Research Article

CELL BIOLOGY

Splice variants of mitofusin 2 shape the endoplasmic reticulum and tether it to mitochondria

Science

23 Jun 2023

Vol 380, Issue 6651

DOI: 10.1126/science.adh9351IF: 44.7 Q1

Editor’s summary 编辑总结

Inside eukaryotic cells, different types of organelles interact at membrane contact sites that arestabilized by protein bridges called tethers. The mitochondrial fusion protein mitofusin 2 (MFN2) links mitochondria to an unknown partner on the endoplasmic reticulum (ER). Naón et al. found two splice variants of mitofusin 2, ERMIT2 and ERMIN2. ERMIN2 affected ER morphology, and ERMIT2 was localized to the ER and enriched at the contact sites with mitochondria. Treatment with adenovirus encoding for ERMIT2 allowed for the transfer of calcium and lipids between the two organelles and reduced liver damage in models of reduced MFN2 expression. —Stella M. Hurtley
在真核细胞内部，不同类型的细胞器在膜接触位点相互作用，这些接触位点通过称为系链的蛋白质桥来稳定。线粒体融合蛋白 mitofusin 2 (MFN2) 将线粒体与内质网 (ER) 上的未知伙伴连接起来。纳翁等人。发现了线粒体融合蛋白 2 的两个剪接变体，ERMIT2 和 ERMIN2。 ERMIN2 影响 ER 形态，ERMIT2 定位于 ER 并在与线粒体的接触位点富集。在MFN2表达减少的模型中，用编码 ERMIT2 的腺病毒治疗可以实现钙和脂质在两个细胞器之间的转移，并减少肝损伤。 —斯特拉·M·赫特利

Structured Abstract 结构化摘要

INTRODUCTION 介绍

In the cytosol of eukaryotic cells, organelles are closely juxtaposed at membrane contact sites. Membrane contact sites provide hotspots for the metabolic and signaling cascades shared between the interacting organelles. Protein bridges called tethers stabilize membrane contact sites to generate the microenvironment essential for the exchange of metabolites and second messengers. The interaction between the endoplasmic reticulum (ER) and mitochondria at membrane contact sites allows ER-to-mitochondria Ca²⁺ and phosphatidylserine (PS) transfer and serves as a site of autophagy and mitochondrial fission initiation. In mammalian cells, the mitochondrial fusion protein mitofusin 2 (MFN2) also functions as a structural ER-mitochondria tether, but its partner on the ER membrane is unknown.
在真核细胞的胞浆中，细胞器在膜接触位点紧密并置。膜接触位点为相互作用的细胞器之间共享的代谢和信号级联提供了热点。称为系链的蛋白质桥稳定膜接触位点，以产生代谢物和第二信使交换所必需的微环境。内质网 (ER) 和线粒体在膜接触位点之间的相互作用允许 ER 至线粒体 Ca ²⁺和磷脂酰丝氨酸 (PS) 转移，并作为自噬和线粒体裂变起始的位点。在哺乳动物细胞中，线粒体融合蛋白 mitofusin 2 (MFN2) 也起到结构 ER-线粒体系绳的作用，但其在内质网膜上的伴侣尚不清楚。

RATIONALE 基本原理

Interactions between mitofusins on separate mitochondria drive organelle fusion. MFN2 can also support heterotypic organelle interaction, as indicated by the ability of an artificial ER-restricted MFN2 mutant to physically interact with mitofusins on mitochondria. Because of this MFN2 propensity for homotypic interactions, and because mitochondrial dynamics genes are alternatively spliced in variants with divergent subcellular localizations and functions, we hypothesized that the partner of MFN2 on the ER could be a splice variant of the MFN2 gene. We studied MFN2 splice variants dubbed ERMIN2 (ER mitofusin 2) and ERMIT2 (ER mitofusin 2 tether) that we found in human skeletal muscle and mouse fibroblasts.
单独线粒体上的线粒体融合蛋白之间的相互作用驱动细胞器融合。 MFN2 还可以支持异型细胞器相互作用，如人工 ER 限制性 MFN2 突变体与线粒体上的线粒体融合素物理相互作用的能力所表明的那样。由于 MFN2 具有同型相互作用的倾向，并且线粒体动力学基因选择性剪接成具有不同亚细胞定位和功能的变体，我们假设 ER 上 MFN2 的伴侣可能是MFN2基因的剪接变体。我们研究了在人类骨骼肌和小鼠成纤维细胞中发现的称为 ERMIN2 (ER mitofusin 2) 和 ERMIT2 (ER mitofusin 2 tether) 的 MFN2 剪接变体。

RESULTS 结果

ERMIN2 and ERMIT2 were expressed in multiple human tissues and were induced upon stresses that drive ER-mitochondria proximity. We combined imaging and biochemical assays of subcellular localization and found that the MFN2 variants were exclusively at the ER. ERMIT2 and MoV-MFN2 were enriched at the ER-mitochondria interface with their N and C termini exposed to the cytosol. Neither variant was able to correct the disrupted morphology of Mfn2^−/− mitochondria. However, ERMIN2 was essential to shape the ER, and ERMIT2 and MoV-MFN2 were essential to tether it to mitochondria. The coiled coil (CC) domain of ER-located ERMIT2 interacted with the CC regions of the mitochondrial mitofusins, forming a complex that was energetically stable enough to tether the two organelles at least in molecular modeling simulations. Functionally, organelle tethering mediated by ERMIT2 allowed mitochondrial uptake of ER-released calcium in cells. Furthermore, organelle tethering by ERMIT2 allowed for mitochondrial uptake of ER-derived phospholipids in the liver of genetic and diet-induced models of reduced ER-mitochondria juxtaposition. This tethering ultimately helped to alleviate hepatic ER stress and inflammation.
ERMIN2 和 ERMIT2 在多个人体组织中表达，并在驱动 ER 线粒体接近的压力下诱导。我们结合亚细胞定位的成像和生化测定，发现 MFN2 变异仅位于内质网。 ERMIT2 和 MoV-MFN2 富集于 ER-线粒体界面，其 N 和 C 末端暴露于细胞质。这两种变体都无法纠正Mfn2 ^−/−线粒体的破坏形态。然而，ERMIN2 对于形成 ER 至关重要，而 ERMIT2 和 MoV-MFN2 对于将其束缚于线粒体至关重要。位于内质网的 ERMIT2 的卷曲线圈 (CC) 结构域与线粒体线粒体融合蛋白的 CC 区域相互作用，形成一种能量稳定的复合物，至少在分子建模模拟中足以束缚这两个细胞器。从功能上讲，ERMIT2 介导的细胞器束缚允许线粒体摄取细胞内 ER 释放的钙。此外，ERMIT2 的细胞器束缚允许在遗传和饮食诱导的 ER-线粒体并置减少模型的肝脏中线粒体摄取 ER 衍生的磷脂。这种束缚最终有助于减轻肝脏内质网应激和炎症。

CONCLUSION 结论

In this work, we identified alternatively spliced, ER-restricted variants of the single MFN2 gene. These variants acted as the molecular mediators of MFN2 extramitochondrial functions in ER morphology and tethering to mitochondria. We also identified the role of the tethering function of MFN2 in liver pathophysiology. In particular, in the context of nonalcoholic steatohepatitis, ERMIT2 could correct the altered phospholipid transfer from ER to mitochondria and the ensuing hepatic distress. Thus, alternative splicing can extend the function of the mitochondria-shaping machinery beyond the mitochondria, and altered ER-mitochondria communication plays a key role in the pathobiology of metabolic liver diseases.
在这项工作中，我们鉴定了单个MFN2基因的选择性剪接、ER 限制性变体。这些变体充当 MFN2 在内质网形态和线粒体束缚中线粒体外功能的分子介体。我们还确定了 MFN2 的束缚功能在肝脏病理生理学中的作用。特别是，在非酒精性脂肪性肝炎的情况下，ERMIT2 可以纠正从 ER 到线粒体的磷脂转移的改变以及随之而来的肝脏窘迫。因此，选择性剪接可以将线粒体塑造机制的功能扩展到线粒体之外，并且改变的内质网线粒体通讯在代谢性肝病的病理学中发挥着关键作用。

Alternatively spliced, ER-specific *MFN2* variants ERMIN2 and ERMIT2 expand MFN2 function beyond the mitochondria.
选择性剪接的 ER 特异性*MFN2*变体 ERMIN2 和 ERMIT2 将 MFN2 功能扩展到线粒体之外。
(Top) Alternative splicing of the human *MFN2* gene results in two additional transcripts coding for two proteins restricted at the ER. (Bottom left) ERMIT2 is enriched at the ER-mitochondria interface and is the ER partner of mitochondrial mitofusins in tethering, allowing efficient Ca²⁺ and phospholipid transfer between the organelles. (Bottom right) ERMIN2 is not enriched in mitochondria-associated membranes (MAMs), and it regulates ER morphology. IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane; PE, phosphatidylethanolamine.
（上）人类*MFN2*基因的选择性剪接会产生两个额外的转录本，编码两种限制于 ER 的蛋白质。（左下）ERMIT2 在内质网-线粒体界面处富集，是线粒体融合素在束缚中的内质网伴侣，允许细胞器之间有效的 Ca ²⁺和磷脂转移。（右下）ERMIN2 在线粒体相关膜 (MAM) 中不富集，但它调节 ER 形态。 IMM，线粒体内膜； OMM，线粒体外膜； PE，磷脂酰乙醇胺。

Open in viewer

Abstract 抽象的

In eukaryotic cells, different organelles interact at membrane contact sites stabilized by tethers. Mitochondrial mitofusin 2 (MFN2) acts as a membrane tether that interacts with an unknown partner on the endoplasmic reticulum (ER). In this work, we identified the MFN2 splice variant ERMIT2 as the ER tethering partner of MFN2. Splicing of MFN2 produced ERMIT2 and ERMIN2, two ER-specific variants. ERMIN2 regulated ER morphology, whereas ERMIT2 localized at the ER-mitochondria interface and interacted with mitochondrial mitofusins to tether ER and mitochondria. This tethering allowed efficient mitochondrial calcium ion uptake and phospholipid transfer. Expression of ERMIT2 ameliorated the ER stress, inflammation, and fibrosis typical of liver-specific Mfn2 knockout mice. Thus, ER-specific MFN2 variants display entirely extramitochondrial MFN2 functions involved in interorganellar tethering and liver metabolic activities.
在真核细胞中，不同的细胞器在由系链稳定的膜接触位点相互作用。线粒体融合蛋白 2 (MFN2) 充当膜系链，与内质网 (ER) 上的未知伙伴相互作用。在这项工作中，我们确定了MFN2剪接变体 ERMIT2 作为 MFN2 的 ER 束缚伴侣。 MFN2的剪接产生了 ERMIT2 和 ERMIN2，这是两种 ER 特异性变体。 ERMIN2 调节 ER 形态，而 ERMIT2 定位于 ER-线粒体界面，并与线粒体线粒体融合蛋白相互作用以束缚 ER 和线粒体。这种束缚允许有效的线粒体钙离子摄取和磷脂转移。 ERMIT2 的表达改善了肝脏特异性Mfn2敲除小鼠典型的 ER 应激、炎症和纤维化。因此，ER特异性MFN2变体表现出与细胞器间束缚和肝脏代谢活动有关的完全线粒体外MFN2功能。

In eukaryotic cells, individual organelles interact at membrane contact sites (MCSs)—areas of close juxtaposition that facilitate the exchange of ions, metabolites, and lipids (1, 2). The endoplasmic reticulum (ER)–mitochondria juxtaposition epitomizes the importance of MCSs in cell biology. Mitochondria-ER MCSs are required for Ca²⁺ signaling (3, 4), apoptosis (5, 6), mitochondrial fission (7), autophagosome formation (8, 9), phosphatidylserine metabolism (10), and even for nutrient sensing in hypothalamic neurons (11). The ER-mitochondria interface is stabilized by 16- to 30-nm protein bridges between the two organelles (12). The mitochondrial fusion protein mitofusin (MFN) 2 also tethers the two organelles and regulates communication between them (13–22). Several additional ER-mitochondria tethers have also been identified, including the ERMES complex in yeast (23) and the ER VAPB-mitochondrial PTPIP51 couple in mammals (24). These tethers are commonly formed by a protein interacting in trans with its partner on the opposing organelle. However, the molecular partner of MFN2 on the ER that allows tethering without heterotypic organelle fusion is unknown. This uncertainty complicates our understanding of the consequences of MFN2 deficiency, including both mitochondrial and ER alterations (10, 25, 26). It is unclear whether Mfn2 deletion affects the ER because of its role in tethering or because of disrupted mitochondrial dynamics that commonly disturbs the ER (27–29). For example, in nonalcoholic steatohepatitis models, the observed reduction in MFN2 levels can affect mitochondrial dynamics or ER-mitochondria communication—two very different potential pathogenic mechanisms (10). Knowledge of the ER partner of MFN2 could help disentangle them.
在真核细胞中，各个细胞器在膜接触位点 (MCS) 处相互作用，膜接触位点是紧密并置的区域，促进离子、代谢物和脂质的交换 ( 1 , 2 )。内质网 (ER)-线粒体并置体现了 MCS 在细胞生物学中的重要性。线粒体-ER MCS 是 Ca ²⁺信号传导 ( 3 , 4 )、细胞凋亡 ( 5 , 6 )、线粒体裂变 ( 7 )、自噬体形成 ( 8 , 9 )、磷脂酰丝氨酸代谢 ( 10 ) 甚至营养传感所必需的下丘脑神经元（ 11 ）。 ER-线粒体界面通过两个细胞器之间的 16 至 30 nm 蛋白质桥来稳定 ( 12 )。线粒体融合蛋白线粒体融合蛋白 (MFN) 2 也连接两个细胞器并调节它们之间的通讯 ( 13 – 22 )。还鉴定了其他几种 ER-线粒体系链，包括酵母中的 ERMES 复合体 ( 23 ) 和哺乳动物中的 ER VAPB-线粒体 PTPIP51 对 ( 24 )。这些系链通常是由蛋白质与其相对细胞器上的伴侣反式相互作用形成的。然而，ER 上 MFN2 的分子伴侣（允许束缚而无需异型细胞器融合）尚不清楚。这种不确定性使我们对 MFN2 缺乏的后果的理解变得复杂，包括线粒体和 ER的改变 (10,25,26 ) 。目前尚不清楚 Mfn2 缺失是否会影响内质网，因为它在束缚中发挥作用，还是因为线粒体动力学被破坏而影响内质网 ( 27 – 29 )。例如，在非酒精性脂肪性肝炎模型中，观察到的 MFN2 水平降低可能会影响线粒体动力学或 ER-线粒体通讯，这是两种截然不同的潜在致病机制 ( 10 )。了解 MFN2 的 ER 伙伴可以帮助解开它们。

Alternative splicing expands the genome functional repertoire by generating multiple proteins with different intracellular localizations and functions (30). For example, alternative splicing of the mitochondrial fission executor dynamin related protein 1 gene encodes a brain-specific isoform that regulates dendrite formation independently of mitochondrial fission (31, 32).
选择性剪接通过产生具有不同细胞内定位和功能的多种蛋白质来扩展基因组功能库 ( 30 )。例如，线粒体裂变执行器动力相关蛋白 1 基因的选择性剪接编码一种大脑特异性亚型，该亚型独立于线粒体裂变调节树突形成 ( 31 , 32 )。

We decided to look for the partner of MFN2 that enables ER-mitochondrial tethering. Given this propensity of mitochondrial dynamics genes to undergo alternative splicing in variants with different subcellular localizations and functions, and because of MFN2’s propensity to engage in homotypic interactions (33), we hypothesized that a MFN2 gene splice variant could be the ER partner of MFN2.
我们决定寻找 MFN2 的合作伙伴来实现 ER 线粒体束缚。鉴于线粒体动力学基因在具有不同亚细胞定位和功能的变体中进行选择性剪接的倾向，并且由于 MFN2 参与同型相互作用的倾向 ( 33 )，我们假设MFN2基因剪接变体可能是 MFN2 的 ER 伴侣。

Human and mouse mitofusin 2 gene are alternatively spliced
人和小鼠线粒体融合蛋白 2 基因交替剪接

We amplified human skeletal muscle cDNAs by polymerase chain reaction (PCR) using primers within intron 2 and exon 19 of the canonical MFN2 DNA sequence. In addition to the known MFN2 sequence, we identified two shorter amplicons that we cloned and sequenced. The 1330–base pair (bp)–long mRNA of the first variant dubbed ERMIN2 (for ER mitofusin 2) resulted from alternative splicing between exons 3a and 3c and between exons 6a and 15b. The mRNA of the second variant that we called ERMIT2 (for ER mitofusin 2 tether) was 1220 bp long and resulted from alternative splicing between exons 4a and 13b (Fig. 1A
我们使用规范MFN2 DNA 序列的内含子 2 和外显子 19 内的引物，通过聚合酶链反应 (PCR) 扩增人类骨骼肌 cDNA。除了已知的MFN2序列之外，我们还鉴定了两个较短的扩增子，并对其进行了克隆和测序。第一个变体的 1330 个碱基对 (bp) 长的 mRNA 被称为ERMIN2 （ER 线粒体融合蛋白 2），是由外显子 3a 和 3c 之间以及外显子 6a 和 15b 之间的选择性剪接产生的。我们称之为ERMIT2 （ER mitofusin 2 系链）的第二个变体的 mRNA 长 1220 bp，是由外显子 4a 和 13b 之间的选择性剪接产生的（图 1A）). Ribonuclease protection assay (RPA) confirmed that the mRNAs of ERMIN2 and ERMIT2 were expressed in HeLa cells (Fig. 1B
）。核糖核酸酶保护实验（RPA）证实HeLa细胞中表达了ERMIN2和ERMIT2的mRNA（图1B）). We next set up real-time PCR assays to specifically detect mRNAs of ERMIN2, ERMIT2, and MFN2 in human samples (fig. S1A). We found that ERMIN2 accounted for 20 to 25%, whereas ERMIT2 accounted for 7 to 52% of MFN2 mRNA expression in white adipose tissue (WAT), skeletal muscle, and liver from human subjects (fig. S1B). We next monitored whether specific exogenous stresses affected the expression of ERMIN2, ERMIT2, and MFN2. Their mRNA levels were unchanged in starved HeLa cells, whereas they increased in cells exposed to the mitochondrial complex I inhibitor rotenone. The sarcoplasmic ER Ca²⁺ adenosine triphosphatase (SERCA) inhibitor thapsigargin that causes ER stress induced the expression of ERMIN2 and ERMIT2 but not of MFN2 (Fig. 1C
）。接下来，我们建立了实时 PCR 检测来特异性检测人类样本中ERMIN2 、 ERMIT2和MFN2的 mRNA（图 S1A）。我们发现，在人类受试者的白色脂肪组织（WAT）、骨骼肌和肝脏中， ERMIN2占MFN2 mRNA 表达的 20% 至 25%，而ERMIT2占 7% 至 52%（图 S1B）。接下来我们监测特定的外源应激是否影响ERMIN2 、 ERMIT2和MFN2的表达。它们的 mRNA 水平在饥饿的 HeLa 细胞中没有变化，而在暴露于线粒体复合物 I 抑制剂鱼藤酮的细胞中则有所增加。引起 ER 应激的肌浆 ER Ca ²⁺三磷酸腺苷酶 (SERCA) 抑制剂毒胡萝卜素诱导ERMIN2和ERMIT2的表达，但不诱导MFN2的表达（图 1C）). The well-characterized dimerization-dependent green fluorescent protein (ddGFP) ER-mitochondria proximity probe (21) reported higher juxtaposition between these two organelles in cells treated with rotenone and thapsigargin, which suggests that ERMIN2 and ERMIT2 expression was induced when ER-mitochondria proximity increased (fig. S1C).
）。充分表征的二聚化依赖性绿色荧光蛋白 (ddGFP) ER-线粒体邻近探针 ( 21 ) 报告在用鱼藤酮和毒胡萝卜素处理的细胞中这两个细胞器之间有更高的并置，这表明当 ER-线粒体邻近时会诱导ERMIN2和ERMIT2表达增加（图S1C）。

