Autophagy and autophagosome formation -
Moreover, the ER-to-Golgi intermediate compartment ERGIC is also a membrane source for autophagosomes, possibly by supplying membrane for the nucleation of the phagophore, a notion that is supported by the observation that ERES undergo a remodeling and associate with ERGIC upon nutrient starvation Figure 3 [ 47 ].
Recently, it has been shown that cis- Golgi-derived FIPpositive vesicles and ATG16L1-containing endosomal membranes fuse through a mechanism that depends on STX17, the SERCA2 calcium pump, the calcium-regulated membrane tether E-SYT2 and the ER-localized transmembrane calcium regulator SIGMAR1 [ 48 ].
The resulting compartment is a HyPAS, which is important for autophagy initiation and appears to be key in the recognition of diverse cargoes during selective types of autophagy [ 48 ]. It remains to be understood whether the cis- Golgi-derived FIPpositive vesicles are in fact the ERGIC-derived vesicles, and whether the HyPAS may correspond to the recycling endosome-derived vesicles that were found to be positive for ATG16L1 and ULK1.
The autophagy-specific PI3K complex is known as complex I to distinguish it from the PI3K complex II, which is mostly involved in regulating endosomal traffic and functions [ 49 , 50 ].
The phosphatidylinositol 3-phosphate PtdIns3P synthesized by the PI3K complex I plays a key role in the recruitment and assembly of the rest of the ATG machinery see below [ 19 ]. In mammalian cells, the generation of PtdIns3P leads to the recruitment of the ER integral membrane protein DFCP1 that specifically binds this lipid, creating a close association between the external surface of expanding phagophore and the ER [ 53—55 ].
These characteristic ER subdomains are known as omegasomes [ 53 ]. Furthermore, there may be two additional mechanisms for the recruitment of the PI3K complex I that are not mutually exclusive.
The first is that the PI3K complex I may be transported via vesicles derived from ERGIC, to which the COPII-coated vesicles are recruited in a PI3K complex-dependent manner under autophagy-inducing conditions Figure 3 [ 57 ].
In the second, ATG14L and by the extension the PI3K complex I may be recruited to the ER or eventually the HyPAS, by interacting with STX17 Figure 3 [ 58 ].
The following step in the autophagosome biogenesis is the expansion of the phagophore, which requires an enormous supply of lipids and the function of multiple core ATG proteins.
The PtdIns3P present on the phagophore is particularly important for the recruitment of ATG proteins that mediates its expansion [ 6 , 7 ]. Central effectors of this phosphoinositide are the members of the PtdIns3P-binding proteins from the WD-repeat protein interacting with phosphoinositides WIPI protein family, yeast Atg18 and Atg21, and mammalian WIPI1 to WIPI4 [ 59 ].
In yeast, the Atg2—Atg18 complex assembles sequentially at the membrane contact sites MCSs between the ER and the extremities of the phagophore [ 20 , 21 , 61 ]. Its recruitment at this peculiar location depends on its interaction with Atg9 and PtdIns3P Figure 2 [ 61 ].
In mammalian cells, TRAPPC11, a subunit of the human transport protein particle TRAPP III complex [ 62 ], is involved in the recruitment of the complex between ATG2 proteins and WIPI4 Figure 3 [ 63 ].
In vitro experiments have shown that both yeast Atg2 and mammalian ATG2 proteins have membrane tethering and lipid transfer functions [ 64—66 ]. These lipids accumulate on the cytoplasmic leaflet of the phagophore membrane and must be flipped to the luminal leaflets to promote phagophore expansion.
The two interconnected ubiquitin-like conjugation systems drive cargo sequestration, membrane expansion and autophagosome completion. While yeast possesses one Atg8 protein, mammalian cells have six main isoforms of Atg8-like proteins, i.
three LC3 paralogs LC3A, LC3B and LC3C and three GABARAP paralogs GABARAP, GABARAP-L1 and GABARAP-L2 [ 71 ]. These two aromatic residues are important for the autophagosome biogenesis during bulk autophagy in yeast and the degradation efficiency of a major selective cargo p62 for autophagy in mammals [ 76 ].
These properties may also contribute to the formation of a proteinaceous coat by Atg8-PE [ 77 ]. For example, ultrastructural investigations have revealed that mammalian phagophores may have additional MCSs [ 54 , 55 , 80 ].
The mammalian autophagosomes biogenesis occurs at the ER subdomain enriched in both the ER membrane VMP1 and TMEM41B, which modulates ER-phagophore MCSs [ 28 , 80 , 81 ], and the phosphatidylinositol synthase PIS [ 46 ].
Moreover, de novo lipid synthesis is also a possible lipid source for phagophore expansion. A recent study in yeast has revealed that the acyl-CoA synthetase Faa1, which is also localized on the phagophore, activates fatty acids to convey them into the synthesis of phospholipids at the phagophore-ERES MCSs and promotes their incorporation into Atg8-positive membranes [ 82 ].
