentiating myoblasts (Fig. 7A). These results support the notion that TG treatment causes more severe ER Ca2+depletion than DM treatment. Rough ER adjacent to SARC bodies induced by DM or TG treatment (for 25.5 hours) had similar luminal widths, but ER distant from SARC bodies had luminal widths of 40– 80 nm (Fig. 7B), which is similar to rough ER in proliferating (Fig. 7A) and COS7 (30) cells. This further indicates that SARC bodies are connected to rough ER. We designed subsequent experiments to determine if ER luminal spaces reduced after treatment with ER stressors that do not deplete ER Ca2+. ER volumes increased in TUN-treated myoblasts (Supplemental Fig. 2A), with rough ER widths greater than 80 nm (and up to 150 nm), ER CA2+ DEPLETION DURING MYOBLAST DIFFERENTIATION

and roughly double the width of untreated cells. In BFAtreated cells, rough ER widths increased to 150–200 nm (Supplemental Fig. 2A). Overall, these results suggest that following ER stress, ER lumen reductions are Ca2+ depletion specific. BFA treatment induced formation of multilayered vesicular structures on the ER (Supplemental Fig. 1C). These were deemed distinct from SARC bodies as they had larger luminal spaces (25–100 nm) (Supplemental Fig. 2B) and cisternae walls with a wavy appearance, implying these membranes are not intact (Supplemental Fig. 2B). Because BFA inhibits retrograde transport of lipids and proteins from the Golgi apparatus and also induces redistribution of resident Golgi proteins to the ER (31), these membranes may have been modified. On the basis of these detailed structures, together with our inhibitor results (TG, CPA, 2-APB, and dantrolene), we definitively named these novel structures in differentiating myoblasts as SARC bodies. ER Ca2+ release is necessary for muscle differentiation We next determined if ER Ca2+ release is essential for ER stress signaling that induces SARC body formation and differentiation. In these experiments, C2C12 cells were 7

Figure 7. Reduction of ER luminal spaces in SARC bodies. A) Cisternae within SARC bodies. Arrows indicate ribosomes; UT, untreated; (day 5). Data shown are from 1 experiment, representative of 4 independent experiments. Cytosolic regions between cisternae in TG-treated cells (5.5 hours) are labeled by asterisks (*). Data shown are from 1 experiment, representative of 2 independent experiments. Scale bars, 0.1 mm. B) Rough ER luminal spaces in differentiating (DM, upper) and TG-treated (TG, lower) cells are shown at different magnifications. Data shown are from 1 experiment, representative of 2 independent experiments. Left, circles (DM) or asterisks (TG) indicate SARC bodies. Panels a, b, a’, and b’ correspond to the boxed areas (a, b, a’, and b’) indicated in the respective lower magnification photomicrographs. Scale bars, 10 or 1 mm as indicated. Arrows indicate rough ER. Boxed areas a and a’ show rough ER membrane adjacent to a SARC body; boxed areas b and b’ show rough ER membrane distant from a SARC body within the same cell. Insets show cisternae in the boxed areas in panels a, b, a’, and b’ at higher magnification.

transferred to DM containing 2-APB and dantrolene. Resulting blockade of ER Ca2+ release considerably suppressed CHOP induction (Fig. 8A), and CHOP-positive cells decreased from 60% to ,10% (Fig. 8A), suggesting that blockade of ER Ca2+ release inhibits ER stress signaling during myoblast differentiation. Furthermore, SARC bodies were detected in ,5% of cells after incubation in DM, and skeletal muscle myosin was not expressed (Fig. 8B). Under these conditions, cell fusion did not occur (Fig. 8B). These results are consistent with our observations that ER stress signaling is required for myoblast differentiation (2). Taken together, these data suggest that ER Ca2+ depletion is critical for physiologic ER stress signaling in differentiating myoblasts, and presence of SARC bodies is a marker of these events. To detect SARC bodies in vivo, we examined ER morphology in embryonic muscle tissue at the onset of muscle formation on embryonic day 13.5 (2, 32). We had previously observed transient CHOP induction at embryonic day 13.5, and coincident caspase-12 activation has been demonstrated (2). Embryonic day 13.5 muscle tissues (e.g., intercostal muscles) showed heterogeneous myosin expression in the same tissue (Fig. 9A). Moreover, globular structures were detected in these cells (Fig. 9B), 8