Fig. 1. *MFN2* undergoes alternative splicing.
图1 . *MFN2*进行选择性剪接。
(A) Schematic representation of the *MFN2* splicing variants. Different PCR-amplification products obtained from human skeletal cDNA were cloned and sequenced. PCR was performed with primers located on intron 2 and exon 19. Exons are numbered 1 to 19, and the splicing events generate new exons (3a, 3b, 3c, 4a, 4b, 6a, 6b, 13a, 13b, 15a, and 15b). *MFN2* corresponds to sequence NM014874. Variant 1 (*V1-MFN2*, *ERMIN2*) mRNA is 1330 bp long, is produced by exons 3b and 6b to 15a skipping, and it encodes for ERMIN2, a 41-kDa (predicted molecular weight) protein. Variant 2 (*V2-MFN2*, *ERMIT2*) mRNA is 1220 bp long, is produced by exon 4b to 13a skipping, and it encodes ERMIT2, a 43-kDa (predicted M.W.) protein. Exons involved in the alternative splicing process and the domains in the produced proteins are indicated. G1 to G5 are GTPase domain motifs. (B) RPA in HeLa cells. *ERMIN2*- and *ERMIT2*-specific probes covered the 5′- and 3′- flanking sequences of the skipped region, and 20% of their length was a nonmatching sequence as positive control for RNAse digestion. (C) Means ± standard errors (SEs) of mRNA of *MFN2*, *ERMIN2*, and *ERMIT2* levels (normalized to PPIA) in HeLa cells 4 hours after incubation in Earle’s buffered salt solution (EBSS) (starvation), treatment with rotenone (Rot; 5 μM), or thapsigargin (TG; 1 μM). N = 8 independent experiments. *P < 0.05 in a one-way analysis of variance (ANOVA). (D) Equal amounts (40 μg) of lysates from the indicated human adult tissues were separated by SDS-PAGE and immunoblotted using the indicated antibodies. The bottom graph reports means ± SEs of protein/vinculin ratios from N = 3 independent immunoblotting experiments. a.u., arbitrary units.
( A ) *MFN2*剪接变体的示意图。对从人类骨骼 cDNA 获得的不同 PCR 扩增产物进行了克隆和测序。使用位于内含子 2 和外显子 19 上的引物进行 PCR。外显子编号为 1 至 19，剪接事件生成新的外显子（3a、3b、3c、4a、4b、6a、6b、13a、13b、15a 和 15b））。 *MFN2*对应于序列 NM014874。变体 1（ *V1-MFN2* 、 *ERMIN2* ）mRNA 长 1330 bp，由外显子 3b 和 6b 至 15a 跳跃产生，编码 ERMIN2，一种 41 kDa（预测分子量）蛋白质。变体 2（ *V2-MFN2* 、 *ERMIT2* ）mRNA 长 1220 bp，由外显子 4b 到 13a 跳跃产生，编码 ERMIT2，一种 43 kDa（预测分子量）蛋白质。指出了参与选择性剪接过程的外显子和所产生的蛋白质中的结构域。 G1至G5是GTPase结构域基序。 ( B ) HeLa 细胞中的 RPA。 *ERMIN2*和*ERMIT2*特异性探针覆盖了跳过区域的 5' 和 3' 侧翼序列，其长度的 20% 是不匹配序列，作为 RNAse 消化的阳性对照。 ( C ) HeLa 细胞中*MFN2* 、 *ERMIN2*和*ERMIT2*水平的 mRNA 的平均值±标准误差 (SE)（标准化为 PPIA）在厄尔缓冲盐溶液 (EBSS) 中孵育 4 小时（饥饿），用鱼藤酮处理（Rot； 5 μM) 或毒胡萝卜素 (TG; 1 μM)。 N = 8 个独立实验。 * 单向方差分析 (ANOVA) 中P < 0.05。 ( D ) 通过 SDS-PAGE 分离来自所示成人组织的等量 (40 μg) 裂解物，并使用所示抗体进行免疫印迹。底部图表报告了来自N = 3 个独立免疫印迹实验的蛋白质/纽蛋白比率的平均值±SE。 au，任意单位。

Open in viewer

The proteins encoded by ERMIN2 and ERMIT2 were predicted to be shorter than MFN2—partially or completely lacking its guanosine triphosphatase (GTPase) and coiled-coil 1 (CC1) domains but retaining its transmembrane (TM) and coiled-coil 2 (CC2) regions (Fig. 1A
ERMIN2 和 ERMIT2 编码的蛋白质预计比 MFN2 短，部分或完全缺乏鸟苷三磷酸酶 (GTPase) 和卷曲螺旋 1 (CC1) 结构域，但保留其跨膜 (TM) 和卷曲螺旋 2 (CC2) 区域（图1A and fig. S2). Exogenous hemagglutinin (HA)–tagged ERMIN2 and ERMIT2 expressed in HeLa cells were detected at around their theoretical molecular masses—i.e., 41 and 43 KDa—and, like full-length MFN2, were recognized by antibodies raised against MFN2 amino acids 557 to 576 or CC2 domain (fig. S1D). Using the former antibody, we retrieved ERMIN2 and ERMIT2 running at their predicted molecular weights in human skeletal muscle, liver, and WAT extracts. The ratio between ERMIN2 and MFN2 levels was 0.39 in skeletal muscle, 1.8 in liver, and 0.17 in WAT, whereas the ERMIT2/MFN2 ratio was 0.3 to 0.35 in all tissues analyzed (Fig. 1D
和图。 S2）。在 HeLa 细胞中表达的外源血凝素 (HA) 标记的 ERMIN2 和 ERMIT2 在其理论分子质量（即 41 和 43 KDa）左右被检测到，并且与全长 MFN2 一样，被针对 MFN2 氨基酸 557 至 576 产生的抗体识别或 CC2 域（图 S1D）。使用前一种抗体，我们检索了在人类骨骼肌、肝脏和 WAT 提取物中以其预测分子量运行的 ERMIN2 和 ERMIT2。 ERMIN2和MFN2水平之间的比率在骨骼肌中为0.39，在肝脏中为1.8，在WAT中为0.17，而在所有分析的组织中ERMIT2/MFN2比率为0.3至0.35（图1D）).

We next tested whether alternatively spliced, shorter MFN2 versions were present in Mus musculus, where MFN2 also tethers ER to mitochondria. By amplifying cDNA from mouse embryonic fibroblasts (MEFs) using Mfn2 gene open reading frame exon 1 and 19 primers, we obtained and sequenced an amplicon corresponding to a shorter Mfn2 transcript (fig. S3, A to C). This mouse Mfn2 variant (MoV-Mfn2) mRNA encoded for a protein containing the MFN2 TM and CC2 domains and hence is very similar to ERMIT2 (fig. S3, A to C). Thus, human and mouse Mitofusin 2 genes are alternatively spliced to generate proteins shorter than full-length MFN2, expressed in multiple tissues and induced when ER-mitochondria juxtaposition is pharmacologically increased.
接下来我们测试了小家鼠中是否存在选择性剪接的较短 MFN2 版本，其中 MFN2 还将 ER 与线粒体连接。通过使用Mfn2基因开放阅读框外显子 1 和 19 引物扩增小鼠胚胎成纤维细胞 (MEF) 的 cDNA，我们获得了对应于较短Mfn2转录本的扩增子并对其进行了测序（图 S3，A 到 C）。这种小鼠Mfn2变体 (MoV- Mfn2 ) mRNA 编码含有 MFN2 TM 和 CC2 结构域的蛋白质，因此与 ERMIT2 非常相似（图 S3，A 到 C）。因此，人类和小鼠线粒体融合蛋白 2基因交替剪接，产生比全长 MFN2 短的蛋白质，在多种组织中表达，并在药理学上增加 ER-线粒体并置时被诱导。

ERMIN2, ERMIT2, and MoV-MFN2 localize to the ER
ERMIN2、ERMIT2 和 MoV-MFN2 定位于 ER

Given that ERMIN2 and ERMIT2 lack several domains found in full-length MFN2, we investigated whether they were targeted to mitochondria. We first examined the distribution of ERMIN2 or ERMIT2 in subcellular fractions purified from Mfn2 knockout livers (Mfn2^LKO) where we had expressed the two variants using adenovirus vectors. We detected ERMIN2 and ERMIT2 in the ER-enriched light membrane (LM) fraction as well as in mitochondria-associated membranes (MAMs) but not in the pure mitochondria fraction (Fig. 2A
鉴于 ERMIN2 和 ERMIT2 缺乏全长 MFN2 中发现的几个结构域，我们研究了它们是否靶向线粒体。我们首先检查了 ERMIN2 或 ERMIT2 在从Mfn2敲除肝脏 (Mfn2 ^LKO ) 纯化的亚细胞级分中的分布，其中我们使用腺病毒载体表达了这两种变体。我们在富含 ER 的轻膜 (LM) 部分以及线粒体相关膜 (MAM) 中检测到 ERMIN2 和 ERMIT2，但在纯线粒体部分中未检测到（图 2A）). We confirmed that endogenous ERMIN2 was present exclusively in LMs and ERMIT2 was present in LMs and MAMs but not in the pure mitochondria fraction of HeLa cells, which differed from full-length MFN2 that was retrieved in all three fractions (13) (fig. S4). We further tested the selective ER and MAMs localization of ERMIN2 and ERMIT2 by coexpressing GFP-tagged MFN2, ERMIN2, and ERMIT2 with a mitochondrially targeted cyan fluorescent protein (mtCFP) and a dsRED fluorescent protein targeted to the ER (ER-RFP) in Mfn2^−/− MEFs. Although reexpressed MFN2 localized mostly in mitochondria and only partially in the ER, ERMIN2 and ERMIT2 displayed a pattern like that of the ER marker. Upon closer inspection, ERMIN2 was uniformly distributed on the ER, and ERMIT2 displayed a punctate staining that colocalized with ER and mitochondria, compatible with the retrieval of the endogenous protein in MAMs (Fig. 2, C and D
）。我们证实内源性 ERMIN2 只存在于 LM 中，ERMIT2 存在于 LM 和 MAM 中，但不存在于 HeLa 细胞的纯线粒体部分中，这与在所有三个部分中检索到的全长 MFN2 不同 ( 13 )（图 S4））。我们通过将 GFP 标记的 MFN2、ERMIN2 和 ERMIT2 与线粒体靶向青色荧光蛋白 (mtCFP) 和靶向Mfn2中 ER (ER-RFP) 的 dsRED 荧光蛋白共表达，进一步测试了 ERMIN2 和 ERMIT2 的选择性 ER 和 MAM 定位。 ^−/− MEF。尽管重新表达的 MFN2 大部分位于线粒体中，仅部分位于 ER 中，但 ERMIN2 和 ERMIT2 显示出与 ER 标记类似的模式。仔细观察，ERMIN2 均匀分布在 ER 上，ERMIT2 呈现与 ER 和线粒体共定位的点状染色，与 MAM 中内源蛋白的检索相一致（图 2，C 和 D）; individual channels in fig. S5). Similarly, we found GFP-moV-MFN2 at the interface between ER and mitochondria in Mfn2^−/− MEFs (fig. S3, D and E) and endogenous moV-MFN2 in MAMs and LM fractions purified from MEFs (fig. S3F). Thus, ERMIN2 and ERMIT2 localize only at the ER, and ERMIT2 and moV-MFN2 are enriched at the ER-mitochondria interface.
;图中的各个通道S5）。同样，我们在Mfn2 ^−/− MEF 中的 ER 和线粒体之间的界面处发现了 GFP-moV-MFN2（图 S3、D 和 E），并且在从 MEF 纯化的 MAM 和 LM 组分中发现了内源性 moV-MFN2（图 S3F）。因此，ERMIN2和ERMIT2仅定位于ER，而ERMIT2和moV-MFN2在ER-线粒体界面处富集。

Fig. 2. MFN2 variants localize at the ER, not the mitochondria.
图2 . MFN2 变异体定位于内质网，而不是线粒体。
(A and B) Mfn2^LKO mice were injected with adenoviruses encoding ERMIN2 (A) or ERMIT2 (B), and after 72 hours, liver subcellular fractions were prepared. Equal amounts (40 μg) of protein from total extracts (Total), crude (Mito), pure mitochondria (Pure mito), MAMs, and LMs were separated by SDS-PAGE and immunoblotted using antibodies against MFN2, the MAM markers PS1 (presenilin 1) and FACL4 (long-chain acyl-CoA synthetase 4), the outer mitochondrial membrane marker TOM20 (translocase of the outer mitochondrial membrane 20), and the ER marker SERCA2 (sarcoplasmic ER Ca²⁺ ATPase). (C) Representative confocal images of *Mfn2*^−/− MEFs cotransfected with the indicated plasmids (green), mitochondrially targeted CFP (mito; blue) and ER-dsRED (ER; red). Insets are magnified 7×. Scale bars, 10 μm. (D) Averages ± SEMs of Manders’ coefficient of mitochondria-ER pseudocolocalization. Dots indicate individual cells (n = 150) from N = 3 independent experiments. *Mfn2*^−/− MEFs were cotransfected with ER-dsRED or mito-dsRED and the indicated GFP-tagged constructs. *P < 0.05 in a one-way ANOVA with Tukey’s mean comparison.
( A和B )向Mfn2 ^LKO小鼠注射编码ERMIN2(A)或ERMIT2(B)的腺病毒，72小时后，制备肝亚细胞级分。通过 SDS-PAGE 分离来自总提取物 (Total)、粗提物 (Mito)、纯线粒体 (Pure mito)、MAM 和 LM 的等量 (40 μg) 蛋白质，并使用针对 MFN2、MAM 标记物 PS1（早老素）的抗体进行免疫印迹。 1) 和 FACL4（长链酰基辅酶 A 合成酶 4）、线粒体外膜标记 TOM20（线粒体外膜易位酶）膜 20) 和 ER 标记 SERCA2（肌浆 ER Ca ²⁺ ATP 酶）。 ( C ) 与指定质粒（绿色）、线粒体靶向 CFP（mito；蓝色）和 ER-dsRED（ER；红色）共转染的*Mfn2* ^−/− MEF 的代表性共焦图像。插图放大 7 倍。比例尺，10 μm。 ( D ) 线粒体-ER 伪共定位曼德斯系数的平均值±SEM。点表示来自N = 3 个独立实验的单个细胞 ( n = 150)。 *Mfn2* ^−/− MEF 与 ER-dsRED 或 mito-dsRED 以及指定的 GFP 标记构建体共转染。 * 在与 Tukey 均值比较的单向方差分析中， P < 0.05。

Open in viewer

We next explored the determinants of ERMIT2 ER targeting. By coexpressing GFP-tagged ERMIT2 mutants with ER-RFP in Mfn2^−/− MEFs, we found that the ER localization of ERMIT2 was not affected by deletion of the CC2, the pre-CC2 region, or the N-terminal domain (fig. S6, A to C). The low level of mitochondrial colocalization of ERMIT2 was unaffected in these deletion mutants (fig. S6, D and E). Because these results suggested a role for the TM region of ERMIT2/MFN2 as an ER localization signal, we generated an ERMIT2/MFN2 TM region–GFP chimera that indeed localized at the ER (fig. S7, A and B). Addition of CC1 and CC2 to this chimera reduced its ER localization and increased its mitochondrial localization (fig. S7).
接下来我们探讨了 ERMIT2 ER 靶向的决定因素。通过在Mfn2 ^−/− MEF 中共表达 GFP 标记的 ERMIT2 突变体与 ER-RFP，我们发现 ERMIT2 的 ER 定位不受 CC2、CC2 前区域或 N 末端结构域删除的影响（图 1）。 S6，A 至 C)。 ERMIT2 的低水平线粒体共定位在这些缺失突变体中未受影响（图 S6、D 和 E）。因为这些结果表明 ERMIT2/MFN2 的 TM 区域作为 ER 定位信号，我们生成了确实定位于 ER 的 ERMIT2/MFN2 TM 区域 - GFP 嵌合体（图 S7、A 和 B）。在该嵌合体中添加 CC1 和 CC2 减少了其 ER 定位并增加了其线粒体定位（图 S7）。

Topology of ERMIN2 and ERMIT2 at the ER
ER 处的 ERMIN2 和 ERMIT2 拓扑

To determine the topology of ERMIN2 and ERMIT2 at the ER, we devised a SPLIT-GFP assay to detect the orientation of the N terminus of ERMIN2 and ERMIT2. We coexpressed a nonfluorescent cytosolic GFP_1-10 protein and chimeras of GFP₁₁ β-strand fused to the N terminus of MFN2, ERMIN2, or ERMIT2. Fluorescence of the SPLIT-GFP system is reconstituted only if both GFP_1-10 and the GFP₁₁ chimeras are in the same cellular compartment. GFP fluorescence was detected when we coexpressed GFP_1-10 with GFP₁₁-MFN2, GFP₁₁-ERMIN2, and GFP₁₁-ERMIT2, which indicates that the N termini of MFN2, ERMIN2, and ERMIT2 face the cytosol (fig. S8A). To understand whether the C termini of ERMIN2 and ERMIT2 were exposed to the lumen of the ER, we performed limited trypsin proteolysis assays on LM fractions purified from Mfn2^LKO livers infected with adenoviruses expressing ERMIN2 and ERMIT2. If ERMIN2 and ERMIT2 C termini were exposed to the lumen, trypsin would eliminate the epitopes recognized by the antibody against their N terminus, whereas the CC2 epitope would be protected, and vice versa if the N terminus was facing the ER lumen (fig. S8B). Both the N-terminal and the CC2 epitopes were lost upon trypsin treatment, indicating that both the N terminus and the CC2 domain of ERMIN2 and ERMIT2 were accessible to the protease (fig. S8C). Thus, ERMIN2 and ERMIT2 N and C termini face the cytosol (fig. S8D).
为了确定 ERMIN2 和 ERMIT2 在 ER 上的拓扑结构，我们设计了 SPLIT-GFP 测定法来检测 ERMIN2 和 ERMIT2 N 末端的方向。我们共表达了非荧光胞质 GFP _1-10蛋白和融合到 MFN2、ERMIN2 或 ERMIT2 N 末端的 GFP ₁₁ β-链嵌合体。仅当 GFP _1-10和 GFP ₁₁嵌合体位于同一细胞室中时，SPLIT-GFP 系统的荧光才会重建。当我们将 GFP _1-10与 GFP ₁₁ -MFN2、GFP ₁₁ -ERMIN2 和 GFP ₁₁ -ERMIT2 共表达时，检测到 GFP 荧光，这表明 MFN2、ERMIN2 和 ERMIT2 的 N 末端面向细胞质（图 S8A）。为了了解 ERMIN2 和 ERMIT2 的 C 末端是否暴露于 ER 内腔，我们对从感染表达 ERMIN2 和 ERMIT2 的腺病毒的 Mfn2 ^LKO肝脏中纯化的 LM 级分进行了有限的胰蛋白酶蛋白水解测定。如果 ERMIN2 和 ERMIT2 C 末端暴露于内腔，胰蛋白酶将消除针对其 N 末端的抗体识别的表位，而 CC2 表位将受到保护，如果 N 末端面向 ER 内腔，则反之亦然（图 S8B））。胰蛋白酶处理后，N 端和 CC2 表位均丢失，表明 ERMIN2 和 ERMIT2 的 N 端和 CC2 结构域均可被蛋白酶接触（图 S8C）。因此，ERMIN2 和 ERMIT2 N 和 C 末端面向细胞质（图 S8D）。