Mitochondria and lipid droplets are also possible lipid sources for phagophore expansion, but their contribution is probably indirect [ 7 ]. The curvature of the phagophore membrane during its expansion is pivotal for autophagosome biogenesis, especially during bulk autophagy because this event is not guided by the cargo.
Proteins sensing and generating membrane curvature, and the physical properties of the membrane appear to be involved in this process. The yeast Atg12—Atg5—Atg16 complex binds membranes and assembles into a mesh-like structure in association with Atg8 lipidation on artificial vesicles [ 77 , 83 ], which might serve as a scaffold to shape the phagophore.
While the copy number of Atg12—Atg5—Atg16 complexes present at the PAS is too small to cover the entire surface of an autophagosome with an average size [ 84 ], it is possible that these proteins affect the shaping of the phagophore.
This latter notion is supported by the observation that Atg8-PE can also shape the membrane of synthetic vesicles Figure 2 [ 76 ]. Actin filaments do not only recruit ATG proteins in the early stage of autophagosome biogenesis [ 91 ], but they also contribute to the shaping of the phagophore membrane in mammalian cells [ 92 ].
In this process, the actin-capping protein CapZ stimulates actin polymerization by binding to PtdIns3P [ 92 ]. The resulting actin filaments serve as a cytoskeletal scaffold to develop phagophores into autophagosomes with normal morphology. It has been biophysically shown that the highly curved edges of disc-like membranous vesicles, like phagophores, are energetically unstable and this can be a factor that affects their shape [ 93 ].
With the disc expanding, the areas at the edges increase and the disc becomes more unstable [ 93 ], which could be compensated by the spontaneous acquisition of a spherical structure. Based on this model, the physical properties of the lipid bilayer can also be an important factor for the shaping of the phagophore.
To generate autophagosomes with normal sizes, it is important to delay the spontaneous spherical rearrangement of the expanding phagophore. There are two main hypotheses linked to this concept [ 6 , 94 ].
One is that the highly curved edge of the phagophore is stabilized by membrane-binding proteins that are asymmetrically distributed on the inner and outer membrane of the phagophore [ 94 , 95 ].
The other hypothesis is that an alteration of the lipid composition of the phagophore membrane [ 94 ]. This will in turn increase the curvature at the edges of the membrane and consequently accelerate the bending of the phagophore. The phagophore expansion and its bending into a spherical shape lead to a close proximity of its extremities, and the closure of the remaining pore is the last step for the autophagosome formation Figure 1.
This process involves a membrane fission event, which is mediated by the endosomal sorting complexes required for transport ESCRT machinery in both yeast and mammalian cells [ 98— ]. However, this result remains contradictory since in the absence of Vps4, a component of ESCRT machinery, autophagy progresses normally in yeast [ ].
It has also been reported that the Rab5-like GTPase Vps21 and Atg14 are involved in the pore closure [ 98 ], but this remains to be fully understood. Upon completion, autophagosomes undergo a maturation that appears to be concomitant with their detachment from the ER [ 27 , 53 ].
Autophagosome maturation is characterized by the release of most of the ATG proteins from their surface into the cytoplasm for reuse, which also involves the turnover of PtdIns3P by phosphatases from the myotubularin family [ — ].
Atg9 is also present on the surface of the yeast autophagosomes [ 39 , ], but the functional relevance of this remains to be uncovered.
Fusion of autophagosomes with degradative organelles begins with tethering, which involves the homotypic vacuole fusion and protein sorting HOPS tethering complex [ ].
The fusion of autophagosomes with the compartments of the endolysosomal system has been covered in details by recent reviews [ — ]. Importance of the field. Autophagy is essential to maintain cellular homeostasis in countless situations.
Unveiling the mechanism of this process is critical to understand the pathophysiology of autophagy-related diseases and might provide insight into therapeutic interventions. ATG proteins play a central role in autophagosome biogenesis and their functions have been gradually elucidated during last few decades, providing increasing insights into the molecular mechanism of autophagy.
Summary of the current thinking. The signal cascades triggering autophagy and how these orchestrate ATG proteins are becoming better understood. Moreover, increasing evidence suggests that the shaping of the phagophore membrane depends not only on proteins sensing and generating membrane curvature, but probably also on biophysical properties of the membrane.
Future directions. There are still several central questions that remain to be answered. For example, it is largely unclear how the phagophore-ER MCSs are generated, especially in vivo.
VMP1 and TMEM41B [ ], probably do not produce the energy necessary to catalyse lipid transfer. Addressing these questions but also other ones, will help to increase our knowledge and shed new light into the molecular mechanism of autophagy. is supported by ZonMW TOP , Open Competition ENW-KLEIN OCENW.
is supported by a China Scholarship Council PhD fellowship CSC No. The authors thank Yasmina Filali-Mouncef Lazcano, Rubén Gómez-Sánchez, Babu Raman and Emma Zwilling for the critical reading of the manuscript. Sign In or Create an Account. Search Dropdown Menu.
