Reversible interactions between smooth domains of the endoplasmic reticulum and mitochondria are regulated by physiological cytosolic Ca2+ levels.
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.
Goetz
Jacky G
JG
Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver V6T 1Z3, Canada.
Genty
Hélène
H
St-Pierre
Pascal
P
Dang
Thao
T
Joshi
Bharat
B
Sauvé
Rémy
R
Vogl
Wayne
W
Nabi
Ivan R
IR
D000818:Animals; D000911:Antibodies, Monoclonal; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002460:Cell Line; D004285:Dogs; D018871:Endoplasmic Reticulum, Smooth / Q000378:metabolism* / Q000648:ultrastructure; D015759:Ionomycin / Q000494:pharmacology; D051379:Mice; D046529:Microscopy, Electron, Transmission; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D041681:NIH 3T3 Cells; D060285:Receptors, Autocrine Motility Factor; D018121:Receptors, Cytokine / Q000276:immunology / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D018121:Receptors, Cytokine; D015759:Ionomycin; C555002:Amfr protein, mouse; D060285:Receptors, Autocrine Motility Factor; D044767:Ubiquitin-Protein Ligases; D002118:Calcium