Goetz Jacky G, Genty Hélène, St-Pierre Pascal, Dang Thao, Joshi Bharat, Sauvé Rémy, Vogl Wayne, Nabi Ivan R
Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver V6T 1Z3, Canada.
J Cell Sci. 2007 Oct 15;120(Pt 20):3553-64. doi: 10.1242/jcs.03486. Epub 2007 Sep 25.
The 3F3A monoclonal antibody to autocrine motility factor receptor (AMFR) labels mitochondria-associated smooth endoplasmic reticulum (ER) tubules. siRNA down-regulation of AMFR expression reduces mitochondria-associated 3F3A labelling. The 3F3A-labelled ER domain does not overlap with reticulon-labelled ER tubules, the nuclear membrane or perinuclear ER markers and only partially overlaps with the translocon component Sec61alpha. Upon overexpression of FLAG-tagged AMFR, 3F3A labelling is mitochondria associated, excluded from the perinuclear ER and co-distributes with reticulon. 3F3A labelling therefore defines a distinct mitochondria-associated ER domain. Elevation of free cytosolic Ca(2+) levels with ionomycin promotes dissociation of 3F3A-labelled tubules from mitochondria and, judged by electron microscopy, disrupts close contacts (<50 nm) between smooth ER tubules and mitochondria. The ER tubule-mitochondria association is similarly disrupted upon thapsigargin-induced release of ER Ca(2+) stores or purinergic receptor stimulation by ATP. The inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] receptor (IP3R) colocalises to 3F3A-labelled mitochondria-associated ER tubules, and conditions that induce ER tubule-mitochondria dissociation disrupt continuity between 3F3A- and IP3R-labelled ER domains. RAS-transformed NIH-3T3 cells have increased basal cytosolic Ca(2+) levels and show dissociation of the 3F3A-labelled, but not IP3R-labelled, ER from mitochondria. Our data indicate that regulation of the ER-mitochondria association by free cytosolic Ca(2+) is a characteristic of smooth ER domains and that multiple mechanisms regulate the interaction between these organelles.
针对自分泌运动因子受体（AMFR）的3F3A单克隆抗体标记与线粒体相关的平滑内质网（ER）小管。AMFR表达的siRNA下调会减少与线粒体相关的3F3A标记。3F3A标记的ER结构域不与网质蛋白标记的ER小管、核膜或核周ER标记重叠，仅部分与转位子成分Sec61α重叠。在FLAG标签的AMFR过表达时，3F3A标记与线粒体相关，被排除在核周ER之外，并与网质蛋白共分布。因此，3F3A标记定义了一个独特的与线粒体相关的ER结构域。用离子霉素提高游离胞质Ca(2+)水平会促进3F3A标记的小管与线粒体解离，并且通过电子显微镜判断，会破坏平滑ER小管与线粒体之间的紧密接触（<50 nm）。在毒胡萝卜素诱导的ER Ca(2+)储存释放或ATP对嘌呤能受体刺激后，ER小管与线粒体的关联同样会被破坏。肌醇（1,4,5）-三磷酸[Ins(1,4,5)P(3)]受体（IP3R）与3F3A标记的与线粒体相关的ER小管共定位，并且诱导ER小管与线粒体解离的条件会破坏3F3A和IP3R标记的ER结构域之间的连续性。RAS转化的NIH-3T3细胞基础胞质Ca(2+)水平升高，并且显示3F3A标记的而非IP3R标记的ER与线粒体解离。我们的数据表明，游离胞质Ca(2+)对ER-线粒体关联的调节是平滑ER结构域的一个特征，并且多种机制调节这些细胞器之间的相互作用。