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Skip Nav Destination Close navigation menu Article navigation. Volume 50, Issue 1. Previous Article Next Article. All Issues. Cover Image Cover Image. Autophagosome formation mechanism.
Competing Interests. Open Access. Authors Contribution. Article Navigation. Review Article January 25 Molecular regulation of autophagosome formation In Collection Cell death and survival. Yan Hu Yan Hu. Department of Biomedical Sciences of Cells and Systems, Molecular Cell Biology Section, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
This Site. Google Scholar. Fulvio Reggiori Fulvio Reggiori. Correspondence: Fulvio Reggiori f. reggiori umcg. Author and Article Information. Publisher: Portland Press Ltd. Received: November 22 Revision Received: December 21 Accepted: January 04 Online ISSN: This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.
Biochem Soc Trans 50 1 : 55— Article history Received:. Revision Received:. Get Permissions. toolbar search Search Dropdown Menu. toolbar search search input Search input auto suggest. Figure 1. View large Download slide.
Figure 2. The molecular mechanisms of the early stages of autophagosome biogenesis during bulk autophagy in S.
Figure 3. The molecular mechanisms of the early stages of autophagosome biogenesis during bulk autophagy in mammals. Figure 4.
The authors declare that there are no competing interests associated with the manuscript. and F. have conceived the review and written the manuscript. AIM Atg8 protein-interacting motif. ERES ER exit sites. ERGIC ER-to-Golgi intermediate compartment. ESCRT endosomal sorting complexes required for transport.
GUVs giant unilamellar vesicles. HyPAS hybrid pre-autophagosomal structure. LIR LC3-interacting region. mTORC1 mammalian TORC1. PAS phagophore assembly sites.
PE phosphatidylethanolamine. PI3K phosphatidylinositol 3-kinase. TRAPP transport protein particle. Watch what you self- eat: autophagic mechanisms that modulate metabolism. Search ADS. A diversity of selective autophagy receptors determines the specificity of the autophagy pathway.
FIP claw domain binding to p62 promotes autophagosome formation at ubiquitin condensates. The cargo receptor NDP52 initiates selective autophagy by recruiting the ULK complex to cytosol-invading bacteria.
The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation. Convergence of multiple autophagy and cytoplasm to vacuole targeting components to a perivacuolar membrane compartment prior to de novo vesicle formation.
Fine mapping of autophagy-related proteins during autophagosome formation in Saccharomyces cerevisiae. ER exit sites are physical and functional core autophagosome biogenesis components.
Vac8 spatially confines autophagosome formation at the vacuole in S. The carboxy terminus of yeast Atg13 binds phospholipid membrane via motifs that overlap with the Vac8-interacting domain. Atg21 organizes Atg8 lipidation at the contact of the vacuole with the phagophore.
Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles. As is currently understood, autophagosome biogenesis requires a series of biochemical reactions whereby two conjugation systems converge to create a multimeric protein assembly that nucleates formation of the autophagosome Yorimitsu and Klionsky, Because omegasomes colocalized with components of both conjugation systems Atg5 and MAP-LC3 , they would be well-suited for nucleating formation of such a particle, and the underlying ER membrane would provide a good source of the lipids that are used in the conjugation step.
PI 3 P enriched in omegasomes would also be important for attracting and localizing effectors such as Atg18 Guan et al. DFCP1 is another such effector whose function is currently unknown. Based on the fact that DFCP1 does not have a counterpart in yeast, and especially in Drosophila melanogaster , which exhibits normal autophagic responses Rusten et al.
Although DFCP1 is not essential for autophagy, the pool of PI 3 P that it binds must be given the genetic and pharmacological data that reveal PI 3 P formation as an essential requirement in autophagosome formation Blommaart et al.
In particular, overexpression of a FYVE domain has been shown to lead to sequestration of endosomal PI 3 P and redistribution of EEA1 from endosomes to the cytosol Byfield et al.
In the case of DFCP1, both a myc- and a GFP-tagged construct expressed at levels higher than fold over the endogenous protein inhibited autophagosome formation. The ER in eukaryotic cells is highly compartmentalized Levine and Rabouille, but contains little PI 3 P under normal conditions Gillooly et al.
How then is PI 3 P formed on the ER during starvation? We saw that vesicles containing Vps34 were frequently associated with the earliest discernible omegasome spots and continued associating as the omegasomes expanded. These Vps34 vesicles were positive for LAMP-2, which indicates that they are late endosomes or lysosomes, and were always found in the vicinity of the ER, frequently moving long distances along the ER strands.