ERMIN2 regulates ER morphology
ERMIN2 调节 ER 形态

To functionalize ERMIN2 and ERMIT2, we first analyzed mitochondrial and ER morphology in Mfn2^−/− MEFs reconstituted with MFN2, ERMIN2, and ERMIT2. Inspection of volume-rendered three-dimensional (3D) reconstructions of z-stacks of confocal images of mtRFP revealed that, in contrast to MFN2, ERMIN2 or ERMIT2 did not rescue Mfn2^−/− mitochondrial morphology (Fig. 3, A and B
为了功能化 ERMIN2 和 ERMIT2，我们首先分析了用 MFN2、ERMIN2 和 ERMIT2 重建的Mfn2 ^−/− MEF 中的线粒体和 ER 形态。对 mtRFP 共焦图像z堆栈的体积渲染三维 (3D) 重建的检查表明，与 MFN2 相比，ERMIN2 或 ERMIT2 并没有挽救Mfn2 ^−/−线粒体形态（图 3、A 和 B）). Expression of MFN2 and ERMIN2 corrected the ER morphology of Mfn2^−/− cells (Fig. 3C
）。 MFN2 和 ERMIN2 的表达纠正了Mfn2 ^−/−细胞的 ER 形态（图 3C）). Fluorescent recovery after photobleaching (FRAP) assays further corroborated the finding that expression of MFN2 and ERMIN2 restored ER connectivity in Mfn2^−/− MEFs. Although ERMIT2 slightly ameliorated ER morphology, it did not improve ER connectivity (Fig. 3, C and D
）。光漂白后荧光恢复 (FRAP) 测定进一步证实了 MFN2 和 ERMIN2 的表达恢复了Mfn2 ^−/− MEF 中 ER 连接的发现。尽管 ERMIT2 稍微改善了 ER 形态，但它并没有改善 ER 连接性（图 3，C 和 D）). We then turned to a loss-of-function approach using small interfering RNAs (siRNAs) to specifically down-regulate ERMIN2 or ERMIT2 in HeLa cells (fig. S9). Depletion of ERMIN2 or ERMIT2 did not affect mitochondrial morphology (Fig. 3, E and F
）。然后，我们转向使用小干扰 RNA (siRNA) 的功能丧失方法来特异性下调 HeLa 细胞中的 ERMIN2 或 ERMIT2（图 S9）。 ERMIN2 或 ERMIT2 的耗尽并不影响线粒体形态（图 3、E 和 F). Conversely, ERMIN2 down-regulation altered peripheral ER morphology and diminished ER interconnectivity, as determined by FRAP analysis (Fig. 3G
）。相反，根据 FRAP 分析确定，ERMIN2 下调改变了外周 ER 形态并减弱了 ER 互连性（图 3G）). Thus, ERMIN2 and ERMIT2 do not modulate mitochondrial morphology, and endogenous ERMIN2 is required for ER connectivity even when MFN2 is present.
）。因此，ERMIN2 和 ERMIT2 不调节线粒体形态，即使存在 MFN2，内源性 ERMIN2 也是 ER 连接所必需的。

Fig. 3. ERMIN2 regulates ER morphology.
图3 . ERMIN2 调节 ER 形态。
(A) Representative volume-rendered 3D reconstructions of confocal z-stacks of *Mfn2*^−/− MEFs cotransfected with the indicated HA-tagged constructs and mt-dsRED. Scale bars, 10 μm. (B) Means ± SEs of mitochondrial fragmentation (N = 3 independent experiments, n = 50 cells per condition) in experiments as in (A). *P < 0.05 in a one-way ANOVA with Tukey’s mean comparison among EV and the other conditions. (C) Experiments were performed as in (A), except that *Mfn2*^−/− MEFs were cotransfected with ER-YFP. Scale bars, 10 μm. (D) ER-YFP fluorescence recordings from FRAP experiments in *Mfn2*^−/− MEFs cotransfected with ER-YFP and the indicated HA-tagged plasmid. After 1 s, the ROI was photobleached by illumination at 100% laser power, and then recording continued for the duration of the experiments. Data are means ± SEs of three independent experiments (12 cells per condition). (E) Representative Volume Viewer 3D reconstructions of confocal z-stack images of HeLa cells cotransfected with mt-dsRED, ER-YFP, and the indicated siRNA. Scale bars, 10 μm. (F) Means ± SEs of mitochondrial aspect ratio (N = 3 independent experiments, n = 27 cells per condition) in experiments as in (E). (G) ER-YFP fluorescence recordings from ER-YFP FRAP experiments in HeLa cells cotransfected with the ER-YFP and the indicated siRNA. Experiments were performed as in (D). Data are means ± SEs from three independent experiments (12 cells per condition).
( A ) 与指示的 HA 标记构建体和 mt-dsRED 共转染的*Mfn2* ^−/− MEF 的共焦z堆栈的代表性体积渲染 3D 重建。比例尺，10 μm。 ( B ) 实验中线粒体碎片的平均值±SE（ N = 3 个独立实验，每个条件n = 50 个细胞），如 (A) 所示。 * 单向方差分析中P < 0.05，与 EV 和其他条件之间的 Tukey 均值比较。 ( C ) 实验如 (A) 中所述进行，不同之处在于*Mfn2* ^-/- MEF 与 ER-YFP 共转染。比例尺，10 μm。 ( D ) ER-YFP 荧光记录，来自与 ER-YFP 和指示的 HA 标记质粒共转染的*Mfn2* ^−/− MEF 中的 FRAP 实验。 1 秒后，通过 100% 激光功率照射对 ROI 进行光漂白，然后在实验期间继续记录。数据为三个独立实验的平均值±SE（每个条件12个细胞）。 ( E ) 用 mt-dsRED、ER-YFP 和所示 siRNA 共转染的 HeLa 细胞的共焦z堆栈图像的代表性 Volume Viewer 3D 重建。比例尺，10 μm。 ( F ) 线粒体纵横比的平均值±SE（ N = 3 个独立实验，每个条件n = 27 个细胞），如 (E) 中的实验。 ( G ) ER-YFP 荧光记录，来自用 ER-YFP 和所示 siRNA 共转染的 HeLa 细胞中的 ER-YFP FRAP 实验。实验如(D)中那样进行。数据为来自三个独立实验的平均值±SE（每个条件12个细胞）。

Open in viewer

ERMIT2 and moV-MFN2 tether ER to mitochondria
ERMIT2 和 moV-MFN2 将 ER 与线粒体连接

Given that ERMIT2 is found in MAMs and does not regulate ER or mitochondrial morphology, we posited that it participated in ER-mitochondria tethering (13). We performed several orthogonal assays to verify this hypothesis. First, we evaluated juxtaposition between the two organelles in volume-rendered 3D reconstructions of z-axis stacks of confocal images of mtRFP and ER–yellow fluorescent protein (YFP). Reexpression of MFN2 as well as of ERMIT2 in Mfn2^−/− MEFs improved ER-mitochondria pseudocolocalization. Conversely, ERMIN2 that restored ER morphology did not modify ER-mitochondria juxtaposition (Fig. 4, A and B
鉴于 ERMIT2 存在于 MAM 中并且不调节 ER 或线粒体形态，我们假设它参与 ER-线粒体束缚 ( 13 )。我们进行了几次正交测定来验证这一假设。首先，我们评估了 mtRFP 和 ER-黄色荧光蛋白 (YFP) 共焦图像z轴堆栈体积渲染 3D 重建中两个细胞器之间的并置情况。 MFN2 以及 ERMIT2 在Mfn2 ^−/− MEF 中的重新表达改善了 ER 线粒体假共定位。相反，恢复 ER 形态的 ERMIN2 并未改变 ER-线粒体并置（图 4，A 和 B）; individual channels in fig. S10). Second, the fluorescence resonance energy transfer (FRET)–based ER-mitochondria proximity probe (FEMP) (16) showed that expression of MFN2 and ERMIT2 in Mfn2^−/− MEFs increased steady-state (basal) and maximal ER-mitochondria juxtaposition (obtained by a brief rapamycin pulse to engage the FEMP FKBP-FRB dimerization motif), whereas ERMIN2 increased only basal FEMP signal (Fig. 4C
;图中的各个通道S10）。其次，基于荧光共振能量转移（FRET）的内质网线粒体邻近探针（FEMP）（ 16 ）表明， MFN2 ^-/− MEF 中 MFN2 和 ERMIT2 的表达增加了稳态（基础）和最大内质网线粒体并置（通过短暂的雷帕霉素脉冲接合 FEMP FKBP-FRB 二聚化基序获得），而 ERMIN2 增加仅基础 FEMP 信号（图 4C). Third, we quantified by flow cytometry the percentage of Mfn2^−/− cells positive to the ddGFP ER-mitochondria proximity probe (16, 21). Although ERMIN2 did not significantly increase the fraction of Mfn2^−/− ddGFP⁺ cells, whereas expression of MFN2 or ERMIT2 led to a 2- and 1.75-fold increase in ddGFP positivity (Fig. 4, D and E
）。第三，我们通过流式细胞术定量了 ddGFP ER-线粒体邻近探针阳性的Mfn2 ^−/−细胞的百分比 ( 16 , 21 )。虽然 ERMIN2 没有显着增加Mfn2 ^−/− ddGFP ⁺细胞的比例，但 MFN2 或 ERMIT2 的表达导致 ddGFP 阳性率增加 2 倍和 1.75 倍（图 4、D 和 E）). Live confocal imaging confirmed that expression of MFN2 and ERMIT2, but not of ERMIN2, in Mfn2^−/− cells led to a punctate ddGFP signal, reminiscent of ER-mitochondria contacts (fig. S11). Fourth, we ultrastructurally analyzed ER-mitochondria contacts in transmission electron microscopy images from where we calculated the ER-mitochondria contact coefficient (ERMICC) that computes the average ER-mitochondria distance and interaction length over the mitochondrial perimeter (16). MFN2 and ERMIT2, but not ERMIN2, increased ER-mitochondria juxtaposition and ERMICC in Mfn2^−/− cells (Fig. 4, F and G
）。活体共聚焦成像证实， Mfn2 ^-/−细胞中 MFN2 和 ERMIT2 的表达（但不是 ERMIN2）导致点状 ddGFP 信号，让人想起 ER-线粒体接触（图 S11）。第四，我们对透射电子显微镜图像中的内质网线粒体接触进行了超微结构分析，从中计算了内质网线粒体接触系数（ERMICC），该系数计算了线粒体周长上的平均内质网线粒体距离和相互作用长度（ 16 ）。 MFN2 和 ERMIT2，但不是 ERMIN2，增加了Mfn2 ^−/−细胞中的 ER-线粒体并置和 ERMICC（图 4，F 和 G）). MFN2 and ERMIT2 decreased the average ER-mitochondria distance measured from >70 interorganellar interactions occurring in the ≤30-nm range in Mfn2^−/− cells (fig. S12). In parallel, we analyzed whether moV-MFN2, which resembles ERMIT2, was also involved in ER-mitochondria tethering. Expression of moV-MFN2 in Mfn2^−/− cells increased ER-mitochondria juxtaposition, as reflected by increased pseudolocalization of fluorescent proteins targeted to ER and mitochondria (fig. S2, G and H). Thus, expression of ERMIT2 and moV-MFN2 increases ER-mitochondria juxtaposition.
）。 MFN2 和 ERMIT2 降低了Mfn2 ^−/−细胞中 ≤30 nm 范围内发生的 >70 个细胞间相互作用测量的平均 ER-线粒体距离（图 S12）。同时，我们分析了类似于 ERMIT2 的 moV-MFN2 是否也参与 ER 线粒体束缚。 Mfn2 ^-/−细胞中 moV-MFN2 的表达增加了 ER-线粒体并置，这反映在针对 ER 和线粒体的荧光蛋白的假定位增加（图 S2、G 和 H）。因此，ERMIT2和moV-MFN2的表达增加了ER-线粒体并置。

Fig. 4. ERMIT2 reconstitutes ER-mitochondria interaction in *Mfn2*^−/− MEFs.
图4 . ERMIT2 在*Mfn2* ^−/− MEF 中重建 ER-线粒体相互作用。
(A) Representative volume-rendered 3D reconstructions of confocal z-stacks of *Mfn2*^−/− MEFs cotransfected with ER-YFP, mt-dsRED, and plasmids coding for the indicated HA-tagged proteins. Yellow indicates pseudocolocalization of the two organelles. (B) Means ± SEs of Manders’ coefficient (n = 3 independent experiments, 30 cells per condition per experiment; open dots) of ER-mitochondria pseudocolocalization in experiments as in (A). *P < 0.05 in a one-way ANOVA with Tukey’s mean comparison among the indicated groups. (C) Means ± SEs of basal (B) and maximal (M) FEMP FRET values (n = 6 independent experiments, >200 cells per condition per experiment; open dots). *P < 0.05 in a one-way ANOVA with Tukey’s means comparison between the indicated groups. (D) Dot plots of ddGFP fluorescence versus SSC in the mCherry⁺ gated population from flow cytometric analysis of *Mfn2*^−/− MEFs cotransfected for 48 hours with ddGFP A-B monomers, cytosolic mCherry, and the indicated plasmids. (E) Means ± SEs of the percentage of ddGFP⁺ cells (n = 6 independent experiments; open dots) in experiments as in (D). *P < 0.05 in a one-way ANOVA with Tukey’s means comparison between the indicated groups. (F) Representative electron microscopy images of *Mfn2*^−/− MEFs cotransfected with GFP and the indicated plasmid. GFP-positive cells were sorted, fixed, processed, and analyzed by transmission electron microscopy (TEM). Scale bars, 500 nm. (G) Means ± SEs of ERMICC (n = 50 to 182 measurements per condition; open dots) from N = 3 independent experiments as in (A). *P < 0.05 in a one-way ANOVA with Tukey’s means comparison between the indicated groups.
( A ) 与 ER-YFP、mt-dsRED 和编码所示 HA 标记蛋白的质粒共转染的*Mfn2* ^−/− MEF 的共焦z堆叠的代表性体积渲染 3D 重建。黄色表示两个细胞器的假共定位。 ( B ) 实验中 ER 线粒体假共定位的曼德斯系数平均值 ± SE（ n = 3 个独立实验，每个实验每个条件 30 个细胞；空心点），如 (A) 中的实验。 * 单向方差分析中P < 0.05，与指定组之间的 Tukey 平均值比较。 ( C ) 基础 (B) 和最大 (M) FEMP FRET 值的平均值 ± SE（ n = 6 个独立实验，每个实验每个条件 >200 个细胞；空心点）。 * 单向方差分析中P < 0.05，与指定组之间的 Tukey 均值比较。 ( D ) 对用 ddGFP AB 单体、胞质 mCherry 和所示质粒共转染 48 小时的*Mfn2* ^−/− MEF 进行流式细胞分析，得出 ddGFP 荧光与 mCherry ⁺门控群体中 SSC 的点图。 ( E ) 实验中 ddGFP ⁺细胞百分比的平均值 ± SE（ n = 6 个独立实验；空心点），如 (D) 中所示。 * 单向方差分析中P < 0.05，与指定组之间的 Tukey 均值比较。 ( F ) 与 GFP 和所示质粒共转染的*Mfn2* ^−/− MEF 的代表性电子显微镜图像。通过透射电子显微镜（TEM）对 GFP 阳性细胞进行分类、固定、处理和分析。比例尺，500 nm。 ( G ) ERMICC 的平均值 ± SE（每个条件n = 50 至 182 次测量；空心点），来自N = 3 个独立实验，如 (A) 所示。 * 单向方差分析中P < 0.05，与指定组之间的 Tukey 均值比较。

Open in viewer

ERMIT2 requires mitofusins on mitochondria to tether them to the ER
ERMIT2 需要线粒体上的线粒体融合素将其连接到 ER

A feature of heterotypic tethers is their interaction in trans with homo- or heterotypic partners on the surface of the opposite organelle. Given the propensity of MFNs to engage in homo- and hetero-oligomerization (34), we tested whether mitochondrial MFNs were the mitochondrial partners of ERMIT2. First, we addressed whether ERMIT2 (and ERMIN2) were able to interact with MFN1 and/or MFN2 in pull-down experiments. GFP-tagged ERMIN2 or ERMIT2 immunoprecipitated V5-tagged MFN2^ActA and Flag-tagged MFN1 that are restricted to mitochondria (13), although ERMIN2 immunoprecipitated these mitochondrial Mfns somewhat less efficiently (Fig. 5A
异型系链的一个特征是它们与相反细胞器表面的同型或异型伴侣进行反式相互作用。鉴于 MFN 参与同源和异源寡聚化的倾向 ( 34 )，我们测试了线粒体 MFN 是否是 ERMIT2 的线粒体伴侣。首先，我们讨论了 ERMIT2（和 ERMIN2）是否能够在下拉实验中与 MFN1 和/或 MFN2 相互作用。 GFP 标记的 ERMIN2 或 ERMIT2 免疫沉淀 V5 标记的 MFN2 ^ActA和 Flag 标记的 MFN1，仅限于线粒体（ 13 ），尽管 ERMIN2 免疫沉淀这些线粒体 Mfns 的效率稍低（图 5A）). Next, we used genetics to verify whether this biochemical interaction was functionally relevant for ER-mitochondria tethering. We expressed MFN2, ERMIN2, and ERMIT2 in MEFs lacking Mfn1 and Mfn2 (Mfn1, Mfn2^−/−). MFN2 and ERMIT2 were unable to increase ER-mitochondria tethering unless coexpressed with MFN1 that is found only on the surface of mitochondria (13) (Fig. 5, B and C
）。接下来，我们利用遗传学来验证这种生化相互作用是否与内质网线粒体束缚在功能上相关。我们在缺乏Mfn1和Mfn2的 MEF 中表达 MFN2、ERMIN2 和 ERMIT2（ Mfn1 、 Mfn2 ^−/− ）。 MFN2 和 ERMIT2 无法增加 ER 线粒体束缚，除非与仅在线粒体表面发现的 MFN1 共表达 ( 13 )（图 5、B 和 C）). Thus, ER ERMIT2 interacts with mitochondrial MFN1 for ER-mitochondria tethering.
）。因此，ER ERMIT2 与线粒体 MFN1 相互作用，实现 ER 线粒体束缚。

