When is a vesicle not just a vesicle: mitochondrial spheroids and mitochondrial autophagosomes
© Cook et al.; licensee BioMed Central Ltd. 2014
Received: 23 September 2014
Accepted: 21 October 2014
Published: 14 November 2014
To the Editor
The commentary by Ding and Eskelinen “Do mitochondria donate membrane to form autophagosomes or undergo remodeling to form mitochondrial spheroids?” on our recently published manuscript raises several important points that we wish to address. To do so, we here include several key experiments to clarify further that the mitochondrial vesicles observed in Cook et al. are likely to be autophagosomes , rather than “mitochondrial spheroids”.
Ding et al. previously showed that when mouse embryonic fibroblasts are treated with the mitochondrial de-coupler agent CCCP, a structure they called a “mitochondrial spheroid” develops. These authors concluded that the spheroids are not autophagosomes because they still form in ATG5−/− and ATG7−/− embryonic fibroblasts . However, autophagy is a complex pathway involving multiple mechanisms of activation. While ATG5 and ATG7 can play an important role in autophagosome formation, they are not obligatory in all cases. For example, autophagy can be activated in an ATG5/ATG7 independent manner involving ULK1 and Rab9 .
In our previous publication, we demonstrated by immuno-gold electron microscopy, that mitochondria form vesicles that stain positive for LC3, suggesting that these vesicles are likely to be autophagosomes . Microtubule associated protein 1 light chain 3 (MAPLC3, LC3) is lipidated and incorporated into the autophagosomal membrane and is often used as a means to identify appropriate structures as autophagosomes and not lysosomes . ATG7 can play a critical role in LC3 processing and autophagosome formation. Coupled with our new data included here, showing that ATG7 inhibition prevented mitochondrial vesicle formation, these observations further support our original conclusion that the vesicles are most likely to be autophagosomes. We also showed that the mitochondria forming autophagosomes stain positive for parkin. Quantification of parkin immuno-gold EM showed elevated levels of parkin in the cytosol and also increased parkin labeling on mitochondria-forming vesicles. These data imply that the mitochondrial vesicles represent a novel form of mitophagy. Moreover, inhibition of parkin by RNAi prevented an ICI (the antiestrogen known as Fulvestrant or Faslodex)-mediated reduction of mitochondrial content, supporting a role of parkin in mitochondrial clearance .
Consideration of cellular context is usually critical in the interpretation of much cell and molecular biologic data. Cellular signaling in cancer is often altered to favor proliferation and survival. While Ding and Eskelinen questioned our study due to differences they observed between parkin and mitofusin regulation, it would not be unusual for mitochondrial vesication to be controlled differently between a human ER + breast cancer cell line and a mouse embryonic fibroblast cell line.
The observations from western hybridizatons were confirmed in EM images taken from LCC9 cells transfected with mitofusin-1 siRNA (Figure 3C). Quantification of EM micrographs indicates that mitofusin-1 inhibition had no effect on autophagosome number (Figure 3D) when compared with control siRNA transfected cells. However, mitofusin-1 knockdown inhibited mitochondrial vesicle formation (Figure 3E). These data suggest that mitofusin-1 plays a critical role in the development of mitochondrial autophagosomes with no effect on the classical autophagosome formation pathways. These new data, coupled with our previous data, strongly support our original interpretation that, in ER + breast cancer cells, mitochondria donate their cellular membrane material to form autophagosomes and that this occurs in an ATG7 and mitofusin-1 dependent manner.
Finally, we appreciate that this is a controversial area and that others may choose to arrive at different conclusions from the same data. We appreciate the opportunity the journal has provided to contrast our interpretations with those of Ding and Eskelinen. We also look forward to the publication of additional studies that may better delineate the nature and physiological relevance of both autophagosomes and what appear to be the closely related “mitochondrial spheroid” structures.
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