The simplest hypothesis for our observations is that Vps34 is delivered to the ER via some type of vesicular transport step, although we cannot rule out the possibility that Vpspositive vesicles interact directly with the ER, allowing Vps34 to generate PI 3 P on the ER in trans.
Remarkably, this proposed mode of action of Vps34 is analogous to the situation in yeast. The single autophagosome produced in yeast during autophagy localizes near the vacuole, in close proximity to Vps34 protein, which also localizes to the vacuole Obara et al.
Previous work has shown that Vps34 is essential for initiation of the autophagic response Kihara et al. These observations suggest that a subpopulation of Vps34 molecules is activated during amino acid starvation, whereas the majority of the protein is inhibited.
In support of this, work in yeast has suggested the existence of two Vps34 complexes, with one involved in endosomal function and the other in autophagy Kihara et al. The mammalian homologue of Atg6, beclin 1, has been shown to be essential for induction of autophagy Liang et al.
Given that omegasome formation requires Vps34 and beclin function, a possible mechanism for PI 3 P formation is that a Vpspositive vesicle interacting with the ER delivers Vps34 to its binding partner, beclin, which is already there. Based on our results and on extensive previous work, we propose the following working model for the biogenesis of at least certain types of autophagosomes Fig.
Early during amino acid starvation, PI 3 P begins to accumulate in a part of a flat ER cisterna green in the vicinity of Vpscontaining vesicles. This PI 3 P-enriched membrane helps to localize autophagy-related proteins red , and a mixed cisterna is created.
Topologically, the PI 3 P-enriched membrane omegasome is always found encircling the autophagosomal membrane, but its exact connection to the ER is still not known Fig.
This mixed cisterna continues to expand, with the PI 3 P- and the autophagosomal-enriched membranes remaining spatially distinct but continuous with each other. Our model thus far is similar to the pathway of isolation membrane expansion previously proposed, with the difference being that in our view, all of this takes place on a PI 3 P-enriched membrane platform.
The final step in previous models of autophagosome formation involves fusion of the isolation membrane into a double membrane vesicle by an unknown mechanism. Again, our proposal is similar, but we would suggest that fusion of the flat isolation membrane is aided by the PI 3 P outer layer: when omegasomes reach their maximum size, inward budding would generate a prefusion autophagosome intermediate whereby the PI 3 P-enriched membrane marks the point of eventual closure of the double membrane vesicle.
Indeed, live imaging showed that the last discernible pool of PI 3 P during omegasome collapse decorates the edge of the autophagosome. Our proposed model is not in conflict with previous observations on autophagosome biogenesis, but it does place emphasis on the generation of PI 3 P as a very critical early event.
In our view, PI 3 P is a regulator of this pathway by being the determining factor for the localization of autophagosome induction. In addition, our proposal is more consistent with the maturation model of autophagosome biogenesis and suggests that the ER plays an important role in this process both by providing the site for omegasome formation and the membrane used.
The antibodies used in the course of this work were: mouse anti-GST GeneTex, Inc. Kreis, European Molecular Biology Laboratory, Heidelberg, Germany , rat anti-ER proteins primarily BiP, a gift from G.
Butcher, Babraham Institute, Cambridge, England, UK , rabbit anti-GFP Invitrogen , 9E10 mouse anti-myc, rabbit anti—MAP-LC3 Santa Cruz Biotechnology Inc. Moremen, University of Georgia, Athens, Georgia , rabbit anti-Vps34 Invitrogen , rabbit anti—beclin 1 Santa Cruz Biotechnology, Inc.
Truncated mutants of DFCP1 residues —, —, —, and — were cloned into pEGFPC2 using standard techniques. Site-directed mutagenesis to generate point mutants within DFCP1 was performed using standard PCR techniques, and all constructs were verified by DNA sequencing.
The pECFP-ER plasmid which contains CFP flanked by an ER insertion signal and a KDEL C-terminal ER retention signal was a kind gift of C.
Taylor University of Cambridge, Cambridge, England, UK. The pdsRED-ER plasmid similar to the CFP one was obtained from Clontech Laboratories, Inc.
To generate the GFP-ER-FYVE reporters, the FYVE domain of FENS-1 Ridley et al. The exact orientation in this reporter is important: GFP-ER-FYVE works, whereas GFP-FYVE-ER does not.
The spacer region in this construct amino acids starting with GGGS and ending with ESNS is extremely important for its function. A shorter spacer results in a construct that stays in the ER without translocating to punctate structures.
Truncated DFCP1 constructs — and WA — DFCP1 were also cloned into pGEX 4T-1 GE Healthcare using standard techniques. The oligonucleotides were annealed and ligated into the pSilencer vector Ambion containing either an RNA U6 or H1 promoter using standard techniques.