Fig. 5. ERMIT2 interacts with mitochondrial mitofusins, tethering ER to mitochondria.
图5 。 ERMIT2 与线粒体线粒体融合蛋白相互作用，将 ER 束缚于线粒体。
(A) *Mfn1*^−/−, *Mfn2*^−/− MEFs were cotransfected with the indicated plasmids and after 24 hours lysed. Equal amounts (400 μg) of protein were immunoprecipitated (IP) using the indicated antibodies, and immunoprecipitates were separated by SDS-PAGE and immunoblotted using the indicated antibodies. (B) Representative volume-rendered 3D reconstructions of confocal z-stacks of *Mfn1*^−/−, *Mfn2*^−/− MEFs cotransfected with ER-YFP, mt-dsRED, and plasmids coding for the indicated HA-tagged proteins. Where indicated (+MFN1), MEFs were also cotransfected with MFN1. (C) Means ± SEs of Manders’ coefficient (n = 3 independent experiments, 15 cells per condition per experiment) (open dots, EV; closed dots, +MFN1) of ER-mitochondria pseudocolocalization in experiments as in (B). *P < 0.05 in a one-way ANOVA with Tukey’s mean comparison among the indicated groups.
( A ) *Mfn1* ^−/− 、 *Mfn2* ^−/− MEF 与所示质粒共转染，24 小时后裂解。使用指定的抗体对等量（400 μg）的蛋白质进行免疫沉淀（IP），并通过 SDS-PAGE 分离免疫沉淀物并使用指定的抗体进行免疫印迹。 ( B ) 与 ER-YFP、mt-dsRED 和编码所示 HA 标记蛋白的质粒共转染的*Mfn1* ^−/− 、 *Mfn2* ^−/− MEF 的共焦z堆叠的代表性体积渲染 3D 重建。在标明 (+MFN1) 的情况下，MEF 也与 MFN1 共转染。 ( C ) 实验中 ER 线粒体假共定位的 Manders 系数平均值 ± SE（ n = 3 个独立实验，每个实验每个条件 15 个细胞）（空心点，EV；实心点，+MFN1），如 (B) 中的实验。 * 单向方差分析中P < 0.05，与指定组之间的 Tukey 平均值比较。

Open in viewer

Coiled-coil domains mediate ERMIN2/ERMIT2-mitofusin interaction
卷曲螺旋结构域介导 ERMIN2/ERMIT2-线粒体融合蛋白相互作用

We generated multiple deletion mutants of MFN1, MFN2, ERMIN2, and ERMIT2 to address which domains were required for their heterotypic interaction. A MFN2 mutant containing the CC1, TM, and CC2 domains, as well as MFN2 mutants alternatively containing the CC1 or the CC2 domain, immunoprecipitated ERMIN2 and ERMIT2 (Fig. 6A
我们生成了 MFN1、MFN2、ERMIN2 和 ERMIT2 的多个缺失突变体，以解决它们异型相互作用所需的结构域。含有CC1、TM和CC2结构域的MFN2突变体，以及交替含有CC1或CC2结构域的MFN2突变体，免疫沉淀的ERMIN2和ERMIT2（图6A）). If we added the GTPase domain to the MFN1 and MFN2 mutants containing only the CC1 region, they comparably immunoprecipitated ERMIN2 and ERMIT2 (Fig. 6B
）。如果我们将 GTPase 结构域添加到仅包含 CC1 区域的 MFN1 和 MFN2 突变体中，它们会相应地免疫沉淀 ERMIN2 和 ERMIT2（图 6B）). Thus, MFN1 and MFN2 interact with ERMIN2 or ERMIT2 through their CC1 or CC2 domains. Reciprocally, an ERMIT2 mutant lacking the pre-TM domain still immunoprecipitated MFN1, whereas upon deletion of its CC2 domain, MFN1 binding was lost (Fig. 6C
）。因此，MFN1和MFN2通过它们的CC1或CC2域与ERMIN2或ERMIT2相互作用。相反，缺乏 pre-TM 结构域的 ERMIT2 突变体仍然免疫沉淀 MFN1，而在删除其 CC2 结构域后，MFN1 结合消失（图 6C）). ERMIT2 and ERMIN2 also interacted in HeLa cells as well as in MEFs lacking Mfn1, Mfn2, or both mitofusins (fig. S13A). We further tested the emerging interaction paradigm between the C-terminal domains of ERMIT2 or ERMIN2 and the CC1 or CC2 domains of mitochondrial mitofusins in an in silico structural modeling analysis. Guided by the AlphaFold 2 predicted structures of Mfn1 and ERMIT2, the available MFN1 CC2 homodimer crystal structure (35, 36), the experimentally determined dimerization interface, and sequence alignment, we created a model of the MFN2-ERMIT2 complex where the CC1 domain of MFN2 coiled around the CC2 domain of ERMIT2. Using in silico mutagenesis, we prepared an identical model of the Mfn1-ERMIT2 complex. We verified the stability of the atomistic models anchored to model ER and mitochondrial membranes by relaxing the 1.2M-atom systems in a long molecular dynamics simulation in explicit solvent. The number of contacts between residues of Mfn1 CC1 and ERMIT2 CC2 was consistently stable, which suggests that this interaction can tether ER and mitochondria even at a surface-to-surface distance of 20 nm (Fig. 6D
）。 ERMIT2 和 ERMIN2 也在 HeLa 细胞以及缺乏Mfn1 、 Mfn2或两种丝裂融合素的 MEF 中相互作用（图 S13A）。我们在计算机结构建模分析中进一步测试了 ERMIT2 或 ERMIN2 的 C 端结构域与线粒体线粒体融合素的 CC1 或 CC2 结构域之间的新兴相互作用范例。在 AlphaFold 2 预测的 Mfn1 和 ERMIT2 结构、可用的 MFN1 CC2 同二聚体晶体结构 ( 35 , 36 )、实验确定的二聚化界面和序列比对的指导下，我们创建了 MFN2-ERMIT2 复合物的模型，其中 CC1 结构域MFN2 缠绕在 ERMIT2 的 CC2 结构域周围。使用计算机诱变，我们制备了 Mfn1-ERMIT2 复合物的相同模型。我们通过在显式溶剂中的长分子动力学模拟中松弛 1.2M 原子系统，验证了锚定到模型 ER 和线粒体膜的原子模型的稳定性。 Mfn1 CC1 和 ERMIT2 CC2 残基之间的接触数量始终稳定，这表明这种相互作用即使在 20 nm 的表面距离下也可以束缚 ER 和线粒体（图 6D）). A contact-based affinity prediction performed on an isolated CC1-CC2 pair yielded almost identical stability for the Mfn1-ERMIT2 and the MFN2-ERMIT2 complexes (estimated affinity: MFN2-ERMIT2, −12.5 kcal/mol; Mfn1-ERMIT2, −12.6 kcal/mol; fig. S13, B and C). Thus, this simulation shows that the affinity of these complexes is well within the range required for membrane tethering, which suggests how ERMIT2 can tether ER to mitochondria through MFN1 in the absence of MFN2.
）。对分离的 CC1-CC2 对进行基于接触的亲和力预测，得出 Mfn1-ERMIT2 和 MFN2-ERMIT2 复合物几乎相同的稳定性（估计亲和力：MFN2-ERMIT2，-12.5 kcal/mol；Mfn1-ERMIT2，-12.6 kcal /mol；图S13、B和C)。因此，该模拟表明这些复合物的亲和力完全在膜束缚所需的范围内，这表明 ERMIT2 如何在没有 MFN2 的情况下通过 MFN1 将 ER 束缚于线粒体。

Fig. 6. Ermit2-mitofusins interact via their coiled-coil domains.
如图。 6 . Ermit2-mitofusins 通过其卷曲螺旋结构域相互作用。
(A to C) HeLa cells were cotransfected with plasmids encoding for the indicated constructs and after 24 hours lysed. Schemes depict the domains found in the transfected chimeras. Equal amounts (400 μg) of protein were immunoprecipitated (IP) using the indicated antibodies, and immunoprecipitates were separated by SDS-PAGE and immunoblotted using the indicated antibodies. (D) Structural modeling of the interaction between a truncated version of MFN1 and ERMIT2. The initial knowledge-based model was relaxed in a long, fully atomistic molecular dynamics simulation, revealing one of the possible arrangements of the GTPase domain. The magnified region shows the interface between MFN1 (green) CC1 and ERMIT2 (orange) CC2 domain.
( A至C ) HeLa 细胞与编码所示构建体的质粒共转染，并在 24 小时后裂解。方案描绘了在转染的嵌合体中发现的结构域。使用指定的抗体对等量（400 μg）的蛋白质进行免疫沉淀（IP），并通过 SDS-PAGE 分离免疫沉淀物并使用指定的抗体进行免疫印迹。 ( D ) MFN1 和 ERMIT2 的截短版本之间相互作用的结构建模。最初的基于知识的模型在长时间的、完全原子分子动力学模拟中得到了放松，揭示了 GTPase 结构域的一种可能排列。放大区域显示 MFN1（绿色）CC1 和 ERMIT2（橙色）CC2 结构域之间的界面。

Open in viewer

ERMIT2 licenses Ca²⁺ and phospholipid transfer from ER to mitochondria
ERMIT2 许可 Ca ²⁺和磷脂从 ER 转移至线粒体

We finally verified whether ERMIT2 like MFN2 influenced two facets of ER-mitochondria communication: mitochondrial uptake of ER-released Ca²⁺ (37, 38) and lipid transfer (2, 10, 39). MFN2, ERMIN2, or ERMIT2 lowered steady-state ER Ca²⁺ levels measured in empty vector (EV) transfected Mfn2^−/− MEFs (13, 16) (fig. S14, A and B). We titrated the inositol triphosphate (IP₃)–generating agonist adenosine 5′-triphosphate (ATP) to induce the same ER Ca²⁺ release in Mfn2^−/− MEFs transfected with all the vectors used in this study (40, 41) (fig. S14, C and D). Upon equalized ER Ca²⁺ release, MFN2 and ERMIT2, but not ERMIN2, increased mitochondrial Ca²⁺ uptake compared with EV (Fig. 7, A and B
我们最终验证了 ERMIT2 是否像 MFN2 一样影响 ER-线粒体通讯的两个方面：线粒体摄取 ER 释放的 Ca ²⁺ ( 37 , 38 ) 和脂质转移 ( 2 , 10 , 39 )。 MFN2、ERMIN2 或 ERMIT2 降低了在空载体 (EV) 转染的Mfn2 ^−/− MEF ( 13 、 16 ) 中测量的稳态 ER Ca ²⁺水平（图 S14、A 和 B）。我们滴定了肌醇三磷酸 (IP ₃ ) – 产生激动剂腺苷 5'-三磷酸 (ATP)，以在用本研究中使用的所有载体转染的Mfn2 ^−/− MEF 中诱导相同的 ER Ca ²⁺释放 ( 40 , 41 ) (图 S14、C 和 D）。在均衡 ER Ca ²⁺释放后，与 EV 相比，MFN2 和 ERMIT2（但不是 ERMIN2）增加了线粒体 Ca ²⁺摄取（图 7，A 和 B）). A genetically encoded ATP probe targeted to the mitochondrial matrix (3) indicated that MFN2 and ERMIT2 expression increased the Ca²⁺ uptake–dependent priming of mitochondrial ATP generation (16, 42) (Fig. 7, C and D
）。靶向线粒体基质的基因编码 ATP 探针 ( 3 ) 表明 MFN2 和 ERMIT2 表达增加了线粒体 ATP 生成的 Ca ²⁺摄取依赖性启动 ( 16 , 42 )（图 7、C 和 D）). Thus, ERMIT2 expression in Mfn2^−/− cells licenses mitochondrial uptake of ER-released Ca²⁺ and Ca²⁺-primed mitochondrial ATP production that depends on matrix Ca²⁺-activated Krebs’ cycle dehydrogenases (43).
）。因此， Mfn2 ^−/−细胞中的 ERMIT2 表达允许线粒体摄取 ER 释放的 Ca ²⁺和 Ca ²⁺引发的线粒体 ATP 生成，这取决于基质 Ca ²⁺激活的克雷布斯循环脱氢酶 ( 43 )。

Fig. 7. ERMIT2 licenses ER-mitochondria Ca²⁺ and lipid transfer in vitro and in vivo.
图7 . ERMIT2 许可 ER-线粒体 Ca ²⁺和体外和体内脂质转移。
(A) Representative mitochondrial aequorin (mt-AEQ) measurements of [Ca²⁺]_mit in response to ATP (0.2 mM) in *Mfn2*^−/− MEFs cotransfected with the indicated plasmid and mt-AEQ. Averages ± SEMs peak mitochondrial Ca²⁺: EV = 3.04 ± 0.27, N = 5; MFN2 = 9.30 ± 1.14, N = 8; ERMIN2 = 5.58 ± 0.63, N = 7; ERMIT2 = 10.79 ± 1.42, N = 6. P < 0.05 in a one-way ANOVA with Tukey’s test for the EV-MFN2, EV-ERMIT2, and ERMIT2-ERMIN2 pairs. (B) Means ± SEs of the area under the curve (AUC) in experiments as in (A) (n = 10 independent experiments). *P < 0.05 in a one-way ANOVA with Tukey’s mean comparison among the indicated conditions. (C) Representative traces of mitochondrial ATP measurements in *Mfn2*^−/− MEFs cotransfected with mt-luciferase and the indicated plasmids. Where indicated, cells were perfused with ATP (0.2 mM) to initiate IP3-mediated ER Ca²⁺ release. (D) Means ± SEs of the AUC in experiments as in (D) (n = 10 independent experiments). *P < 0.05 in a one-way ANOVA with Tukey’s mean comparison among the indicated conditions. (E and F) Five days after tail-vein injection of adenoviruses encoding LacZ or ERMIT2 in Mfn2^f/f or Mfn2^LKO mice, livers were explanted and means ± SEMs ³H–L-Ser incorporation into PS (E) and PE (F) in hepatic mitochondria-associated ER-enriched fractions (n = 3 to 6 mice per condition; open dots) were measured. *P < 0.05 in a one-way ANOVA with Tukey’s mean comparison among the indicated conditions. (G and H) Wild-type mice were fed with chow or MCD for 3 weeks. On day 7, mice were tail-vein injected with adenoviruses encoding LacZ or ERMIT2 (n = 7 to 11 mice per group). At the end of the diet protocol, livers were explanted and means ± SEMs ³H–L-Ser incorporation into PS (G) and PE (H) in hepatic mitochondria-associated ER-enriched fractions were measured. *P < 0.05 in a one-way ANOVA with Tukey’s mean comparison among the indicated conditions. DPM, disintegrations per minute.
( A ) 在用所示质粒和 mt-AEQ 共转染的*Mfn2* ^−/− MEF 中，对 [Ca ²⁺ ] _mit响应 ATP (0.2 mM) 的代表性线粒体水母发光蛋白 (mt-AEQ) 测量。平均值 ± SEM 峰值线粒体 Ca ²⁺ ：EV = 3.04 ± 0.27， N = 5； MFN2=9.30±1.14， N =8； ERMIN2=5.58±0.63， N =7； ERMIT2 = 10.79 ± 1.42， N = 6。使用 Tukey 对 EV-MFN2、EV-ERMIT2 和 ERMIT2-ERMIN2 对进行单向方差分析时， P < 0.05。 ( B ) 实验中曲线下面积 (AUC) 的平均值 ± SE（ n = 10 个独立实验）。 * 单向方差分析中P < 0.05，与指定条件之间的 Tukey 平均值比较。 ( C ) 用 mt-荧光素酶和所示质粒共转染的*Mfn2* ^−/− MEF 中线粒体 ATP 测量的代表性痕迹。如有指示，用 ATP (0.2 mM) 灌注细胞以启动 IP3 介导的 ER Ca ²⁺释放。 ( D ) 实验中 AUC 的平均值 ± SE（ n = 10 个独立实验）。 * 单向方差分析中P < 0.05，与指定条件之间的 Tukey 平均值比较。 ( E和F ) 在 Mfn2 ^f/f或 Mfn2 ^LKO小鼠中尾静脉注射编码 LacZ 或 ERMIT2 的腺病毒五天后，移植肝脏，平均值±SEM ³ H-L-Ser 掺入 PS (E) 和 PE ( F) 测量了肝线粒体相关的 ER 富集部分（每个条件n = 3 至 6 只小鼠；空心点）。 * P < 0。05 在单向方差分析中与指定条件之间的 Tukey 均值比较。 ( G和H ) 野生型小鼠用食物或 MCD 喂养 3 周。第 7 天，对小鼠进行尾静脉注射编码 LacZ 或 ERMIT2 的腺病毒（每组n = 7 至 11 只小鼠）。在饮食方案结束时，移植肝脏并测量平均值±SEM ³ H-L-Ser 掺入肝线粒体相关 ER 富集部分中的 PS (G) 和 PE (H)。 * 单向方差分析中P < 0.05，与指定条件之间的 Tukey 平均值比较。 DPM，每分钟崩解次数。

Open in viewer

In the liver, phosphatidylserine (PS) is synthesized in the ER and transferred to mitochondria where it is then converted to phosphatidylethanolamine (PE). In Mfn2^LKO hepatocytes, PS synthesis and transfer to mitochondria are impaired, resulting in ER stress and inflammation (10). We thus measured whether ERMIT2 expression normalized lipid transfer between ER and mitochondria. In control (LacZ) infected Mfn2^LKO mice, hepatic incorporation of L-serine (L-Ser) into PS and PE was reduced. When we infected Mfn2^LKO livers with ERMIT2 adenoviruses (fig. S15A), L-Ser incorporation into PE and PS was corrected (Fig. 7, E and F
在肝脏中，磷脂酰丝氨酸 (PS) 在 ER 中合成并转移到线粒体，然后在线粒体中转化为磷脂酰乙醇胺 (PE)。在 Mfn2 ^LKO肝细胞中，PS 合成和向线粒体的转移受损，导致 ER 应激和炎症 ( 10 )。因此，我们测量了 ERMIT2 表达是否使 ER 和线粒体之间的脂质转移正常化。在对照 (LacZ) 感染 Mfn2 ^LKO小鼠中，肝脏将 L-丝氨酸 (L-Ser) 掺入 PS 和 PE 中减少。当我们用 ERMIT2 腺病毒感染 Mfn2 ^LKO肝脏时（图 S15A），L-Ser 掺入 PE 和 PS 中被纠正（图 7、E 和 F）). In addition, in the Mfn2^LKO cohort (10), ERMIT2 reduced the accumulation of H₂O₂ (fig. S15B), reduced the levels of ER stress markers phosphorylated eIF2α and CHOP (fig. S15, C and D), and reduced the levels of the inflammation marker tumor necrosis factor–α (TNF-α) (fig. S15E). Thus, ERMIT2 sustains ER to mitochondria lipid transfer and corrects the liver ER stress and inflammation observed in the Mfn2^LKO mice. Mice fed a methionine-choline–deficient (MCD) high-fat diet develop a nonalcoholic steatohepatitis (NASH)–like condition, an orthogonal model of liver ER stress and inflammation compared with MFN2 deficiency (10) (fig. S16A). In the MCD-fed mice, we observed altered hepatic L-Ser labeling of PS and PE (Fig. 7, G and H
）。此外，在Mfn2 ^LKO队列中（ 10 ），ERMIT2减少了H ₂ O ₂的积累（图S15B），降低了ER应激标记物磷酸化eIF2α和CHOP的水平（图S15，C和D），并减少了炎症标志物肿瘤坏死因子-α (TNF-α) 的水平（图 S15E）。因此，ERMIT2 维持 ER 向线粒体脂质转移，并纠正 Mfn2 ^LKO小鼠中观察到的肝脏 ER 应激和炎症。饲喂蛋氨酸胆碱缺乏（MCD）高脂饮食的小鼠会出现类似非酒精性脂肪性肝炎（NASH）的病症，这是与 MFN2 缺乏相比的肝脏 ER 应激和炎症的正交模型（ 10 ）（图 S16A）。在 MCD 喂养的小鼠中，我们观察到 PS 和 PE 的肝脏 L-Ser 标记发生改变（图 7，G 和 H）). Adenoviruses encoding ERMIT2 delivered at day 7 to mice undergoing a 3-week MCD feeding protocol increased ERMIT2 but not MFN2 levels (fig. S16, A and B). ERMIT2 delivery corrected L-Ser labeling of PE and PS (Fig. 7, G and H
）。编码 ERMIT2 的腺病毒在第 7 天递送给接受 3 周 MCD 喂养方案的小鼠，增加了 ERMIT2，但没有增加 MFN2 水平（图 S16、A 和 B）。 ERMIT2 递送校正了 PE 和 PS 的 L-Ser 标记（图 7，G 和 H) and reduced the accumulation of markers of ER stress (fig. S16, C and D) and inflammation (fig. S16E) without altering body weight or glycemia (fig. S15, F and G). Thus, ERMIT2 sustains ER to mitochondria lipid transfer and corrects the liver ER stress and inflammation in genetic and environmental mouse models of reduced liver Mfn2 expression, highlighting the importance of ER-mitochondria juxtaposition in models of NASH.
）并减少 ER 应激标志物（图 S16、C 和 D）和炎症（图 S16E）的积累，而不改变体重或血糖（图 S15、F 和 G）。因此，在肝脏Mfn2表达减少的遗传和环境小鼠模型中，ERMIT2维持ER到线粒体的脂质转移，并纠正肝脏ER应激和炎症，凸显了ER-线粒体并置在NASH模型中的重要性。