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particularly in myosin-negative regions (Fig. 9A, arrowheads). Most of these globular structures were donutshaped and indistinguishable from SARC bodies found in vitro (Fig. 9B). Closer examination confirmed that these globular structures were less abundant in myosinpositive cells (Fig. 9C, region 1) than in myosin-negative (less differentiated) cells (Fig. 9C, region 2). Cells with higher myosin levels had more intense ERTracker signal, suggesting ER development during myogenesis (Fig. 9D). This is in accordance with expansion of the ER to surround myofibrils. Altogether, these results suggest that SARC bodies are transiently formed during the early stage of muscle development, as observed in vitro. SARC bodies observed in vivo were slightly smaller (1–2 mm) than those in culture, potentially reflecting scaling of organelle size to cell volume (33), as in vivo myoblasts are much smaller than cultured C2C12 cells (major axes: approximately 10 mm vs. 40 mm). DISCUSSION Structural features of SARC bodies (including pseudoconcentric ER cisternae and narrow luminal spaces) are

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Figure 8. ER Ca2+ depletion is important for myoblast differentiation. A) CHOP induction in C2C12 cells cultured in DM (day 2) with or without combined 2-APB and dantrolene. CHOP immunostaining (upper); DAPI staining (lower); GM, growth medium. Scale bars, 50 mm. Percentage of CHOP-positive cells are shown in the bar graph (right). Scale bars represent the average of 3 independent experiments. Data shown are from 1 experiment, representative of 3 independent experiments performed with different batches of cells. B) The effect of 2-APB and dantrolene on SARC body formation and myoblast differentiation. Left, ERTracker staining (day 9); middle, phase contrast (day 8); right, myosin immunostaining (day 4). Scale bars, 20 mm (left); 200 mm (middle, right). Data shown are from 1 experiment, representative of 2 independent experiments performed with different batches of cells.

specifically observed in Ca2+ depleted ER treated with TG or CPA, but not with ER stressors that act through different mechanisms. Appearance of SARC bodies in mononucleated myoblasts indicates that Ca2+ depletion begins prior to cell fusion and supports the notion that transient Ca2+ depletion from the ER occurs prior to myoblast fusion and is critical for myoblast differentiation. Previous reports have shown that Ca2+ signaling dependent on increased cytosolic Ca2+ regulates fusion of myoblasts into multinucleated myotubes (34–36). Moreover, it has been suggested that increased cellular Ca2+ uptake prior to myoblast fusion induces calcineurin (37), which initiates skeletal muscle differentiation by activating MEF2 and MyoD (38). A proposed mechanism for increased Ca2+ in differentiating myoblasts is influx of extracellular Ca2+ through T-type alpha1H Ca2+ channels (36). Our current findings suggest the importance of ER Ca2+ depletion, in addition to increased intracellular Ca2+, as an early event of intracellular Ca2+ dynamics during myoblast differentiation. Although ER Ca2+ depletion is potentially detrimental to cells, it may be necessary to generate the required ER stress for myoblast differentiation. Because ER Ca2+ release increases ER CA2+ DEPLETION DURING MYOBLAST DIFFERENTIATION

cytosolic Ca2+, ER Ca2+ depletion may also contribute, at least partially, to cytosolic Ca2+ signaling activation. Investigating the potential contribution is worthwhile. Our results also indicate that ER Ca2+ depletion causes physiologic ER stress during myogenesis. In a previous study, we found that ER stress in differentiating myoblasts specifically activates the ER stress sensor protein, activation transcription factor 6 (ATF6), 1 of 3 major ER stress sensors (2). Activated ATF6 acts as a transcription factor (39) with a dominant role in ER stress signaling during myoblast differentiation (2). Accordingly, our present findings suggest that ER Ca2+ depletion specifically activates ATF6 during terminal myoblast differentiation. Similar to TG-treated cells, SARC body formation followed GFP-STIM1 clustering in differentiating myoblasts. However, in contrast to TG-treated cells, SARC body formation did not begin several hours after disappearance of clusters in differentiating cells, but was detectable after 2 days at the earliest. It should be noted that ER Ca2+ depletion caused by ER stressors activates all 3 branches of ER stress signaling, while only the ATF6 branch is activated in differentiating cells (2). The long time lapse between 9