Constructs were verified by DNA sequencing. pEGFPC2 expressing iFYVE of FENS-1 was a gift from P. Hawkins Babraham Institute. pEGFPC1 LC3 was a gift from T. Yoshimori Osaka University, Osaka, Japan. To create the RFP version, LC3 was cloned into the pmRFPC1 vector using standard techniques.
pEGFPC1 expressing the PX domain from p40 phox was a gift from C. Ellson Babraham Institute. COS-7 cell lines were transiently transfected with diethylaminoethyl-dextran as described previously Manifava et al.
HEK cell lines were transfected using FuGENE-6 and FuGENE HD transfection reagent Roche according to the manufacturer's instructions. We used predesigned oligonucleotides from Thermo Fisher Scientific SMART pool to reduce expression levels of Vps34, beclin, and DFCP1.
Cells were transfected using dharmafect-1 and examined 72 h later. Throughout the paper, we refer to amino acid starvation, although it should be noted that other growth factors normally present in the medium are also absent.
However, we determined that the DFCP1 response was only evident during amino acid withdrawal, i. For amino acid starvation, cells were washed once with prewarmed PBS and then twice with prewarmed starvation medium mM NaCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 5 mM glucose, and 20 mM Hepes, pH 7.
Note that this medium lacks amino acids as well as potassium; in preliminary experiments, we found it to give faster starvation responses in HEK cells. The duration of the starvation treatment varied between experiments as indicated.
PI 3-kinase was inhibited by the addition of either 10 nM wortmannin or μM LY to the cell medium. Autophagy was inhibited by the addition of 10 mM 3-methyladenine to the cell medium. Duration of the treatments varied between experiments. When MDC was used, a stock was made in starvation medium at 0.
Cells for immunofluorescence were grown on glass coverslips and fixed in 3. Staining for immunofluorescence and digital photography were done as described previously Manifava et al. Purified GST-tagged DFCP1 proteins were precleared by centrifugation at 14, g for 10 min and mixed with microsomes in siliconized Eppendorf tubes in a total volume of μl for 15 min at 37°C.
After centrifugation, the pellets were analyzed by SDS-PAGE. COS-7 cells transiently transfected with the appropriate constructs were lysed in lysis buffer 50 mM Tris-HCl, pH 8. Binding to PI 3 P-coupled beads was done as described previously Ridley et al.
Cells expressing GFP-DFCP1 and grown on coverslips were starved for 60 min and washed extensively with PBS. They were then perforated with nitrocellulose as described previously Simons and Virta, and fixed with formaldehyde.
Ellson followed by monoclonal anti-GST antibodies and TRITC-conjugated goat anti—mouse secondary. Images were captured with a confocal microscope FV; Olympus using a 60× 1.
Samples triple labeled with GFP, TRITC, and Cy5 were imaged using a sequential scan setting using excitation light at , , and nm, respectively. Two imaging systems were used to capture images of live cells. For both systems, cells were plated onto mm-diameter glass coverslips BDH and transiently transfected with the relevant constructs, then individual coverslips were secured in an imaging chamber with 2 ml of cell medium or starvation medium added as indicated.
The assembled imaging chamber was fitted into a heated stage on the microscope, and cells were maintained at 37°C. The UltraView LCI confocal was equipped with a × 1. Emission was collected using — nm GFP and — DsRed band-pass filters. CFP, GFP, and mRFP were excited using — nm, — nm, and — nm band-pass filters, respectively.
Emission was collected using — nm CFP , — nm GFP , and — nm mRFP band-pass filters. Deconvolution was performed using Autodeblur MediaCybernetics , and 3D reconstructions were made using Volocity software PerkinElmer where indicated. Samples for EM were washed three times in ice-cold EM preparation buffer PBS, 0.
Cells were fixed in 3. For preembedding labeling, cells were stained with primary antibodies and protein—A gold before a second fixation step and embedding in epon. For cryo-EM, cells were scraped and centrifuged at rpm for 5 min at RT, and the cell pellet was processed for immunogold labeling with rabbit anti-GFP antibodies Invitrogen using standard techniques.
We measured cells in three separate experiments to determine levels of punctate structures or the extent of colocalization. These measurements were done on randomly selected fields of view.
In addition, all data reported that show differences in puncta formation were verified qualitatively in blind fashion by an independent observer. S1 shows the response of three PI 3 P-binding proteins to starvation and concludes that DFCP1 is the most appropriate reporter for colocalization with autophagosomes.
S2 shows that overexpression of WT DFCP1 at high levels inhibits autophagosome formation, whereas its down-regulation by siRNA does not inhibit this response. S3 shows additional examples of colocalization of GFP-DFCP1 with autophagy-related proteins MAP-LC3 and Atg5 and the ER.
S4 shows additional examples of localization of DFCP1 by immuno-EM. S5 shows that autophagy as measured by three independent assays proceeds normally in the cell lines used throughout this work.
Video 1 shows double imaging of GFP-TM-FYVE and dsRED-ER during starvation. Video 2 shows single imaging of GFP-DFCP1 during starvation.