Discussion 讨论

The mechanism by which MFN2 tethers ER to mitochondria has been elusive. In contrast to other tether pairs or complexes (23, 44, 45), MFN2 lacked a known ER partner, and its ER-mitochondria tethering function was explained by its retrieval also on the ER, from where it interacted in trans with mitochondrial MFN2 or MFN1 (13, 16). Our results identify a MFN2 splice variant enriched at the ER interface with mitochondria as the ER tethering partner of MFN2.
MFN2 将 ER 与线粒体连接的机制尚不清楚。与其他系链对或复合物 ( 23 , 44 , 45 ) 相比，MFN2 缺乏已知的 ER 伴侣，其 ER-线粒体系链功能可以通过其在 ER 上的恢复来解释，从那里它与线粒体 MFN2 反式相互作用或MFN1（13、16 ）。我们的结果确定了MFN2剪接变体在 ER 界面富集，线粒体作为 MFN2 的 ER 束缚伴侣。

The identification of extramitochondrial MFN2 splice variants can explain the pleiotropic effects of Mfn2 ablation on mitochondrial and ER morphology and on ER-mitochondria tethering (11, 13, 15, 16, 22, 26, 46). Although MFN2, like the mitochondrial outer membrane proteins BAK (5), BCL-2 (47), and cytochrome b₅ (48), can also localize at the ER, the model of ER sculpting and tethering in trans by a “mislocalized” fraction of MFN2 did not explain how a fusion GTPase could participate in mitochondrial and ER fusion and at the same time tether ER and mitochondria without fusing them. The identification of ERMIN2 and ERMIT2, two alternatively spliced, ER-restricted MFN2 variants, contributes to clarifying these issues. ERMIN2 that retains part of the MFN2 GTPase domain restores Mfn2^−/− ER connectivity, which suggests that this variant can participate in ER fusion like the ER-specific GTPase atlastin (49). ERMIT2 tethers the ER to mitochondria by interacting with MFN1 and/or MFN2 on the mitochondrial outer membrane. The structural organization of ERMIT2 seems adequate to support this function: ERMIT2 lacks the GTPase domain essential for mitochondrial fusion (34) but retains the CC2 domain (33) that is required for the interaction with CC1 or CC2 of mitochondrial mitofusins. In silico modeling supports this paradigm and highlights that the ERMIT2-MFN1/2 interaction can indeed stabilize the juxtaposition of the two organelles.
线粒体外 MFN2 剪接变体的鉴定可以解释Mfn2消融对线粒体和 ER 形态以及 ER-线粒体束缚的多效性影响 ( 11 , 13 , 15 , 16 , 22 , 26 , 46 )。尽管 MFN2 与线粒体外膜蛋白 BAK ( 5 )、BCL-2 ( 47 ) 和细胞色素 b ₅ ( 48 ) 一样也可以定位于 ER，但 ER 塑造和束缚的模型是通过“错误定位”反式进行的。 MFN2 的部分没有解释融合 GTPase 如何参与线粒体和 ER 融合，同时在不融合的情况下束缚 ER 和线粒体他们。 ERMIN2 和 ERMIT2（两种选择性剪接的 ER 限制性 MFN2 变体）的鉴定有助于澄清这些问题。保留部分 MFN2 GTPase 结构域的 ERMIN2 恢复了Mfn2 ^−/− ER 连接，这表明该变体可以像 ER 特异性 GTPase atlastin 一样参与 ER 融合 ( 49 )。 ERMIT2 通过与线粒体外膜上的 MFN1 和/或 MFN2 相互作用将 ER 束缚于线粒体。 ERMIT2 的结构组织似乎足以支持这一功能：ERMIT2 缺乏线粒体融合所必需的 GTPase 结构域 ( 34 )，但保留了与线粒体线粒体融合素的 CC1 或 CC2 相互作用所需的 CC2 结构域 ( 33 )。计算机模型支持这种范式，并强调 ERMIT2-MFN1/2 相互作用确实可以稳定两个细胞器的并置。

Functionally, ERMIT2 corrected the ER-mitochondria communication defects caused by reduced MFN2 levels. ERMIT2 was sufficient to license mitochondrial uptake of Ca²⁺ released from the ER when we equalized the amount of agonist-induced ER Ca²⁺ release. We surmise that ERMIT2 operates by allowing the formation of the high Ca²⁺ microdomains at the interface between the two organelles required for efficient mitochondrial Ca²⁺ uptake (37, 38, 50). In addition, ERMIN2 and ERMIT2 also normalized ER Ca²⁺ in Mfn2-deficient cells by modulating the facets of Ca²⁺ signaling altered by Mfn2 deletion: capacitative Ca²⁺ entry, SERCA activity, or ER Ca²⁺ leak (40, 41).
从功能上来说，ERMIT2 纠正了因 MFN2 水平降低而导致的 ER 线粒体通讯缺陷。当我们平衡激动剂诱导的 ER Ca ²⁺释放量时，ERMIT2 足以允许线粒体摄取从 ER 释放的 Ca ²⁺ 。我们推测 ERMIT2 的运作方式是允许在线粒体有效吸收 Ca ²⁺所需的两个细胞器之间的界面处形成高 Ca ²⁺微结构域 ( 37 , 38 , 50 )。此外，ERMIN2 和 ERMIT2 还通过调节因Mfn2缺失而改变的 Ca ²⁺信号传导的各个方面，使Mfn2缺陷细胞中的 ER Ca ²⁺正常化：电容性 Ca ²⁺进入、SERCA 活性或 ER Ca ²⁺泄漏 ( 40 , 41 ）。

In vivo ERMIT2 delivery to the liver corrected most of the hepatic alterations associated with Mfn2 genetic ablation or depletion in a diet-induced model of NASH. ERMIT normalized PS transfer from the ER to mitochondria, a process linked to alterations in lipid metabolism, ER stress, inflammation, and fibrosis (10), which suggests that it participates in PS transfer to mitochondria and in documenting the importance of MFN2-mediated ER-mitochondria tethering in NASH.
在饮食诱导的 NASH 模型中，体内 ERMIT2 递送至肝脏纠正了与Mfn2基因消融或耗竭相关的大部分肝脏改变。 ERMIT 标准化 PS 从 ER 到线粒体的转移，这一过程与脂质代谢、ER 应激、炎症和纤维化的改变有关 ( 10 )，这表明它参与 PS 转移到线粒体并记录 MFN2 介导的 ER 的重要性- NASH 中的线粒体束缚。

Several pathogenic MFN2 mutations causing defects in axonal trafficking of mitochondria and resulting in the peripheral neuropathy Charcot-Marie-Tooth (CMT) 2A (51–53) affect the CC2 region found also in the ER-specific variants (54). Thus, altered ER-mitochondria tethering or ER morphology might be a common feature of CMT2A and hereditary sensory neuropathy type I caused by mutations in atlastin that control ER fusion (49, 55) and restored by the small molecules normalizing CMT2A neuromuscular dysfunction (52).
几种致病性MFN2突变会导致线粒体轴突运输缺陷，并导致周围神经病夏科-马里-图思 (CMT) 2A ( 51 – 53 ) 影响 CC2 区域，这些突变也存在于 ER 特异性变异中 ( 54 )。因此，ER-线粒体束缚或 ER 形态的改变可能是 CMT2A 和 I 型遗传性感觉神经病的共同特征，这些特征是由控制 ER 融合的 atlastin 突变引起的 ( 49 , 55 )，并通过使 CMT2A 神经肌肉功能障碍正常化的小分子恢复 ( 52 ) 。

Overexpression of MFN2 in acute myeloid leukemia (AML) causes excessive ER-mitochondria tethering that contributes to venetoclax resistance, reversed by a small molecule binding the CC1-CC2 MFN2 interface and reducing ER-mitochondria juxtaposition (56). Thus, the discovery of MFN2 ER-specific variants offers pathobiological and therapeutic insights into metabolic and neuromuscular diseases and cancer. Our work elucidates the importance of alternative splicing of mitochondrial dynamics genes for their function (31, 57, 58) and subcellular localization (32, 59) and suggests that alternative splicing provides a mechanism for how a single mitochondrial gene can modulate manifold biological processes.
急性髓系白血病 (AML) 中 MFN2 的过度表达会导致 ER-线粒体过度束缚，从而导致 Venetoclax 耐药，而结合 CC1-CC2 MFN2 界面并减少 ER-线粒体并置的小分子可逆转这种情况 ( 56 )。因此，MFN2 ER 特异性变体的发现为代谢和神经肌肉疾病以及癌症提供了病理生物学和治疗见解。我们的工作阐明了线粒体动力学基因的选择性剪接对其功能 ( 31 , 57 , 58 ) 和亚细胞定位 ( 32 , 59 ) 的重要性，并表明选择性剪接为单个线粒体基因如何调节多种生物过程提供了一种机制。

Materials and methods 材料和方法

Molecular biology and real-time PCR
分子生物学和实时 PCR

For ERMIN2 and ERMIT2 amplification, PCRs were performed using human skeletal muscle cDNA and the following primers: (i) Mfn2 intron2: 5′-AAGATCTCTCAGCATCCAAAAA-3′ and (ii) Mfn2 exon19: 5′-ATGGCACTTAGGGCTGGCAGCA-3′.
对于ERMIN2和ERMIT2扩增，使用人骨骼肌 cDNA 和以下引物进行 PCR：（i）Mfn2 内含子 2：5'-AAGATCTCTCAGCATCCAAAAA-3' 和（ii）Mfn2 外显子 19：5'-ATGGCACTTAGGGCTGGCAGCA-3'。

Different products were cloned into Topo-TA vector (Invitrogen) and sequenced with M13 forward and reverse primers using BigDye 3.1 terminator (Applied Biosystem). Inserts were subcloned in frame into the mammalian expression vectors pCMV-HA and pEGFP (Clontech) using Bgl II (NEB) and Asp 718 (Roche) enzymes.
将不同的产物克隆到Topo-TA载体(Invitrogen)中，并使用BigDye 3.1终止子(Applied Biosystem)用M13正向和反向引物进行测序。使用 Bgl II (NEB) 和 Asp 718 (Roche) 酶将插入物框内亚克隆到哺乳动物表达载体 pCMV-HA 和 pEGFP (Clontech) 中。

Mouse variant amplification was performed using cDNA from MEFs and the primers 5′-AAGCTTGGACAGGTGGAGTCA-3′ and 5′-CAACCAGCCAGCTTTATTCC-3′. Different motility products were cloned into pCR8 Topo-GW vector (Invitrogen). First-Strand cDNA synthesis was performed using 4 μg of total RNA and the specific Mfn2 primer 5′-TGGCAAGAAGGGAGGCAAGTC-3′ and oligo dT primers incubated at 50°C for 4 hours with SuperScript IV Reverse Transcriptase (Invitrogen).
使用 MEF 的 cDNA 和引物 5'-AAGCTTGGACAGGTGGAGTCA-3' 和 5'-CAACCAGCCAGCTTTATTCC-3' 进行小鼠变异扩增。将不同的运动产物克隆到pCR8 Topo-GW载体(Invitrogen)中。使用 4 μg 总 RNA 和特异性 Mfn2 引物 5'-TGGCAAGAAGGGAGGCAAGTC-3' 和寡聚 dT 引物（与 SuperScript IV 逆转录酶 (Invitrogen) 一起在 50°C 下孵育 4 小时）进行第一链 cDNA 合成。

Split super-folder GFP (sfGFP) previously engineered for efficient self-complementation (10) breaks the sequence of sfGFP between the 10th and the 11th β-strand into two parts: GFP_1-10 and GFP₁₁, a short, 16–amino acid peptide (60). The GFP_1-10 fragment, which contains the three residues that constitute the GFP chromophore, is nonfluorescent because chromophore maturation requires the conserved E222 residue located on GFP₁₁. Upon complementation, the reconstituted GFP becomes fluorescent after the chromophore maturation reaction is completed. GFP₁₁ were fused to the N terminus of ERMIN2 or ERMIT2 and coexpressed with GFP_1-10.
分裂超级文件夹 GFP (sfGFP) 先前设计用于有效的自我互补 ( 10 )，将第 10 条和第 11 条 β 链之间的 sfGFP 序列分成两部分：GFP _1-10和 GFP ₁₁ （短的 16-氨基）酸性肽( 60 )。 GFP _1-10片段包含构成 GFP 生色团的三个残基，它是非荧光的，因为生色团成熟需要位于 GFP ₁₁上的保守 E222 残基。互补后，在发色团成熟反应完成后，重构的 GFP 会发出荧光。 GFP ₁₁融合至ERMIN2或ERMIT2的N 末端并与GFP _1-10共表达。

pcDNA3.1-GFP(1-10) (Addgene plasmid no. 70219) and pEGFP-GFP11-Clathrin light chain (Addgene plasmid no. 70217) were from Bo Huang. GFP₁₁ was generated by digestion of pEGFP-GFP11-Clathrin light chain using BglII and BclI. GFP₁₁-Mfn2, GFP₁₁-Ermin2 and GFP₁₁-Ermit2 were generated using Infusion (Takara Technology) and the following primers: (i) Vector: 5′-TAGGGATCCACCGGATCTAGATAA-3′ and 5′-GCCTGTAATCCCAGCAGCATTTAC-3′. (ii) Mfn2, Ermin2, and Ermit2: 5′-GCTGGGATTACAGGCGGGATGTCCCTGCTCTTC-3′ and 5′-TCCGGTGGATCCCTAGGGCTATCTGCTGGGCTG-3′. Mt-RFP, ER-RFP, ER-YFP, FEMP, ddGFP, mtCFP, and aequorins (AEQs) were previously described (13, 16, 61).
pcDNA3.1-GFP(1-10) (Addgene 质粒编号 70219) 和 pEGFP-GFP11-Clathrin 轻链 (Addgene 质粒编号 70217) 来自 Bo Huang。 GFP ₁₁是通过使用 BglII 和 BclI 消化 pEGFP-GFP11-网格蛋白轻链而生成的。使用 Infusion (Takara Technology) 和以下引物生成 GFP ₁₁ -Mfn2、GFP ₁₁ -Ermin2 和 GFP ₁₁ -Ermit2：(i) 载体：5'-TAGGGATCCACCGGATCTAGATAA-3' 和 5'-GCCTGTAATCCCAGCAGCATTTAC-3'。 (ii) Mfn2、Ermin2 和 Ermit2：5'-GCTGGGATTACAGGCGGGATGTCCCTGCTCTTC-3' 和 5'-TCCGGTGGATCCCTAGGGCTATCTGCTGGGCTG-3'。先前描述了 Mt-RFP、ER-RFP、ER-YFP、 FEMP、ddGFP、mtCFP 和水母发光蛋白 (AEQ) (13、16、61 ) 。

siRNA against ERMIN2 and ERMIT2 were synthesized from the following sequences: 5′-GTGATGTGGCCCAACTCTA-3′ and 5′-GACATGATAGATGGCTTGA-3′, respectively; scrambled siRNA was used as control. All siRNAs were obtained from Sigma. Experiments were performed 24 hours after transfection.
针对ERMIN2和ERMIT2 的siRNA 由以下序列合成：分别为 5'-GTGATGGTGGCCCAACTCTA-3' 和 5'-GACATGATAGATGGCTTGA-3'；乱序的siRNA用作对照。所有 siRNA 均获自 Sigma。转染后24小时进行实验。

For RNA isolation, human and mouse tissues were immediately frozen. RNA from liver tissues was extracted using a protocol combining TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and RNAeasy minikit columns (Qiagen, Alameda, CA, USA), following the manufacturer’s instructions. RNA was reverse transcribed with the SuperScript RT IV kit (Invitrogen, Carlsbad, CA, USA). PCR was performed using the ABI Prism 7900 HT real-time PCR machine (Applied Biosystems, USA) and the SYBR Green PCR Master Mix (Applied Biosystems, USA). Measurements were normalized to PPIA (Cyclophilin A), ARP, or β-actin.
为了分离 RNA，立即冷冻人类和小鼠组织。使用结合 TRIzol 试剂（Invitrogen，卡尔斯巴德，加利福尼亚州，美国）和 RNAeasy 微型试剂盒柱（Qiagen，阿拉米达，加利福尼亚州，美国）的方案，按照制造商的说明从肝组织中提取 RNA。使用 SuperScript RT IV 试剂盒（Invitrogen，卡尔斯巴德，加利福尼亚州，美国）对 RNA 进行逆转录。使用 ABI Prism 7900 HT 实时 PCR 机（Applied Biosystems，美国）和 SYBR Green PCR Master Mix（Applied Biosystems，美国）进行 PCR。测量结果标准化为 PPIA（亲环蛋白 A）、ARP 或 β-肌动蛋白。

Absolute cDNA quantification in real-time, PCR was performed using a standard curve method. For standard curve construction, the amount of plasmid MFN2, ERMIN2, and ERMIT2 was measured using Qubit fluorometric quantification (ThermoFisher scientific) and copy number was calculated using the online application Calculator for determining the number of copies of a template (URI Genomics and Sequencing Center).
使用标准曲线法进行 PCR 实时绝对 cDNA 定量。对于标准曲线构建，使用 Qubit 荧光定量（ThermoFisher Scientific）测量质粒MFN2 、 ERMIN2和ERMIT2的量，并使用在线应用程序计算器计算拷贝数，以确定模板的拷贝数（URI Genomics and Sequencing Center））。