Figure 9. SARC bodies transiently appear in developing muscle tissue of embryos on embryonic day 13.5. SARC bodies were detected in 3 mouse embryos, and myosin staining performed with 2 of the embryos. A) Myosin immunostaining in intercostal muscle tissues. Arrows indicate areas where the majority of cells express myosin; triangles indicate areas where myosin-positive and -negative cells coexist to a similar extent. Scale bar, 200 mm. B) SARC bodies transiently appear in developing muscle tissues (embryonic day 13.5). Bottom panel shows SARC bodies in boxed areas at higher magnification. Scale bars, 10 or 2 mm as indicated. C) SARC body density is negatively correlated with myosin expression level. Myosin expression was either higher (top) or lower (bottom) in the same tissue. Merged images show ER-Tracker staining (green) and myosin immunostaining (red). Right panels show the images in boxed areas (1 and 2) at higher magnification. Scale bars, 20 mm. D) ER development in myosinpositive cells. ER-Tracker staining in the upper half of the photomicrograph appears brighter than in the lower half. Scale bars, 20 mm.

STIM1 cluster disappearance and SARC body formation in differentiating cells may depend on differences in the mode of ER stress signaling, which may affect efficiency in transcriptomic or proteomic changes required for SARC body formation. STIM1 behavior under differentiation conditions suggests less marked Ca2+ depletion. Nevertheless, it is possible that Ca2+ depletion ensures the ER exceeds a critical point for this organelle, allowing transmission of ER stress signaling. We found that pharmacologically induced SARC bodies are efficiently formed at low doses (e.g., 10 nM TG), yet less efficiently at higher doses (e.g., 1 mM; data not shown), suggesting that the critical point for ER Ca2+ levels may be within a small window. Hence, further studies are required to determine the precise mechanisms underlying transient ER Ca2+ depletion during normal differentiation. Because ryanodine receptors increase during myogenesis (40), their abundance may enhance ER Ca2+ release. In addition, many proteins regulate either SERCA or Ca2+ channels in myoblast cells. For example, ER membrane 10

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proteins (e.g., sarcolipin and phospholamban) have been identified as SERCA inhibitors (41). Moreover, several anti-apoptotic proteins (members of the Bcl-2 family and Bax inhibitor 1) regulate IP3R, and thereby contribute to calcium homeostasis (42, 43). Micro-RNAs also regulate ER Ca2+ signaling via transcriptional control (44). In addition, IP3R-dependent Ca2+ signaling is induced by IGF1 (45), which is involved in myogenesis control (46). It would be interesting to investigate the role of these potential candidates in regulating programmed Ca2+ depletion at an early stage of myoblast fusion. Recent studies have shown that cellular stresses, or responses to these stresses, modify gene expression and play various physiologic roles during successful differentiation. For example, X-box binding protein 1 mediates the inositol requiring enzyme 1a branch of ER stress signaling (4), and is critical for differentiation of protein secreting plasma cells (47). In addition to ER stress, potentially harmful mitochondrial reactive oxygen species regulate a wide range of physiologic phenomena (48). Absence of