Video 3 shows triple imaging of GFP-DFCP1, mRFP-MAP-LC3, and CFP-ER during starvation. Video 4 shows double imaging of GFP-DFCP1 and dsRED-ER during starvation using TIRFM imaging.
Video 5 shows a 3D reconstruction of GFP-DFCP1 and mRFP-MAP-LC3 at different planes of rotation. Video 6 shows double imaging of GFP-DFCP1 and mRFP-MAP-LC3 during starvation. Video 7 shows triple imaging of GFP-DFCP1, mRFP-Vps34, and CFP-ER during starvation.
This work was supported by the Biotechnology and Biological Sciences Research Council BBSRC. Axe was the recipient of a BBSRC special committee studentship on live imaging.
Roderick is a Royal Society University Research Fellow. Response of three PI 3 P-binding proteins to starvation.
A HEK cells were transfected with plasmids encoding GFP-DFCP1, GFP-FYVE domain from FENS-1, or GFP-PX domain from p40 PHOX. Note that all three proteins translocated to punctate structures upon starvation, but the nature and number of these structures were distinct.
B HEK cells were cotransfected with plasmids encoding RFP-MAP-LC3 and GFP-tagged DFCP1, FYVE domain, or PX domain as indicated.
Note that overexpression of DFCP1 reduced the amount of MAP-LC3—containing autophagosomes, but that those that were formed colocalized with DFCP1. There was less colocalization of MAP-LC3 with the FYVE and PX domain constructs. Images on the bottom show enlarged views of the boxed regions.
Bars, 20 µm. Overexpression of WT DFCP1 at high levels inhibits autophagosome formation, whereas down-regulation of DFCP1 by RNAi does not inhibit autophagosome formation. A—D Effects of overexpression.
A Normal HEK cells or clonal cell lines derived from HEK, and overexpressing WT or DM DFCP1 were transfected with GFP-MAP-LC3. Note that WT DFCP1 partially inhibited formation of MAP-LC3 autophagosomes, whereas the DM behaved very similarly to the parental HEK cells. In general, cells expressing WT DFCP1 had fewer punctate structures containing MAP-LC3 than the cells shown here; in all cases, these punctate structures were smaller than in parental cells or in cells expressing DM DFCP1.
B Normal HEK cells, clone , expressing WT DFCP1 and a clone expressing the DM were left untreated or starved for 45 or 90 min as indicated. At the end of starvation, lysates were prepared and immunoblotted for endogenous MAP-LC3 or myc for DFCP1.
Note that in HEK cells and in cells expressing the DM, MAP-LC3 was converted to the type II form during starvation; this was partially inhibited by WT DFCP1.
The numbers underneath the bottom panel indicate the amount of MAP-LC3 type II form as a percentage of total MAP-LC3 type I and type II forms in each lane. C Normal HEK cells or clonal cell lines derived from HEK and overexpressing WT or DM DFCP1 were transfected with GFP-MAP-LC3.
The cells were examined for the appearance of MAP-LC3—positive autophagosomes under control or starvation conditions, and the percentage of MAP-LC3—positive cells with more than five punctate structures were counted. Note that WT DFCP1 partially inhibited formation of MAP-LC3 autophagosomes, whereas the DM-expressing cells appear very similar to the parental HEK cells.
D Cells from the clone see previous panels expressing WT DFCP1 were transfected with GFP-MAP-LC3 and pSilencer vectors with different polymerase III promoters H1 or U6 as indicated expressing either a control siRNA or siRNA specific for DFCP1. The cells were examined for the appearance of MAP-LC3—positive autophagosomes as above.
Note that down-regulation of overexpressed DFCP1 restored starvation response of MAP-LC3. The insert shows levels of DFCP1 after transfection with various siRNA-expressing plasmids as indicated.
Based on these results, the C siRNA was selected for the cotransfection experiments shown in the graph. E—H Effects of down-regulation. E HEK cells were treated with siRNA against DFCP1 or with a control siRNA. Note that the DFCP1 oligonucleotide reduces expression of endogenous DFCP1 substantially.
F After siRNA treatment as in E, cells were starved for 60 or 90 min as indicated, and lysates were immunoblotted with antibodies to endogenous MAP-LC3. Note that the starvation-specific type II form is evident for both control and DFCP1 siRNA samples. G Cells were treated with siRNA as in E and F, and starved as indicated, but one sample was additionally treated with 10 nM wortmannin during starvation.
Note that this treatment inhibits MAP-LC3 type II formation in both samples. H Cells treated as above were starved in the presence or absence of bafilomycin A1. Note that the type II MAP-LC3 form is evident in both sets of samples, and it is similarly enhanced by bafilomycin A1 treatment.