Real-time PCR was performed using a QuantStudio 6 Real-Time PCR system (ThermoFisher) using Applied biosystems power Sybr green PCR master mix (AB Applied Bioscience) and the following primers: (i) MFN2: 5′-GACTGAGCTGGGCGTGGTGG-3′ and 5′-GTAAACCTGCTGCTCCCGAGC-3′. (ii) ERMIN2: 5′-GAACCAGCTGGCCCATGCCCT-3′ and 5′-TCCTGGGTGAGCGAGCCCTG-3′. (iii) ERMIT2: 5′-TCCCTGCTCTTCTCTCGATG-3′ and 5′-ATTCTTATAAACCTTGAGGACT-3′.
使用 QuantStudio 6 实时 PCR 系统 (ThermoFisher) 使用 Applied Biosystems power Sybr green PCR 主混合物 (AB Applied Bioscience) 和以下引物进行实时 PCR：(i) MFN2 ：5'-GACTGAGCTGGGCGTGGTGG-3' 和5'-GTAAACCTGCTGCTCCCGAGC-3'。 (ii) ERMIN2 ：5'-GAACCAGCTGGCCCATGCCCT-3'和5'-TCCTGGGTGAGCGAGCCCTG-3'。 (iii) ERMIT2 ：5'-TCCCTGCTCTTCTCTCGATG-3'和5'-ATTCTTATAAACCTTGAGGACT-3'。

PCR was run at standard conditions: 50°C for 2 min, predenaturation at 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 s, followed by annealing plus extension at 60°C for 1 min. Melting curve analysis was performed following the manufacturer’s instructions.
PCR 在标准条件下运行：50°C 2 分钟，95°C 预变性 10 分钟，95°C 变性 15 秒，40 个循环，然后在 60°C 退火加延伸 1 分钟。按照制造商的说明进行熔解曲线分析。

RPA 机器人程序自动化

Probes were designed for ERMIN2 and ERMIT2 so that they covered the flanking sequences at 5′- and -3′ of the skipped region and a nonmatching sequence representing ~20% of the probe length. PCR was performed using primers that incorporate a SP6 and T7 phage promoter sequence. PCR products were cloned into pGEMT Easy Vector (Promega) and sequenced. Cloned probes were linearized with Sac II restriction enzyme. Retrotranscription and antisense labeled probes were performed using T7 or SP6 phage RNA polymerases (MaxiScript Kit, Ambion) labeled with [α-³²P] UTP (Perkin Elmer), following manufacturer’s instructions. Deoxyribonuclease (DNase) digestion of template was achieved by phenol/chloroform extraction precipitation with NH₄Acetate and linear acrylamide as carrier. Hybridization and digestion were performed using RPA-III Kit (Ambion), Target RNA and positive control RNA obtained from overexpressing cells were hybridized with 4.4 × 10⁵ cpm of probe overnight at 56°C. Ribonuclease (RNase) digestion of unhybridized RNA was performed at 33°C for 60 min with RNase A/T1 mix. Separation and detection of protected fragments were carried out using 8 M urea denaturing 5% polyacrylamide gel. The dried gel was exposed at −80°C overnight using an x-ray film (Amersham) or a Molecular Dynamics (Amersham Pharmacia) PhosphoImager plate, which was then scanned with a Typhoon 9200 Bio-imaging analyzer (Molecular dynamics, Amersham Pharmacia Biotech, Piscataway, NJ).
探针是为ERMIN2和ERMIT2设计的，因此它们覆盖了跳过区域的 5'- 和 -3' 侧翼序列以及代表探针长度约 20% 的非匹配序列。使用包含 SP6 和 T7 噬菌体启动子序列的引物进行 PCR。 PCR 产物被克隆到 pGEMT Easy Vector (Promega) 中并测序。克隆的探针用 Sac II 限制性内切酶线性化。使用标记有[α- ³² P] UTP (Perkin Elmer)的T7或SP6噬菌体RNA聚合酶(MaxiScript Kit，Ambion)按照制造商的说明进行逆转录和反义标记探针。以NH ₄乙酸盐和线性丙烯酰胺为载体，通过苯酚/氯仿萃取沉淀实现模板的脱氧核糖核酸酶(DNase)消化。使用RPA-III试剂盒(Ambion)进行杂交和消化，将从过表达细胞获得的目标RNA和阳性对照RNA与4.4×10 ⁵ cpm的探针在56℃下杂交过夜。使用 RNase A/T1 混合物在 33°C 下对未杂交的 RNA 进行核糖核酸酶 (RNase) 消化 60 分钟。使用8M尿素变性5%聚丙烯酰胺凝胶进行受保护片段的分离和检测。使用 X 射线胶片 (Amersham) 或 Molecular Dynamics (Amersham Pharmacia) PhosphoImager 板将干燥的凝胶在 -80°C 下曝光过夜，然后用 Typhoon 9200 生物成像分析仪（Moleculardynamics, Amersham Pharmacia Biotech）扫描，新泽西州皮斯卡塔韦）。

Cell culture 细胞培养

HeLa and SV40 immortalized wild-type (WT), Mfn2^−/−, Mfn1^−/−Mfn2^−/− MEFs were grown in Dulbecco’s minimum essential medium (DMEM) (Gibco) supplemented with 10% (vol/vol) fetal bovine serum (FBS) (Gibco), 1× nonessential amino acids, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco). Cells were maintained at 80 to 90% confluency. Microscopy experiments were performed at 70% confluency. MEFs were cultured and transfected as described previously using Transfectin (Biorad) or Lipoafectamine 2000 (Invitrogen), following the manufacturer’s instructions (13). siRNA transfection was performed at a final concentration of 250 nM using Oligofectamine (Invitrogen). For imaging studies, 1.6 × 10⁴ cells were seeded onto 24-mm round glass coverslips or 4 × 10³ cells onto 13-mm round glass coverslips.
HeLa 和 SV40 永生化野生型 (WT)、 Mfn2 ^−/− 、 Mfn1 ^−/− Mfn2 ^−/− MEF 在补充有 10% (vol/vol) 胎牛血清的 Dulbecco 最低必需培养基 (DMEM) (Gibco) 中生长(FBS) (Gibco)，1× 非必需氨基酸，2 mM L-谷氨酰胺，100 U/ml青霉素和 100 μg/ml 链霉素 (Gibco)。细胞保持在80%至90%的汇合度。显微镜实验在70%汇合时进行。按照制造商的说明，使用 Transfectin (Biorad) 或 Lipofectamine 2000 (Invitrogen) 如前所述培养和转染 MEF ( 13 )。使用Oligofectamine (Invitrogen)以250nM的终浓度进行siRNA转染。对于成像研究，将 1.6 × 10 ^{4 个}细胞接种到 24 毫米圆形玻璃盖玻片上，或将 4 × 10 ^{3 个}细胞接种到 13 毫米圆形玻璃盖玻片上。

Imaging 影像学

For confocal imaging of live cells, 2 × 10⁵ cells seeded onto 24-mm round glass coverslips, treated as indicated, were placed on the stage of a Nikon Eclipse TE300 inverted microscope equipped with a PerkinElmer Ultraview LCI confocal system, a piezoelectric z axis motorized stage (Pifoc, Physik Instrumente), and an Orca ER 12-bit CCD camera (Hamamatsu Photonics). Acquisition, deconvolution, 3D reconstruction, volume rendering of the stacks, as well as analysis of the mitochondria-to-ER interaction were performed as described previously (16). For colocalization analyses, images stacks were acquired using a Leica SP5 inverted microscope equipped with confocal or Leica TCS SP2 AOBS, using a 63X1.4 NA Plan Apo objective. Cells were excited using 488 nm Ar/ArKr and 561 DPSS (diode pump solid state) lasers.
对于活细胞的共焦成像，将 2 × 10 ^{5 个}细胞接种到 24 毫米圆形玻璃盖玻片上，按照指示进行处理，然后将其放置在配备 PerkinElmer Ultraview LCI 共焦系统、压电 z 轴的 Nikon Eclipse TE300 倒置显微镜的载物台上电动平台（Pifoc、Physik Instrumente）和 Orca ER 12 位 CCD 相机（Hamamatsu Photonics）。如前所述进行采集、反卷积、3D 重建、堆栈的体积渲染以及线粒体与 ER 相互作用的分析 ( 16 )。对于共定位分析，使用配备共焦或 Leica TCS SP2 AOBS 的 Leica SP5 倒置显微镜和 63X1.4 NA Plan Apo 物镜获取图像堆栈。使用 488 nm Ar/ArKr 和 561 DPSS（二极管泵浦固态）激光器激发细胞。

For FEMP FRET imaging, unless differently specified, 9000 Mfn2^−/− MEFs/ml were seeded on a Cell carrier 384-well plate (PerkinElmer) and transfected as indicated. After 24 hours, cells were transfected with FEMP cDNA using Genjet (SignaGen) according to manufacturer’s instructions, and after further 24 hours, cells were imaged using an Operetta CLS High Content imaging system (Perkin Elmer) using the following filters: CFP (excitation 410 to 430, emission 460 to 500), YFP (excitation 490 to 510, emission 520 to 560), and YFPFRET (excitation 410 to 430, emission 520 to 560). To image the maximum FRET intensity (FRET_max), cells were treated with 100 nM Rapamycin for 15 min and then fixed with 1% formaldehyde for 10 min. Images were analyzed using PerkinElmer Harmony 3.5 image analysis software. The YFP channel was chosen to mark the region of interest (ROI), and around each ROI, a second boundary was drawn to measure the background (bg) intensity. FRET_basal and FRET_max were calculated as
对于 FEMP FRET 成像，除非另有说明，否则将 9000 个Mfn2 ^−/− MEF/ml 接种到细胞载体 384 孔板 (PerkinElmer) 上并按指示进行转染。 24小时后，根据制造商的说明，使用Genjet (SignaGen)用FEMP cDNA转染细胞，再过24小时后，使用Operetta CLS高内涵成像系统(Perkin Elmer)使用以下滤光片对细胞进行成像：CFP(激发410)至 430，发射 460 至 500），YFP（激发 490 至 510，发射 520 至560）和 YFPFRET（激发 410 至 430，发射 520 至 560）。为了对最大 FRET 强度 (FRET _max ) 进行成像，将细胞用 100 nM 雷帕霉素处理 15 分钟，然后用 1% 甲醛固定 10 分钟。使用 PerkinElmer Harmony 3.5 图像分析软件对图像进行分析。选择 YFP 通道来标记感兴趣区域 (ROI)，并在每个 ROI 周围绘制第二个边界来测量背景 (bg) 强度。 FRET_基础值和 FRET_最大值计算如下

FRET = \frac{(F_{YFP} {×FRET}_{cell} - F_{YFP} {×FRET}_{bg})}{(F_{CFPcell} - F_{CFPbg})}

and the FRET ratio was calculated as
FRET 比率计算为

{FRET}_{ratio} = \frac{({FRET}_{max} - {FRET}_{basal})}{({FRET}_{basal})}

FRAP

After 24 hours, cells plated and transfected with erYFP as described were incubated in HBSS and placed on the stage of a laser scanning microscope (TCS SP5, Leica). Using the LasAF software (Leica), regions measuring 16 μm² were manually defined to be bleached avowing the perinuclear region. The selected cells showed a moderate expression of erYFP. To bleach the YFP fluorescence, one z-plane was bleached for a total of 3 s using 100% power of the 488-nm laser line with a 63X, 1.4 NA objective. The postbleaching images were taken at 1-s intervals for a total of 90 s. Intensities of the photobleached regions were measured and normalized to the intensities of the same region before photobleaching (photobleaching effect) and the neighboring regions at the same time point (auto-bleaching effect during the recording).
24小时后，将按所述铺板并用erYFP转染的细胞在HBSS中孵育并置于激光扫描显微镜（TCS SP5，Leica）的载物台上。使用LasAF软件(Leica)，手动定义测量16μm ^{2 的}区域以漂白核周区域。所选择的细胞显示erYFP的中等表达。为了漂白 YFP 荧光，使用 63X、1.4 NA 物镜的 488 nm 激光线的 100% 功率，将一个z平面漂白总共 3 秒。漂白后图像以 1 秒的间隔拍摄，总共 90 秒。测量光漂白区域的强度，并将其归一化为光漂白前同一区域的强度（光漂白效应）和同一时间点相邻区域的强度（记录期间的自动漂白效应）。

Morphometric and MCS analysis
形态测量和 MCS 分析

For morphometric analysis of mitochondria, the length of the major axis and the roundness index of each identified object were calculated. Cells were scored with elongated mitochondria when >50% of the objects in the image (i.e., mitochondria) displayed a major axis longer than 3 μm and a roundness index below 0.3 (maximum value is 1). Alternatively, mitochondria aspect ratio (AR) was calculated from automatically thresholded images in ImageJ (NIH). For analysis of the mitochondria-ER interaction, images of cells expressing mtRFP or erYFP were processed using the automatic threshold function of ImageJ, followed by deconvolution, 3D reconstruction, and surface rendering using the VolumeJ plugin of ImageJ. The interaction between the ER and mitochondria was analyzed using Manders’ colocalization coefficient.
对于线粒体的形态测量分析，计算了每个识别对象的长轴长度和圆度指数。当图像中 >50% 的物体（即线粒体）显示长轴超过 3 μm 且圆度指数低于 0.3（最大值为 1）时，对细胞进行细长线粒体评分。或者，线粒体纵横比 (AR) 是根据 ImageJ (NIH) 中的自动阈值图像计算的。为了分析线粒体-ER 相互作用，使用 ImageJ 的自动阈值功能处理表达 mtRFP 或 erYFP 的细胞图像，然后使用 ImageJ 的 VolumeJ 插件进行反卷积、3D 重建和表面渲染。使用曼德斯共定位系数分析内质网和线粒体之间的相互作用。

For ddGFP imaging, Mfn2^−/− MEFs were cotransfected with the indicated plasmids and with ddGFP-A and ddGFP-B. Forty-eight hours after transfection, ddGFP fluorescence was acquired as previously described (16).
对于 ddGFP 成像，将Mfn2 ^−/− MEF 与所示质粒以及 ddGFP-A 和 ddGFP-B 共转染。转染后四十八小时，如前所述获得 ddGFP 荧光 ( 16 )。

Subcellular fractionation
亚细胞分离

Subcellular fractions of HeLa cells (10⁹) were obtained as described (13). Cells were washed with phosphate-buffered saline (PBS), suspended in IB (200 mM sucrose, 1 mM EGTA-Tris, and 10 mM Tris-MOPS, pH 7.4), and then disrupted by dounce homogenization. The homogenate was spun at 800 × g for 10 min. The supernatant was recovered and further centrifuged for 10 min at 8000 × g. The resulting pellet (mitochondrial fraction) was collected while the supernatant was further spun for 30 min at 100,000 × g. The pellet was discarded, and the supernatant was centrifuged for 1 hour at 100,000 × g. The resulting pellet is the LM fraction, and the supernatant is the cytosolic fraction. The mitochondrial fraction was further purified by centrifuging twice at 8000 × g for 10 min. The obtained pellet was purified by centrifugation at 95,000 × g for 30 min on a 30% Percoll gradient in IB. The mitochondrial layer obtained was washed free of Percoll and resuspended in IB. MAMs were identified as an intermediate layer between the LMs and the mitochondrial fraction on the Percoll gradient, as previously described. Forty micrograms of protein was separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted as indicated in the figure legends.
如( 13 )所述获得HeLa细胞的亚细胞级分(10 ⁹ )。用磷酸盐缓冲盐水 (PBS) 洗涤细胞，悬浮于 IB（200 mM 蔗糖、1 mM EGTA-Tris 和 10 mM Tris-MOPS，pH 7.4）中，然后通过杜恩斯匀浆破碎。将匀浆以 800 × g旋转 10 分钟。回收上清液并进一步以 8000 × g离心 10 分钟。收集所得沉淀（线粒体部分），同时将上清液进一步以 100,000 × g旋转 30 分钟。弃去沉淀，将上清液以 100,000 × g离心 1 小时。所得沉淀是 LM 级分，上清液是胞质级分。通过以 8000 × g离心两次 10 分钟进一步纯化线粒体部分。通过在 IB 中的 30% Percoll 梯度上以 95,000 × g离心 30 分钟来纯化获得的沉淀。将获得的线粒体层洗去 Percoll 并重悬于 IB 中。如前所述，在 Percoll 梯度上，MAM 被确定为 LM 和线粒体部分之间的中间层。通过 SDS-聚丙烯酰胺凝胶电泳 (SDS-PAGE) 分离出 40 微克蛋白质，并进行免疫印迹，如图图例所示。

Liver fractions were purified as previously described (62, 63). After homogenization of ~1 g of liver with a Teflon potter in IB (225 mM mannitol, 75 mM sucrose, 0.5% BSA, 0.5 mM EGTA, and 30 mM Tris-HCl, pH 7.4), cellular debris and nucleus were removed with two centrifugations at 740 × g for 5 min. A small volume of supernatant was taken, this was called the homogenate fraction. Crude mitochondria were collected by centrifugation at 9000 × g for 10 min, and the pellet was resuspended in Mitochondria Buffer (MB) (250 mM mannitol, 5 mM HEPES and 0.5 mM EGTA, pH 7.4). We then proceeded as described for subcellular fractionation of cells. Proteins were determined using the PierceTM BCA Protein Assay kit (ThermoFisher scientific).
如前所述纯化肝脏级分（62、63 ）。在 IB（225 mM 甘露醇、75 mM 蔗糖、0.5% BSA、0.5 mM EGTA 和 30 mM Tris-HCl，pH 7.4）中用 Teflon potter 将约 1 g 肝脏匀浆后，用两个740 × g离心 5 分钟。取少量上清液，称为匀浆部分。通过以 9000 × g离心 10 分钟收集粗线粒体，并将沉淀重悬于线粒体缓冲液 (MB)（250 mM 甘露醇、5 mM HEPES 和 0.5 mM EGTA，pH 7.4）中。然后我们按照细胞亚细胞分离所述进行。使用 PierceTM BCA 蛋白质测定试剂盒（ThermoFisher Scientific）测定蛋白质。

Trypsin protection assay 胰蛋白酶保护测定

Adenoviruses Ad-ERMIN2 and Ad-ERMIT2 (1 × 10⁹ inclusion-forming units per mouse) were tail-vein injected to reach the liver of Mfn2^LKO mice. LMs (1 mg/ml) were isolated from livers 3 days after adenovirus delivery and incubated with the indicated trypsin concentrations for 15 min on ice. SBTI (5 mg/ml) was added and incubated for 20 min on ice. Laemmli sample buffer was added, and samples were boiled for 5 min and separated by 12% SDS-PAGE.
腺病毒 Ad- ERMIN2和 Ad- ERMIT2 （每只小鼠 1 × 10 ⁹包涵体形成单位）通过尾静脉注射到达Mfn2 ^LKO小鼠的肝脏。腺病毒递送后 3 天从肝脏中分离 LM (1 mg/ml)，并与指定浓度的胰蛋白酶在冰上孵育 15 分钟。添加 SBTI (5 mg/ml) 并在冰上孵育 20 分钟。添加Laemmli样品缓冲液，将样品煮沸5分钟并通过12% SDS-PAGE分离。

