Mitochondria directly donate their membrane to form autophagosomes during a novel mechanism of parkin-associated mitophagy
© Cook et al.; licensee BioMed Central Ltd. 2014
Received: 13 November 2013
Accepted: 6 February 2014
Published: 27 March 2014
Autophagy (macroautophagy), a cellular process of “self-eating”, segregates damaged/aged organelles into vesicles, fuses with lysosomes, and enables recycling of the digested materials. The precise origin(s) of the autophagosome membrane is unclear and remains a critical but unanswered question. Endoplasmic reticulum, mitochondria, Golgi complex, and the plasma membrane have been proposed as the source of autophagosomal membranes.
Using electron microscopy, immunogold labeling techniques, confocal microscopy, and flow cytometry we show that mitochondria can directly donate their membrane material to form autophagosomes. We expand upon earlier studies to show that mitochondria donate their membranes to form autophagosomes during basal and drug-induced autophagy. Moreover, electron microscopy and immunogold labeling studies show the first physical evidence of mitochondria forming continuous structures with LC3-labeled autophagosomes. The mitochondria forming these structures also stain positive for parkin, indicating that these mitochondrial-formed autophagosomes represent a novel mechanism of parkin-associated mitophagy.
With the on-going debate regarding autophagosomal membrane origin, this report demonstrates that mitochondria can donate membrane materials to form autophagosomes. These structures may also represent a novel form of mitophagy where the mitochondria contribute to the formation of autophagosomes. This novel form of parkin-associated mitophagy may be a more efficient bio-energetic process compared with de novo biosynthesis of a new membrane, particularly if the membrane is obtained, at least partly, from the organelle being targeted for later degradation in the mature autolysosome.
KeywordsBreast cancer Mitochondria Autophagy Mitophagy Parkin Antiestrogen resistance Fulvestrant Imatinib Estrogen receptor-α
Autophagy involves the segregation of subcellular material into double membrane structures (autophagosomes) that then fuse with lysosomes (autolysosomes) wherein the cellular cargo is subsequently degraded by lysosomal hydrolases. This process facilitates the digestive degradation of aged, damaged, or unneeded organelles including mitochondria, Golgi complex, and endoplasmic reticulum . Understanding of the autophagic machinery has advanced; however the primary source of the phospholipid bilayer that creates the autophagosome membrane has remained unclear [2, 3].
The difficulty in identifying the origin of cellular material donated to form autophagosome membranes reflects the inability of specific markers for each subcellular organelle to carry over to autophagosomes. Thus, various organelles have been proposed to be autophagosome membrane donors including the plasma membrane, endoplasmic reticulum, Golgi complex, mitochondria, and even a de novo generation model [2, 3]. The endoplasmic reticulum was originally implicated by studies reporting the concurrent presence of rough endoplasmic reticulum integral membrane proteins both in autophagosome membrane preparations and electron microscopy images [4, 5]. However, contradictory data emerged indicating only 30% of all autophagosomes are associated with the endoplasmic reticulum, suggesting the involvement of other organelles in the formation of autophagosomes . More recently, the outer mitochondrial membrane was proposed to serve as a donor source for starvation-induced autophagosome formation . Time-lapse photography data suggested that the early autophagy protein ATG5 and the autophagosomal marker LC3 translocate to puncta localized on mitochondria, and that labeled outer mitochondrial membrane protein concurrently marked both autophagosomes and mitochondria in data obtained following serum starvation of a rat kidney cell line [8, 9]. However, this study is limited because of the primary use of confocal microscopy and the general observation that localization is to be anticipated since the mitochondria are engulfed within mature autophagosomes during mitophagy. The resolution provided by electron microscopy (EM) is needed to directly show autophagosome structures, their content, and their special relationships with mitochondria; this evidence has been notably lacking. We show, for the first time, visual evidence of the contribution of mitochondrial membrane donation to autophagosome formation in both basal and drug-induced autophagy in a human breast cancer cell line. Moreover, these mitochondria donating membranes to form autophagosomes stain positive for the mitophagy-related protein parkin, suggesting a novel mechanism of mitophagy whereby the mitochondria contribute to autophagosome formation, other than being engulfed by the forming autophagosome .
Materials and methods
The following materials were obtained as indicated: Imatinib and ICI 182,780 (Tocris Bioscience, Ellisville, MO); penicillin and Improved Minimal Essential Medium (IMEM; Gibco Invitrogen BRL, Carlsbad, CA); bovine calf charcoal stripped serum (CCS) (Equitech-Bio Inc, Kerrville, TX); Lipofectamine RNAiMax reagent (Invitrogen); Estrogen receptor-α (ER) shRNA (Evrogen, Moscow, Russia); GFP-LC3 (Addgene, Cambridge, MA); EndoTracker Red, Golgi-RFP, MitoTracker-GFP, MitoTracker-RFP (Invitrogen); Cyto-ID Autophagosome detection kit (Enzo Life Sciences); LC3B and parkin antibody (Cell Signaling Technology, Danvers, MA); PINK1 and parkin siRNA (Origene, Rockville, MD).
LCC9 breast carcinoma cells were grown in phenol-red free IMEM media containing 5% CCS. Cells were grown at 37°C in a humidified, 5% CO2:95% air atmosphere. Cells were plated in 10 cm dishes and treated with 0.1% v/v ethanol vehicle, 100 nM fulvestrant, or 10 μM Imatinib for 72 hours, or transfected with four shRNA constructs targeting ER-α. EM was performed as previously described . Briefly, cells were pelleted and fixed with 2.5% glutaraldehyde and postfixed with 0.5% osmium tetroxide. Cells were then dehydrated and embedded in Spurs epoxy resin. Embedded cells were cut into ultrathin sections (90 nm), double-stained with uranyl acetate and lead citrate, and viewed with a Philips CM10 transmission electron microscope (Phillips Electronics). Autophagosome number and size were quantified using ImageJ software.