Colocalization of GFP-DFCP1 with autophagy-related proteins and the ER. A HEK clones expressing GFP-DFCP1 were left untreated or starved for 60 min and then stained for endogenous MAP-LC3. Note that both GFP-DFCP1 and MAP-LC3 translocated to punctate structures during starvation. A series of colocalization examples during starvation, which are analogous to the interactions seen during live imaging, are shown in panels a—h of the merged image and as separate magnified panels.
B HEK cells or clones expressing GFP-DFCP1 were left untreated or starved for 60 min and then stained for endogenous Atg5. Note that in both cell types, Atg5 became more punctate during starvation, and these punctate structures colocalized with GFP-DFCP1.
These images were obtained using wide-field microscopy. C Additional examples of the colocalization of GFP-DFCP1 with Atg5 after amino acid starvation using confocal microscopy.
A series of colocalization examples are shown magnified in panels 1—3. D HEK clones expressing GFP-DFCP1 were transfected with dsRed-ER and imaged live by confocal microscopy during amino acid starvation.
Shown in panels A—E are examples from several such videos, where the DFCP1 omegasomes coincide with ER regions boxed areas. Localization of DFCP1 by immuno-EM. HEK cells expressing GFP-DFCP1 were starved for 45 min and examined in the EM.
A—C Epon sections after preembedding labeling for DFCPI-GFP using 10 nm gold. The arrowheads indicate labeling on membrane-associated structures adjacent to putative autophagic vacuoles A and B or an electron-dense large vesicle C, asterisks.
D—J Thawed cryosections double-labeled for anti—GFP-DFCPI 10 nm gold, arrowheads and anti-PDI 5 nm gold, small black arrows. All these figures show putative autophagic vacuoles asterisks labeled for DFCPI-GFP as electron-dense vesicles adjacent to PDI-labeled ER membranes.
In E and H, DFCPI is localized to the same membranes that label for PDI. In E, G, H, and I, it is evident that the periphery of the putative autophagic vacuole is surrounded by a membrane cisterna. Opposing large black arrows in A, B, E, G, H, and I indicate double membrane cisterna, which is characteristic of autophagosomes.
A quantitative analysis was performed by systematically sampling these double-labeled cryosections. For this, — gold particles labeling GFP-DFCP1 were systematically sampled from three separate grids, a method recently developed by Lucocq et al. Lucocq, J.
Habermann, S. Watt, J. Backer, T. Mayhew, and G. These gold particles were allocated to the following structures: Golgi Three independent assays for autophagy in the cell lines used throughout this work.
Parental HEK cells or stable lines expressing GFP-DFCP1 on its own or together with mRFP-Vps34 were either left untreated or starved for 60 min as indicated. The cells were then stained for endogenous MAP-LC3 or endogenous Atg5 A , or lysates were prepared in the presence or absence of bafilomycin A1 and examined for MAP-LC3 by immunoblotting B.
Note that both MAP-LC3 and Atg5 became punctate during starvation in all cell types but see Fig. In addition, the MAP-LC3 type II form, which is indicative of autophagic response, was also increased in all cell types.
Identification and partial characterization of an ER-targeting domain within DFCP1. A Sequential truncations of DFCP1 identified an internal domain necessary and sufficient to specify localization in the ER.
Residues in red and underlined are required for ER localization. B The GFP-[—] construct localized to the ER in HEK cells, whereas mutants within this domain shown here is a WA construct were cytosolic. Selected residues important in ER targeting were also mutagenized in the full-length protein as indicated.
All samples were costained with antibodies to calnexin CLNX , an ER protein. Bound material was recovered by centrifugation.
D Microsomes as in C were treated on ice with the indicated units of trypsin before incubation with the GST-[—] domain and centrifugation. Note that β-COP, a peripheral protein found on microsomes, was almost completely digested by trypsin; under these conditions, binding of the DFCP1 fragment to microsomes was not changed.
Starvation-induced and PI 3 P-dependent translocation of DFCP1 to punctate structures partially colocalizing with autophagosomes.
A Lysates from cells expressing WT DFCP1 or the DM were incubated with Affigel beads coupled to PI 3 P; WT DFCP1 bound to PI 3 P, whereas the DM did not. B Stable HEK cell lines expressing WT DFCP1 or the DM were left untreated or starved of amino acids for 90 min. Cells were then fixed and stained for DFCP1.
C HEK cells stably expressing WT DFCP1 were starved alone or in the presence of 3-methyladenine MA or wortmannin wortm as indicated before fixation and staining for DFCP1. D Starved HEK cells expressing DFCP1 were costained with markers to the ER such as calnexin and KDEL. E HEK cells stably expressing WT DFCP1 were transfected with GFP-MAP-LC3 and starved for 90 min as indicated.
Note that in cells expressing WT DFCP1, both MAP-LC3 and DFCP1 translocated to punctate structures upon starvation, some of which colocalized. Bars: A—C and E 20 μm; D 0.