Immunoprecipitation 免疫沉淀

Forty microliters of DynaBeads were crosslinked with 3 μl of anti-GFP (Invitrogen) or 4 μl of anti-HA (Roche) using BS3 (Sulfo-DSS, Thermo Scientific Pierce). Total protein extracts (400 μg) were dissolved in RIPA buffer (1% TritonX-100, 0.1% SDS, 0.5% deoxycholate, 0.15 M NaCl, 5 mM Tris, pH8) and incubated with antibody-crosslinked Dynabeads protein G (Thermo fisher Scientific) for 16 hours 4°C in rotation. After three washes for 30 min at 4°C in rotation with PBS 0.2Tween 20%, immunoprecipitates were boiled in 40 μl elution buffer [glycine 50 mM 2X NuPage loading buffer (Invitrogen) 4% β-mercaptoethanol]. Proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies.
使用 BS3（Sulfo-DSS，Thermo Scientific Pierce）将 40 微升 DynaBeads 与 3 μl 抗 GFP（Invitrogen）或 4 μl 抗 HA（Roche）交联。将总蛋白提取物 (400 μg) 溶解在 RIPA 缓冲液（1% TritonX-100、0.1% SDS、0.5% 脱氧胆酸盐、0.15 M 氯化钠、5 mM Tris、pH8）中，并与抗体交联的 Dynabeads 蛋白 G (Thermo Fisher Scientific) 一起孵育) 4°C 旋转 16 小时。在 4°C 下用 PBS 0.2Tween 20% 轮流洗涤 3 次 30 分钟后，将免疫沉淀物在 40 μl 洗脱缓冲液 [甘氨酸 50 mM 2X NuPage 上样缓冲液 (Invitrogen) 4% β-巯基乙醇] 中煮沸。通过 SDS-PAGE 分离蛋白质并使用所示抗体进行免疫印迹。

Immunoblotting 免疫印迹

Homogenates for Western blot analyses were obtained from either cell cultures or tissues. Cells (1.8 × 10⁶ or 80% of confluence) were harvested 24 hours (for siRNA experiments) or 48 hours after transfection and lysed in 150 mM NaCl, 1% Nonidet P-40/0, 0.25% deoxycholate, 1 mM EDTA, and 50 mM Tris, pH 7.4 in the presence of complete protease inhibitor mixture (Sigma-Aldrich).
用于蛋白质印迹分析的匀浆是从细胞培养物或组织中获得的。转染后 24 小时（用于 siRNA 实验）或 48 小时收获细胞（1.8 × 10 ⁶或 80% 汇合），并在 150 mM NaCl、1% Nonidet P-40/0、0.25% 脱氧胆酸盐、1 mM EDTA、和 50 mM Tris，pH 7.4（在完全蛋白酶抑制剂混合物存在下）（西格玛-奥德里奇）。

Tissue samples were homogenized in 10 volumes of lysis buffer using a polytron. Homogenates were rotated for 1 hour at 4°C in an orbital shaker and centrifuged at 13,000 rpm for 15 min at 4°C.
使用 Polytron 将组织样品在 10 体积的裂解缓冲液中均质化。将匀浆液在定轨摇床中于 4°C 下旋转 1 小时，并在 4°C 下以 13,000 rpm 离心 15 分钟。

Forty micrograms of extracted proteins was separated by 4 to 12% Bis-Tris SDS-PAGE (NuPAGE; Invitrogen), transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories), and probed using the following antibodies: actin (1:2000; EMD Millipore); rabbit polyclonal anti Mfn2 (1:1000, homemade raised against a peptide located between amino acids 557 and 576 of the C terminus of Mfn2); anti-Mfn2 (1:1000; Abcam ab 50838, against Mfn2 amino acids 38 to 55 or Abnova H00009927-M3, against Mfn2 amino acids 661 to 675); chicken anti-Mfn1 (1:1000, Abcam); mouse anti-GFP (1:1000, Invitrogen); rabbit anti-Tom20 (1:5000, Santa Cruz Biotechnology); mouse anti-VDAC1 (1:1000, ab14734, Abcam); goat anti-LDH (1:500, Rockland); goat anti-FACL4 (Epitomic S0101); rabbit anti-calreticulin (Crt, 1:750, Upstate); rabbit anti-Calnexin (1:1000; Stressgen); rat anti-HA (1:1000; Roche); and mouse anti-SERCA2 (1:1000; Thermo Fisher MA3-919). Peroxidase-conjugated anti-mouse, anti-goat, or anti-rabbit immunoglobulins were used as secondary antibodies. The following antibodies were used in tissue extracts: Mfn2 (1:1000, Abcam); p-eIF2α; eIF2α; p-PERK; PERK (1:1000, Cell Signaling); CHOP (1:1000, Santa Cruz Biotechnologies); β-actin (1:10,000, Sigma); and α-tubulin (1:8000, Sigma). The specific proteins were detected by the ECL Western blotting detection analysis system (Amersham). Densitometry was performed using ImageJ (NIH).
40 微克提取的蛋白质通过 4 至 12% Bis-Tris SDS-PAGE（NuPAGE；Invitrogen）分离，转移到聚偏二氟乙烯膜（Bio-Rad Laboratories）上，并使用以下抗体进行探测：肌动蛋白（1:2000；EMD）密理博）；兔多克隆抗 Mfn2（1:1000，针对位于 Mfn2 C 末端氨基酸 557 和 576 之间的肽自制）；抗-Mfn2（1:1000；Abcam ab 50838，针对 Mfn2 氨基酸 38 至 55 或 Abnova H00009927-M3，针对 Mfn2 氨基酸 661 至 675）；鸡抗 Mfn1（1:1000，Abcam）；小鼠抗 GFP（1:1000，Invitrogen）；兔抗 Tom20（1:5000，Santa Cruz Biotechnology）；小鼠抗 VDAC1（1:1000，ab14734，Abcam）；山羊抗 LDH（1:500，Rockland）；山羊抗 FACL4 (Epitomic S0101)；兔抗钙网蛋白（Crt，1:750，Upstate）；兔抗 Calnexin（1:1000；Stressgen）；大鼠抗HA（1:1000；罗氏）；和小鼠抗 SERCA2（1:1000；Thermo Fisher MA3-919）。使用过氧化物酶缀合的抗小鼠、抗山羊或抗兔免疫球蛋白作为二抗。组织提取物中使用了以下抗体：Mfn2（1:1000，Abcam）； p-eIF2α； eIF2α； p-PERK； PERK（1:1000，细胞信号传导）； CHOP（1:1000，圣克鲁斯生物技术公司）； β-肌动蛋白（1:10,000，Sigma）；和 α-微管蛋白（1:8000，Sigma）。采用ECL Western blotting检测分析系统(Amersham)检测特异蛋白。使用 ImageJ (NIH) 进行密度测定。

Aequorin measurements of Ca²⁺ concentration
Ca ²⁺浓度的水母发光蛋白测量

For ER Ca²⁺ measurement, cells grown on 13-mm round glass coverslips at 50% confluence were cotransfected with erAEQ and the plasmids indicated. For reconstituting erAeq, the luminal [Ca²⁺]_ER was reduced by incubating cells for 5 min at 37°C with Krebs-Ringer bicarbonate (KRB) supplemented with 1% FBS, 1 mM EGTA, 10 μM ionomycin, and 0.1 mM 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ). Cells were then washed three times with KRB containing 1 mM EGTA and 5% FBS and reconstituted for 3 hours at 4°C with KRB supplemented with 0.1 mM tBuBHQ, 1 mM EGTA, and 5 μM Coelentarazine N. All AEQ measurements were carried out in KRB. Agonists and other drugs were added to the same medium.
对于 ER Ca ²⁺测量，将生长在 13 毫米圆形玻璃盖玻片上、汇合度为 50% 的细胞与 erAEQ 和所示质粒共转染。为了重建 erAeq，将细胞与补充有 1% FBS、1 mM EGTA、10 μM 离子霉素和 0.1 mM 2 的 Krebs-Ringer 碳酸氢盐 (KRB) 在 37°C 下孵育 5 分钟，从而降低_管腔[Ca ²⁺ ] ER ,5-二-(叔丁基)-1,4-苯并氢醌（tBuBHQ）。然后用含有 1 mM EGTA 和 5% FBS 的 KRB 洗涤细胞 3 次，并用补充有 0.1 mM tBuBHQ、1 mM EGTA 和 5 μM 腔肠素 N 的 KRB 在 4°C 下重构 3 小时。所有 AEQ 测量均在KRB。将激动剂和其他药物添加到同一培养基中。

For cytosolic and mitochondrial Ca²⁺ measurement, cells were cotransfected with CytAEQ and mtAEQ and the indicated plasmid. Reconstitution was performed in KRB containing 1 mM Ca²⁺, and measurement and calibration were performed in KRB containing 100 μM EGTA. ATP was used as IP3R agonist.
对于胞质和线粒体 Ca ²⁺测量，将细胞与 CytAEQ 和 mtAEQ 以及所示质粒共转染。在含有1mM Ca ²⁺的KRB中进行重构，并在含有100μM EGTA的KRB中进行测量和校准。 ATP 用作 IP3R 激动剂。

Measurement of mitochondrial ATP production
线粒体 ATP 产生的测量

Cells cotransfected with mitochondrial luciferase (mtLuc) and the indicated plasmids were constantly perfused with a modified KRB containing 125 mM NaCl, 5 mM KCl, 1 mM Na₃PO₄, 1 mM MgSO₄, 1 mM CaCl₂, 20 mM luciferin, and 20 mM Hepes (pH 7.4 at 37°C). Agonists were perfused in the same buffer containing 20 mM luciferin. Cell luminescence were measured in a luminometer.
用线粒体荧光素酶 (mtLuc) 和指定质粒共转染的细胞不断地用含有 125 mM NaCl、5 mM KCl、1 mM Na ₃ PO ₄ 、1 mM MgSO ₄ 、1 mM CaCl ₂ 、20 mM 荧光素和20 mM Hepes（37°C 时 pH 7.4）。将激动剂灌注在含有 20 mM 荧光素的相同缓冲液中。在光度计中测量细胞发光。

Flow cytometry and sorting
流式细胞术和分选

Analysis of ddGFP fluorescence was performed as described (16, 21). Mfn2^−/− MEFs were first cotransfected with cytosolic mCherry and with ddGFP-A and ddGFP-B. After 24 hours, cells were transfected with the indicated plasmids. Twenty-four hours after the second transfection, GFP and mCherry fluorescence were evaluated by flow cytometry analysis. Acquisition was stopped when 50,000 FL2+ events were reached. Data are shown as dot plot of FL1 versus side scatter derived from FL2+ gated events. Cell size (forward scatter, FSC), cytosolic mCherry level (red signal intensity detected by FL2 channel), and ddGFP fluorescence (GFP signal intensity detected by a 15-mW argon ion laser tuned at 488 nm) analyses were performed on a FacsCalibur Flow Cytometer using CellQuest software (Becton Dickinson Biosciences).
ddGFP 荧光分析按所述进行 ( 16 , 21 )。 Mfn2 ^−/− MEF 首先与胞质 mCherry 以及 ddGFP-A 和 ddGFP-B 共转染。 24小时后，用所示质粒转染细胞。第二次转染后 24 小时，通过流式细胞术分析评估 GFP 和 mCherry 荧光。当达到 50,000 个 FL2+ 事件时停止采集。数据显示为 FL1 与源自 FL2+ 门控事件的侧向散射的点图。在 FacsCalibur Flow 上进行细胞大小（前向散射，FSC）、胞质 mCherry 水平（通过 FL2 通道检测到的红色信号强度）和 ddGFP 荧光（通过调谐至 488 nm 的 15 mW 氩离子激光器检测到的 GFP 信号强度）分析使用 CellQuest 软件（Becton Dickinson Biosciences）的细胞仪。

For sorting using FACSAria (Becton Dickinson Biosciences), cells were analyzed through a 530-nm band-pass filter as they traversed the beam of an argon ion laser (488 nm, 100 mW). Sorted GFP⁺ cells were cultured in DMEM supplemented with 2 mM glutamine, 1 mM penicillin/streptomycin, and 20% (vol/vol) FBS at 37°C in a fully humidified atmosphere of 95% air and 5% CO₂. On the following day, the medium was replaced to remove dead cells. Growing cells were fixed after 8 hours and prepared for EM images.
为了使用 FACSAria（Becton Dickinson Biosciences）进行分选，当细胞穿过氩离子激光束（488 nm，100 mW）时，通过 530 nm 带通滤光片对细胞进行分析。将分选的 GFP ⁺细胞在补充有 2 mM 谷氨酰胺、1 mM 青霉素/链霉素和 20% (vol/vol) FBS 的 DMEM 中于 37°C、95% 空气和 5% CO ₂的完全湿润气氛中培养。第二天，更换培养基以除去死细胞。 8 小时后固定生长的细胞并准备进行 EM 图像。

Electron microscopy 电子显微镜

MEFs cotransfected with GFP and the indicated plasmids and sorted as described above were fixed with 1.25% (vol/vol) glutaraldehyde in 0.1 M sodium cacodylate at pH 7.4 for 1 hour at room temperature. Thin sections were imaged on a Tecnai-20 electron microscope (Philips-FEI).
将用 GFP 和所示质粒共转染并按上述分选的 MEF 用 0.1 M 二甲胂酸钠中的 1.25%（体积/体积）戊二醛在 pH 7.4 下在室温下固定 1 小时。薄切片在 Tecnai-20 电子显微镜 (Philips-FEI) 上成像。

Morphometric measurements were carried out using ImageJ (NIH). For calculations of mitochondria-ER distance, >5 mitochondria per image in 70 images per condition were considered, and the minimal distance of the ER located in a 30- or 20-nm radius from the considered mitochondria was computed. ERMICC was calculated as previously described (16).
使用 ImageJ (NIH) 进行形态测量。为了计算线粒体-ER 距离，考虑了每个条件 70 个图像中每个图像 >5 个线粒体，并计算了位于距所考虑线粒体 30 或 20 nm 半径内的 ER 的最小距离。 ERMICC 的计算方法如前所述 ( 16 )。

Human biopsies 人体活检

Liver biopsies were obtained from NAFLD patients undergoing bariatric surgery; liver biopsies from normal individuals were not collected due to ethical issues. Biopsies of subcutaneous adipose tissue and skeletal muscle were obtained from nonobese subjects. All patients gave written informed consent. The study protocols conformed to the Ethical Guidelines of the 1975 Declaration of Helsinki, revised in 2000, as reflected in a priori approval by the Hospital Sant Joan de Reus (Institutional Review Board, project code: INFLAMED/15-04-30/4prog7), Hospital Joan XXIII, and Human Ethics Committee.
肝活检取自接受减肥手术的 NAFLD 患者；由于伦理问题，没有收集正常人的肝活检。从非肥胖受试者身上获取皮下脂肪组织和骨骼肌的活组织检查。所有患者均签署了书面知情同意书。研究方案符合 1975 年赫尔辛基宣言的道德准则（2000 年修订），这反映在圣琼德雷乌斯医院的事先批准中（机构审查委员会，项目代码：INFLAMED/15-04-30/4prog7）、医院琼二十三世和人类伦理委员会。

Animal care and experiments
动物护理和实验

All animal work was approved and conducted following established guidelines. This project was assessed favorably by the Institutional Animal Care and Use Committee of the Parc Cientific de Barcelona (IACUC-PCB), which considers that the above-mentioned project complies with standard ethical regulations and meets the requirements of current applicable legislation (RD 53/2013 Council Directive; 2010/63/UE; Order 214/1997/GC). Control and Mfn2^LKO mice were littermates and described previously (10). The MCD diet protocol was described previously (10). Mice were kept under a 12-hour light-dark period and provided with a standard chow diet and water ad libitum. Eight-week-old C57Bl6/J males were fed a standard diet. On the experimental day, mice were anesthetized using isoflurane and euthanized by cervical dislocation. Tissues used for RNA purification, protein extraction, or histology were prepared as described (64, 65). Sample size was not predetermined. For the MCD protocol, male and female mice were randomized to chow or MCD diet after a blinded draw of their ID. Researchers were not blinded to the genotype, the insert of the adenovirus injected, or the diet treatment. All animals were included in the analyses.
所有动物工作均经过批准并按照既定指南进行。该项目得到了巴塞罗那科学公园机构动物护理和使用委员会（IACUC-PCB）的好评，认为上述项目符合标准道德法规，并满足现行适用立法（RD 53/ 2013 年理事会指令；2010/63/UE；第 214/1997/GC 号命令）。对照小鼠和 Mfn2 ^LKO小鼠是同窝小鼠，之前已有描述 ( 10 )。 MCD 饮食方案之前已描述过 ( 10 )。将小鼠置于12小时的光照-黑暗期中，并随意提供标准食物和水。八周大的 C57Bl6/J 雄性被喂食标准饮食。在实验当天，使用异氟烷麻醉小鼠并通过颈椎脱位处死小鼠。用于 RNA 纯化、蛋白质提取或组织学的组织按照所述进行制备 ( 64 , 65 )。样本量没有预先确定。对于 MCD 方案，在对雄性和雌性小鼠进行盲法抽签后，将其随机分配到食物或 MCD 饮食中。研究人员并未对基因型、注射的腺病毒插入片段或饮食治疗视而不见。所有动物均包含在分析中。

Adenoviral transduction 腺病毒转导

For adenoviral transduction of 8- to 10-week-old mice, adenoviruses (1 × 10⁹ inclusion-forming units per mouse) were tail-vein injected. Livers were isolated 5 days after adenovirus delivery.
对于8至10周龄小鼠的腺病毒转导，尾静脉注射腺病毒（每只小鼠1×10 ⁹包涵体形成单位）。腺病毒递送后5天分离肝脏。

The following adenoviruses were used in this study: Ad-LacZ, Ad-ERMIN2, and Ad-ERMIT2 (encoding for human ERMIN2 and ERMIT2). ERMIN2 and ERMIT2 were cloned by recombination into the pAdeno-CMV-V5 adenoviral vector (Invitrogen) using the Gateway system. Adenoviruses were generated by transfection of the adenoviral expression vectors in a human embryonic kidney cell line (HEK 293). The adenoviruses generated were then amplified at the Unitat de Producció de Vectors Virals-CBATEG (Universitat Autònoma de Barcelona).
本研究中使用了以下腺病毒：Ad-LacZ、Ad -ERMIN2和 Ad- ERMIT2 （编码人类ERMIN2和ERMIT2 ）。使用 Gateway 系统通过重组将ERMIN2和ERMIT2克隆到 pAdeno-CMV-V5 腺病毒载体（Invitrogen）中。通过在人胚胎肾细胞系（HEK 293）中转染腺病毒表达载体来产生腺病毒。然后，产生的腺病毒在 Unitat de Producció de Vectors Virals-CBATEG（巴塞罗那自治大学）进行扩增。

³H–L-serine incorporation into phospholipids in subcellular fractions
³ H–L-丝氨酸掺入亚细胞组分中的磷脂中