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mitochondrial reactive oxygen species impairs transmission of Notch and b-catenin signals, which are essential for epidermal differentiation and hair follicle development, respectively (49). Taken together with our present data, these studies suggest that various organellespecific cellular stresses, including ER Ca2+ depletion, may transmit differentiation signals in a cell-autonomous manner and act as regulatory mechanisms for differentiation. In this study, we identified transient structures with pseudoconcentric ER cisternae that form during the early stages of myoblast fusion. To the best of our knowledge, this is the first description of globular structures induced by ER Ca2+ depletion (50). SARC body sizes (1–4 mm) were much larger than those of other vesicle structures associated with the ER, including standard COPII vesicles (0.06–0.08 mm), procollagen fibers or chylomicron-specific COPII vesicles (0.3–0.4 mm), COPI vesicles (0.05 mm), and autophagosomes (0.5–1.5 mm). Although autophagosomes are similar to SARC bodies in size, components of autophagosomes such as Atg9A, ULK1, Atg16L, Atg5, Atg12, LC3alpha, LC3beta, GABARAP, GATE16, and Atg8-like protein were not detected in SARC bodies by immunostaining or overexpression of GFP-taggedproteins (data not shown). In addition, SARC bodies were morphologically distinct from autophagosomes, which are double-membrane vesicles. Previous studies by other research groups have shown that ER deformation and ERderived multilayered structures can be artificially formed by overexpression of ER membrane proteins, such as 3-hydroxy-3-methylglutaryl-CoA reductase (51). However, these artificial structures are of smooth ER origin and not accompanied by narrowing of the lumen, which is in contrast to SARC bodies. Myoblast differentiation is accompanied by ER transition to the sarcoplasmic reticulum (SR). SR development is coordinated with appearance of transverse tubules, and junctions form between transverse tubules, the plasma membrane, and SR (40). A classic study found that SR evolves from rough ER (52), although the ER/SR transition is not yet fully understood. An interesting hypothesis may be that the SARC body is a precursor of the SR. The relationship between SARC body formation and SR development during myoblast differentiation should be studied. Unfortunately, it is not easy to investigate SR formation in established cell lines because the SR is poorly developed in vitro (40). Nevertheless, it is unlikely that the SARC body is a building block of the SR because the SARC body structure is quite distinct from that of the SR. SARC bodies are composed of multilayered membranes with very narrow lumens (;20 nm). SR formation apparently begins with multiple tubular projections (single-layered) of the rough ER in various directions (52). Although these tubules are slightly narrower than rough ER, the SR luminal space is 30–50 nm (52), roughly double the SARC body lumen. In addition to these structural differences, the SARC body is transient and disappears without SR formation, at least in vitro. Further investigation of SR formation in vivo is required to determine the relationship between the SARC body and SR. Discovery of ER Ca2+ depletion in differentiating myoblasts was dependent on identification of SARC bodies. Because of their transient nature, SARC bodies may have been overlooked in previous investigations. In addition, ER CA2+ DEPLETION DURING MYOBLAST DIFFERENTIATION

these globular structures could be visualized by ER-Tracker but disappeared following specimen treatment with standard microscopy reagents such as methanol, Triton X-100, and glycerol. Under the same conditions, the reticular ER structure stained by ER-Tracker was maintained (data not shown). Hence, SARC bodies may be fragile and undetectable in the presence of certain chemicals. TG induces SARC body formation, suggesting that SARC bodies may be markers for ER Ca2+ depletion under nonphysiologic conditions. ER Ca2+ depletion alters ER homeostasis, which is potentially harmful under pathologic conditions (53). Therefore, SARC bodies may be markers of decreased ER Ca2+ under various pathologic conditions. Activating ER stress signaling may be an effective means to enhance myofiber formation, because mild treatment of myoblasts with ER stressors enhances myofiber formation (7). Development of treatments for inducing myofiber formation may be facilitated by adopting a controllable method to deplete ER Ca2+ that avoids the potential toxicity of pharmacological ER stressors. In summary, our findings indicate a role for ER Ca2+ depletion in myofiber formation and may contribute to improved therapies for muscle damage or atrophy. The authors thank Tetsuya Tajima and Kaori Higuchi (RIKEN BSI-Olympus Collaboration Center) for technical support with fluorescent microscopy, Takashi Yoshiura (Yokogawa Electric Group) for support with 3-dimensional image reconstruction, and Yasue Ichikawa and Rie Nakazawa for DNA sequencing. K.N. was a Research Fellow of the Japan Society for the Promotion of Science. This work was supported in part by grants from the RIKEN Bioarchitect Research Project (to N.M.), RIKEN Cellular System Program (to N.M.), RIKEN Incentive Research Grant (to N.M.), and Japan Society for the Promotion of Science (to N.M. and K.N.). The authors declare no conflicts of interest.

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