LCC9 cells were transfected with GFP-LC3B and control or ERα shRNA, 0.1% v/v ethanol vehicle, 500 nM ICI, or 10 μM Imatinib and with lentiviral RFP-labeled organelle trackers (endoplasmic reticulum, golgi complex, and mitochondria) for 24 hours. Cells were counterstained with DAPI and confocal microscopy was performed using an Olympus IX-70 confocal microscope (LCCC Imaging Shared Resources) to determine LC3-positive punctate formation and LC3 co-localization with different cellular organelles. LCC9 cells were treated with vehicle, serum starvation, 500 nM ICI, 2 ng/mL tunicamycin, transfected with ATG7 siRNA (negative control), transfected with ERα shRNA, transfected with parkin siRNA, or treated with 10 μM Imatinib for 48 hours. Cells were incubated with MitoTracker-GFP for 24 hours prior to cell harvesting. Cells were collected and treated with a modified monodansylcadaverine. Cells were sorted by flow cytometry to quantify autophagosome and mitochondria number (LCCC Flow Cytometry Shared Resource).
The effect of mitophagy on antiestrogen responsiveness was determined by crystal violet cell density assay. Briefly, 5 x 103 cells/mL LCC9 cell in IMEM containing 5% CCS were transfected with control or PINK1 siRNA and were plated in 24-well tissue culture plates. On day 1 after plating, cells were treated with varying doses of fulvestrant (10 nM-1000 nM). On day 3, medium was aspirated and cells were stained with crystal violet. Cells were permeabilized using citrate buffer and absorbance was read at 660 nm using a plate reader.
To confirm the effect of treatments on autophagy and subcellular localization, western blot hybridization was used to measure LC3-I/LC3-II, p62, PINK1, parkin, and COXIV. Treated cell monolayers were solubilized in lysis buffer, protein was measured using a standard bicinchoninic acid assay, and proteins were size fractionated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Non-specific binding was blocked by incubation with Tris-buffered saline containing 5% powdered milk and 1% Triton X-100. Membranes were incubated overnight at 4°C with primary antibodies, followed by incubation with polyclonal horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000) for 1 hour at room temperature. Immunoreactive products were visualized by chemiluminescence (SuperSignal Femto West, Pierce Biotechnology, Rockford, IL) and quantified by densitometry using the ImageJ digital densitometry software (http://rsbweb.nih.gov/ij/). Protein loading was visualized by incubation of stripped membranes with a monoclonal antibody to β-actin or β-tubulin (1:1000).
All data are presented as the mean ± standard error of the mean (SEM). Statistical differences were evaluated by one way analysis of variance (ANOVA) followed by Dunnett post hoc test. The criterion for statistical significance was set at p < 0.05 prior to initiation of the study.
Results and discussion
Autophagy is thought to occur naturally in most cells, and breast cancer cells often exhibit increased autophagy when compared with immortalized normal breast epithelial cells. Antiestrogen resistant breast cancer cells exhibit a further increase in autophagy when compared with their therapy sensitive counterparts [15, 19, 20]. We cannot exclude the possibility that these higher levels of autophagy in cancer cells result in the use of cellular materials or processes not commonly used in normal cells. Nonetheless, the use of preexisting target organelle membranes is an energy efficient process compared with de novo biosynthesis of a new double membrane, particularly if the membrane is at least partly obtained from the organelle being targeted for later degradation in the mature autolysosome. Moreover, we show that the process of mitochondrial-mediated autophagosome formation also occurs in MCF7 cells (ER+, antiestrogen sensitive breast cancer cells), implying that this phenomenon occurs more broadly than in just the LCC9 variant (Figure 7A). Since autophagy clearly plays an important role in breast cancer progression and therapeutic responsiveness [12, 21, 22], understanding how autophagy occurs may improve our ability to efficiently target this prosurvival pathway.
In conclusion, we show the first physical evidence, by electron microscopy, that mitochondria can supply membrane material during the creation of autophagosomes. We demonstrate that this occurs not only during serum starvation , but also during both basal (in the presence of serum and vehicle) and drug-induced autophagy. We go further to demonstrate that the autophagosomes developing from mitochondria may represent a novel mechanism of parkin-associated mitophagy, where mitochondrial membrane material can be contributed to formation of the developing autophagosome, rather than the autophagosome forming around parkin-labeled mitochondria. While we did not find similar early structures for autophagosomes incorporating other subcellular organelles, the data imply that the autophagic removal of Golgi/secretory vacuoles (crinophagy), endoplasmic reticulum (reticulophagy), and other organelles may also proceed with the contribution of target organelle membrane to formation of the membranes of the subsequent autophagosomes.
Analysis of variance
Autophagy related gene 5
Autophagy related gene 7
Charcoal stripped calf serum
Green fluorescent protein
Faslodex, fulvestrant, ICI 182,780
Microtubule-associated protein light chain 3
Red fluorescent protein
Standard error of the mean
Katherine Cook is supported by a DOD Breast Cancer Research Program Postdoctoral Fellowship (BC112023). This research was also supported in part by awards from the US Department of Health and Human Services (R01-CA131465 and U54-CA149147) to Robert Clarke and from the Intramural Research Program of the NIH/NCI to David Roberts.
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