Liver was homogenized use Teflon-glass homogenizer in Isolation Buffer (IB) (225 mM Mannitol, 25 mM Hepes-KOH, 1 mM EGTA, pH 7.4 and protease inhibitors) at a ratio of 4 ml of IB for every gram of tissue. The homogenate was pelleted for 10 min at 1500 × g at 4°C. The supernatant was transferred to a new tube and pelleted again as above, transferred again to a new tube, and pelleted at 13,000 × g for 20 min at 4°C. This new pellet contained the crude mitochondria fraction, comprising mitochondria and MAM, and was used to measure lipid transfer. The supernatant contained the ER fraction, which was pelleted at 100,000 × g for 1 hour at 4°C. This pellet was used as a control in the assay. One milligram of the fraction was pelleted again and resuspended in 200 μl of PS assay buffer [25 mM Hepes-KOH, 10 mM CaCl₂, 0.4 mM of ³H-Ser (20 to 30 μci/μmol) pH 7.4]. The mixture was incubated for 45 min at 37°C and the reaction was stopped by adding 3 volumes of chloroform:MeOH (2:1). Lipids were extracted using the Folch Method, dried on an N₂ flow, and separated by TLC as described (39, 66).
使用聚四氟乙烯玻璃匀浆器在分离缓冲液 (IB)（225 mM 甘露醇、25 mM Hepes-KOH、1 mM EGTA、pH 7.4 和蛋白酶抑制剂）中以每克组织 4 ml IB 的比例对肝脏进行匀浆。将匀浆在 4°C 下以 1500 × g沉淀 10 分钟。将上清液转移至新管中，并如上所述再次沉淀，再次转移至新管中，并在 4°C 下以 13,000 × g沉淀 20 分钟。这种新沉淀含有粗线粒体部分，包括线粒体和 MAM，用于测量脂质转移。上清液含有 ER 级分，在 4°C 下以 100,000 × g沉淀 1 小时。该沉淀用作测定中的对照。将 1 毫克级分再次沉淀并重悬于 200 μl PS 测定缓冲液 [25 mM Hepes-KOH、10 mM CaCl ₂ 、0.4 mM ³ H-Ser (20 至 30 μci/μmol) pH 7.4]。将混合物在 37°C 下孵育 45 分钟，并通过添加 3 体积的氯仿:MeOH (2:1) 来终止反应。使用Folch方法提取脂质，在N ₂流上干燥，并通过TLC分离，如所述( 39 , 66 )。

Statistical analysis 统计分析

Data are shown as means ± SEM values of the indicated number of independent experiments, unless otherwise noted in the figure legends. Data from individual experiments are plotted as dots except in the AEQ and mt-Luc experiments which represent means ± SEMs of the indicated number of independent experiments. OriginPro 2022 (OriginLab Corp., Northampton MA, USA) was used for statistical analysis. The sample size was predetermined on the basis of published literature and previous laboratory experience. No statistical methods were used to predetermine the sample size. Normal distribution of data was verified by a Shapiro–Wilkinson test. Homogeneity of the variance was verified using Levene’s test. If data fulfilled normality and homoscedasticity criteria, parametric tests were used to evaluate significance. When data exhibited a nonnormal and unequal distribution, nonparametric tests were applied. Used tests are specified in the figure legends. Sample sizes and P values are indicated in the figure legends. P < 0.05 was considered significant.
除非图例中另有说明，数据显示为指定数量的独立实验的平均值±SEM 值。除 AEQ 和 mt-Luc 实验外，各个实验的数据都绘制为点，它们代表指定数量的独立实验的平均值±SEM。使用 OriginPro 2022（OriginLab Corp.，北安普顿马萨诸塞州，美国）进行统计分析。样本量是根据已发表的文献和以前的实验室经验预先确定的。没有使用统计方法来预先确定样本量。数据的正态分布通过夏皮罗-威尔金森检验进行验证。使用 Levene 检验验证方差的同质性。如果数据满足正态性和同方差性标准，则使用参数检验来评估显着性。当数据表现出非正态和不等分布时，应用非参数检验。使用的测试在图例中指定。样本大小和P值在图例中标出。 P < 0.05 被认为是显着的。

Acknowledgments 致谢

We thank V. Hernandez, J. M. Seco, and L. Bardia (Advanced Digital Microscopy, IRB Barcelona) and D. Bach for technical assistance; N. Berrow (Protein Expression, IRB Barcelona) for plasmid generation; J. Comas and R. Alvarez (Cytometry Unit, UB) and R. Seminago and A. Amador (Genomics Unit, UB) for technical assistance; F. Caicci and F. Boldrin (Bioimaging Facility, Department of Biology, University of Padova) for EM samples preparation; and A. Cabrelle (FACS facility, VIMM) for flow cytometry and sorting.
我们感谢 V. Hernandez、JM Seco 和 L. Bardia（高级数字显微镜，IRB 巴塞罗那）和 D. Bach 提供的技术援助； N. Berrow（蛋白质表达，IRB 巴塞罗那）用于质粒生成； J. Comas 和 R. Alvarez（布法罗大学细胞计数单位）以及 R. Seminago 和 A. Amador（布法罗大学基因组学单位）提供技术援助； F. Caicci 和 F. Boldrin（帕多瓦大学生物系生物成像设施）用于 EM 样品制备； A. Cabrelle（FACS 设施，VIMM）用于流式细胞术和分选。

Funding: This study was supported by MINECO PID2019-106209RB-I00 (A.Z.); Generalitat de Catalunya grant 2017SGR1015 (A.Z.); CIBERDEM “Instituto de Salud Carlos III” (A.Z.); Fundación Ramon Areces CIVP18A3942 (A.Z.); Fundación BBVA (A.Z.); Fundació Marató de TV3 20132330 (A.Z.); the European Foundation for the Study of Diabetes (EFSD) (A.Z.); “La Caixa” Foundation (A.Z.); Health Research Grant 2021 LCF/PR/HR21/52410007 (A.Z.); ICREA “Academia” Award Generalitat de Catalunya (A.Z.); institutional funding from MINECO through the Centres of Excellence Severo Ochoa Award (A.Z.); CERCA Programme of the Generalitat de Catalunya (A.Z. and M.O.); MICINN PDI2021-122478NB-I00 (M.O.); Generalitat de Catalunya grant 2021SGR00863, “BioExcel-3: Centre of Excellence for Computational Biomolecular Research” ref. no. 101093290 HORIZON-EUROHPC-JU-2021-COE-01, 101094651 — MDDB — HORIZON-INFRA-2022-DEV-01 (M.O.); European Research Council (ERC) GA282280 (L.S.); Muscular Dystrophy Association (MDA) 4165 (L.S.); EFSD/Novo Nordisk Program for Diabetes Research in Europe (L.S.); Fondation Leducq TNE15004 (L.S.); Ministero dell’Istruzione, dell’Università e della Ricerca RBAP11Z3YA_005 (L.S.); Ministero dell’Istruzione, dell’Università e della Ricerca 2017BF3PXZ (L.S.); Ministero dell’Università e della Ricerca 2020PKLEPN_002 (L.S.); and SOE_0000181, MUR Concession Decree no. 564 of 13/12/2022, CUP C93C22007650006, funded under the National Recovery and Resilience Plan (NRRP), Mission 4, Component 2, Investment 1.2, MUR Call for tender n. 367 of 7/10/2022 funded by the European Union – NextGenerationEU (S.S.).
资助：本研究得到了 MINECO PID2019-106209RB-I00 (AZ) 的支持；加泰罗尼亚政府拨款 2017SGR1015 (AZ)； CIBERDEM“卡洛斯三世健康研究所”（亚利桑那州）；拉蒙·阿雷塞斯基金会 CIVP18A3942（亚利桑那州）； BBVA 基金会（亚利桑那州）； TV3 马拉托基金会 20132330 (AZ)；欧洲糖尿病研究基金会 (EFSD) (AZ)； “La Caixa”基金会（亚利桑那州）；健康研究补助金 2021 LCF/PR/HR21/52410007 (AZ)； ICREA “学术界”奖 Generalitat de Catalunya（亚利桑那州）； MINECO 通过塞韦罗·奥乔亚卓越中心奖 (AZ) 提供机构资助；加泰罗尼亚自治区CERCA计划（亚利桑那州和密苏里州）； MICINN PDI2021-122478NB-I00 (MO)；加泰罗尼亚政府拨款 2021SGR00863，“BioExcel-3：计算生物分子研究卓越中心”参考号。不。 101093290 HORIZON-EUROHPC-JU-2021-COE-01、101094651 — MDDB — HORIZON-INFRA-2022-DEV-01 (MO)；欧洲研究理事会 (ERC) GA282280 (LS)；肌营养不良协会 (MDA) 4165 (LS)； EFSD/诺和诺德欧洲糖尿病研究计划 (LS)； Leducq 基金会 TNE15004 (LS)；教育部长、大学和莱斯卡 RBAP11Z3YA_005 (LS)；教育部长、大学和大学 2017BF3PXZ (LS)；大学和大学部部长 2020PKLEPN_002 (LS)；和 SOE_0000181，MUR 特许令编号。 2022 年 12 月 13 日第 564 号，CUP C93C22007650006，由国家恢复和复原力计划 (NRRP)、任务 4、组成部分 2、投资 1.2、MUR 招标 n. 资助2022 年 7 月 10 日第 367 号决议，由欧盟 – NextGenerationEU (SS) 资助。

Author contributions: Conceptualization: D.N., M.O., A.Z., and L.S. Investigation: D.N., M.I.H.-A., S.S., M.W., S.I., O.M.d.B., A.Q., J.H., M.P., P.A., J.C., L.L., D.S., S.F.-V., J.V., and J.J. Visualization: D.N., M.I.H.-A., S.S., M.W., S.I., O.M.d.B., A.Q., J.H., M.P., P.A., J.C., L.L., D.S., S.F.-V., J.V., J.J., M.O., A.Z., and L.S. Funding acquisition: S.S., M.O., A.Z., and L.S. Project administration: A.Z. and L.S. Supervision: A.Z. and L.S. Writing – original draft: D.N., A.Z., and L.S. Writing – review & editing: D.N., M.I.H.-A., S.S., M.O., A.Z., and L.S.
作者贡献：概念化：DN、MO、AZ 和 LS 调查：DN、MIH-A.、SS、MW、SI、OMdB、AQ、JH、MP、PA、JC、LL、DS、SF-V.、JV和 JJ 可视化：DN、MIH-A.、SS、MW、SI、OMdB、AQ、JH、MP、PA、JC、LL、DS、 SF-V.、JV、JJ、MO、AZ 和 LS 资金获取：SS、MO、AZ 和 LS 项目管理：AZ 和 LS 监督：AZ 和 LS 写作 – 原稿：DN、AZ 和 LS 写作 –审阅和编辑：DN、MIH-A.、SS、MO、AZ 和 LS

Competing interests: The authors declare that they have no competing interests.
竞争利益：作者声明他们没有竞争利益。

Data and materials availability: All data are available in the main text or the supplementary materials. Raw data are available as supplementary materials. Materials are available from the corresponding authors.
数据和材料的可用性：所有数据都可以在正文或补充材料中获得。原始数据可作为补充材料提供。材料可从相应作者处获得。

License information: Copyright © 2023 the authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original US government works. https://www.science.org/about/science-licenses-journal-article-reuse
许可信息：版权所有 © 2023 作者，保留部分权利；独家被许可人美国科学促进会。没有声称拥有美国政府原创作品。 https://www.science.org/about/science-licenses-journal-article-reuse

Supplementary Materials 补充材料

This PDF file includes: 该 PDF 文件包括：

Figs. S1 to S16 无花果。 S1至S16

Download 下载
3.02 MB 3.02MB

Other Supplementary Material for this manuscript includes the following:
本手稿的其他补充材料包括以下内容：

MDAR Reproducibility Checklist
MDAR 再现性检查表

Download 下载
152.64 KB

Data S1 and S2 数据S1和S2

Download 下载
11.77 MB

View/request a protocol for this paper from Bio-protocol.
从Bio-protocol查看/请求本文的协议。

References and Notes 参考文献和注释

M. Eisenberg-Bord, N. Shai, M. Schuldiner, M. Bohnert, A Tether Is a Tether Is a Tether: Tethering at Membrane Contact Sites. Dev. Cell 39, 395–409 (2016).
M. Eisenberg-Bord、N. Shai、M. Schuldiner、M. Bohnert，系绳就是系绳就是系绳：膜接触部位的系绳。开发。细胞39 , 395–409 (2016)。

L. Scorrano, M. A. De Matteis, S. Emr, F. Giordano, G. Hajnóczky, B. Kornmann, L. L. Lackner, T. P. Levine, L. Pellegrini, K. Reinisch, R. Rizzuto, T. Simmen, H. Stenmark, C. Ungermann, M. Schuldiner, Coming together to define membrane contact sites. Nat. Commun. 10, 1287 (2019).
L. Scorrano、MA De Matteis、S. Emr、F. Giordano、G. Hajnóczky、B. Kornmann、LL Lackner、TP Levine、L. Pellegrini、K. Reinisch、R. Rizzuto、T. Simmen、H. Stenmark、 C. Ungermann、M. Schuldiner，共同确定膜接触位点。纳特。交流。 10、1287 （2019）。

Crossref 交叉引用

PubMed 考研

Google Scholar 谷歌学术

L. S. Jouaville, P. Pinton, C. Bastianutto, G. A. Rutter, R. Rizzuto, Regulation of mitochondrial ATP synthesis by calcium: Evidence for a long-term metabolic priming. Proc. Natl. Acad. Sci. U.S.A. 96, 13807–13812 (1999).
LS Jouaville、P. Pinton、C. Bastianutto、GA Rutter、R. Rizzuto，钙调节线粒体 ATP 合成：长期代谢启动的证据。过程。国家。阿卡德。科学。美国96，13807–13812 （1999）。

G. Hajnóczky, L. D. Robb-Gaspers, M. B. Seitz, A. P. Thomas, Decoding of cytosolic calcium oscillations in the mitochondria. Cell 82, 415–424 (1995).

Editor’s summary 编辑总结

Structured Abstract 结构化摘要

INTRODUCTION 介绍

RATIONALE 基本原理

RESULTS 结果

CONCLUSION 结论

Abstract 抽象的

SIGN UP FOR THE AWARD-WINNING SCIENCEADVISER NEWSLETTER订阅屡获殊荣的科学顾问通讯

Human and mouse mitofusin 2 gene are alternatively spliced人和小鼠线粒体融合蛋白 2 基因交替剪接

ERMIN2, ERMIT2, and MoV-MFN2 localize to the ERERMIN2、ERMIT2 和 MoV-MFN2 定位于 ER

Topology of ERMIN2 and ERMIT2 at the ERER 处的 ERMIN2 和 ERMIT2 拓扑

ERMIN2 regulates ER morphologyERMIN2 调节 ER 形态

ERMIT2 and moV-MFN2 tether ER to mitochondriaERMIT2 和 moV-MFN2 将 ER 与线粒体连接

ERMIT2 requires mitofusins on mitochondria to tether them to the ERERMIT2 需要线粒体上的线粒体融合素将其连接到 ER

Coiled-coil domains mediate ERMIN2/ERMIT2-mitofusin interaction卷曲螺旋结构域介导 ERMIN2/ERMIT2-线粒体融合蛋白相互作用

ERMIT2 licenses Ca2+ and phospholipid transfer from ER to mitochondriaERMIT2 许可 Ca 2+和磷脂从 ER 转移至线粒体

Discussion 讨论

Materials and methods 材料和方法

Molecular biology and real-time PCR分子生物学和实时 PCR

RPA 机器人程序自动化

Cell culture 细胞培养

Imaging 影像学

FRAP

Morphometric and MCS analysis形态测量和 MCS 分析

Subcellular fractionation亚细胞分离

Trypsin protection assay 胰蛋白酶保护测定

Immunoprecipitation 免疫沉淀

Immunoblotting 免疫印迹

Aequorin measurements of Ca2+ concentrationCa 2+浓度的水母发光蛋白测量

Measurement of mitochondrial ATP production线粒体 ATP 产生的测量

Flow cytometry and sorting流式细胞术和分选

Electron microscopy 电子显微镜

Human biopsies 人体活检

Animal care and experiments动物护理和实验

Adenoviral transduction 腺病毒转导

3H–L-serine incorporation into phospholipids in subcellular fractions3 H–L-丝氨酸掺入亚细胞组分中的磷脂中

Statistical analysis 统计分析

Acknowledgments 致谢

Supplementary Materials 补充材料

This PDF file includes: 该 PDF 文件包括：

Other Supplementary Material for this manuscript includes the following:本手稿的其他补充材料包括以下内容：

References and Notes 参考文献和注释

(0)eLetters

Information

Published In

Copyright

Article versions

Submission history

Permissions

Acknowledgments

Authors

AffiliationsExpand All

Déborah Naón* https://orcid.org/0000-0002-9726-4664 luca.scorrano@unipd.it

María Isabel Hernández-Alvarez https://orcid.org/0000-0003-2483-7000

Satoko Shinjo https://orcid.org/0000-0002-1063-5961

Milosz Wieczor https://orcid.org/0000-0003-4990-8629

Saska Ivanova https://orcid.org/0000-0003-0080-6680

Olga Martins de Brito

Albert Quintana https://orcid.org/0000-0003-1674-7160

Juan Hidalgo https://orcid.org/0000-0003-0921-1122

Manuel Palacín https://orcid.org/0000-0002-8670-293X

Pilar Aparicio

Juan Castellanos

Luis Lores https://orcid.org/0000-0002-0013-8670

David Sebastián https://orcid.org/0000-0002-7260-3869

Sonia Fernández-Veledo https://orcid.org/0000-0003-2906-3788

Joan Vendrell https://orcid.org/0000-0002-6994-6115

Jorge Joven https://orcid.org/0000-0003-2749-4541

Modesto Orozco https://orcid.org/0000-0002-8608-3278

Antonio Zorzano*,† https://orcid.org/0000-0002-1638-0306 luca.scorrano@unipd.it

Luca Scorrano*,† https://orcid.org/0000-0002-8515-8928 luca.scorrano@unipd.it

Funding Information

Notes

Metrics

Article Usage

Altmetrics

Citations

Cite as

Export citation

View options

SIGN UP FOR THE AWARD-WINNING SCIENCEADVISER NEWSLETTER
订阅屡获殊荣的科学顾问通讯

Human and mouse mitofusin 2 gene are alternatively spliced
人和小鼠线粒体融合蛋白 2 基因交替剪接

ERMIN2, ERMIT2, and MoV-MFN2 localize to the ER
ERMIN2、ERMIT2 和 MoV-MFN2 定位于 ER

Topology of ERMIN2 and ERMIT2 at the ER
ER 处的 ERMIN2 和 ERMIT2 拓扑

ERMIN2 regulates ER morphology
ERMIN2 调节 ER 形态

ERMIT2 and moV-MFN2 tether ER to mitochondria
ERMIT2 和 moV-MFN2 将 ER 与线粒体连接

ERMIT2 requires mitofusins on mitochondria to tether them to the ER
ERMIT2 需要线粒体上的线粒体融合素将其连接到 ER

Coiled-coil domains mediate ERMIN2/ERMIT2-mitofusin interaction
卷曲螺旋结构域介导 ERMIN2/ERMIT2-线粒体融合蛋白相互作用

ERMIT2 licenses Ca²⁺ and phospholipid transfer from ER to mitochondria
ERMIT2 许可 Ca ²⁺和磷脂从 ER 转移至线粒体

Molecular biology and real-time PCR
分子生物学和实时 PCR

Morphometric and MCS analysis
形态测量和 MCS 分析

Subcellular fractionation
亚细胞分离

Aequorin measurements of Ca²⁺ concentration
Ca ²⁺浓度的水母发光蛋白测量

Measurement of mitochondrial ATP production
线粒体 ATP 产生的测量

Flow cytometry and sorting
流式细胞术和分选

Animal care and experiments
动物护理和实验

³H–L-serine incorporation into phospholipids in subcellular fractions
³ H–L-丝氨酸掺入亚细胞组分中的磷脂中

Other Supplementary Material for this manuscript includes the following:
本手稿的其他补充材料包括以下内容：

Affiliations

Déborah Naón^* https://orcid.org/0000-0002-9726-4664 luca.scorrano@unipd.it

Antonio Zorzano^*,† https://orcid.org/0000-0002-1638-0306 luca.scorrano@unipd.it

Luca Scorrano^*,† https://orcid.org/0000-0002-8515-8928 luca.scorrano@unipd.it