FYVE domain tethered peripherally to the ER translocates to starvation-induced punctate structures in a pathway dependent on PI 3 P. B Stable cell lines expressing the four constructs shown in A were left untreated or starved for 45 min before fixation and fluorescence microscopy.
Note that the GFP-ERFYVE construct translocated to punctate structures upon starvation. C Cells expressing GFP-ERFYVE were starved alone or in the presence of wortmannin or BFA as indicated. D Cells expressing GFP-ERFYVE and starved for 45 min were either counterstained for the ER using antibodies to KDEL top or cotransfected with mRFP-MAP-LC3 bottom.
Note that the GFP-ERFYVE punctate structures frequently localized on the ER and showed partial colocalization with LC3. The panels on the right show an enlarged view of the boxed regions on the left. Bars, 10 μm. Double imaging of GFP-FYVE-TM and dsRed-ER during starvation showing formation of GFP-FYVE-TM punctate structures.
HEK cells were transiently transfected with the two constructs and imaged at 1 frame per 10 s for 45 min in starvation medium. The arrow indicates formation and collapse of a ring structure.
Panels on the left show dsRed-ER; right, GFP-FYVE-TM. Also see Fig. The playback rate is 10 frames per second. ER-anchored FYVE domain translocates to starvation-induced punctate structures in a pathway dependent on PI 3 P. All three constructs were expressed transiently in HEK cells, and their localization was examined with or without amino acid starvation for 60 min as indicated.
Note that GFP-TM-FYVE translocated to punctate structures during starvation. B HEK cells were transfected with GFP-FYVE-TM and mRFP-LC3 and starved for 60 min.
Note that puncta of the two reporters colocalize, with the GFP construct frequently encircling mRFP-LC3 membranes arrows.
Insets show enlarged views of the boxed regions. C Live imaging of HEK cells coexpressing GFP-FYVE-TM and dsRED-ER and starved for 60 min. Also see Video 1. D A region indicated in C bottom middle, box is expanded and shown for 28—40 min during starvation. Note the formation and collapse of a ringlike particle.
Arrows mark the particle at early stages. Bars: A—C 20 μm; D 1 μm. Isolation and characterization of stable HEK clones expressing GFP-DFCP1.
A Four clones were screened for GFP-DFCP1 expression inset shows blots of endogenous DFCP1 and GFP-DFCP1 with β-COP as loading control; the fold overexpression of tagged DFCP1 over endogenous DFCP1 is also indicated and for a good response to starvation, as measured by translocation of GFP-MAP-LC3 to punctate structures shown in the graph or acquisition of endogenous LC3-II form last lane of inset.
High levels of DFCP1 inhibited starvation responses especially evident for clone Error bars show the standard deviation from three independent experiments. B Clone cells were left untreated or starved for 45 min in the absence or presence of wortmannin wortm or BFA as indicated, and the distribution of GFP-DFCP1 was examined by fluorescence microscopy.
Note that wortmannin inhibited translocation, whereas BFA was without effect. C Clone cells were treated with siRNA against Vps34 or beclin-1, or with a control siRNA as shown, starved for 60 min, and examined by fluorescence microscopy.
Note that the level of punctate structures is reduced in Vps and beclin-1—reduced cells; this is analyzed for three independent experiments in D error bars show standard deviation.
D, inset The levels of Vps34 and beclin-1 after siRNA treatments. Of peripheral interest for this work is that the reduction of Vps34 during treatment with siRNA for beclin-1 is reproducible. E Clone cells were starved for 60 min. Imaging was at 1 frame per 20 s, and selected frames throughout the sequence are shown.
Also see Video 2. F For a selected time interval starting approximately at 35 min after starvation , a sequence showing formation and collapse of an omegasome arrows is shown. Bars A—C and E 20 μm; F 2 μm. Single imaging of GFP-DFCP1 during starvation showing formation of DFCP1 punctate structures.
Clone cells stably expressing GFP-DFCP1 were imaged at 1 frame per 10 s in starvation medium. Triple imaging of GFP-DFCP1 green , mRFP-MAP-LC3 red , and CFP-ER blue during starvation showing formation of an autophagosomes from an omegasome arrow. Clone cells were transiently transfected with mRFP-MAP-LC3 and CFP-ER, then imaged in starvation medium at 1 frame per 10 s.
The three panels are shown separately in addition to the triple overlay. The playback rate is at 10 frames per second. Relationship of PI 3 P-containing omegasomes with the ER and autophagosomes by live imaging and in fixed cells. A Clone cells were transfected with mRFP-MAP-LC3 red and CFP-ER blue and starved for 60 min.
Imaging was performed at 1 frame per 10 s, and a selected interval within this sequence is shown, starting at 33 min after starvation. Arrows indicate the first discernible omegasome green and autophagosome red occurrences. The enlarged panels are from the two boxed areas and represent views of 1 ER, 2 DFCP1, 3 MAP-LC3, 4 ER-MAPLC3, 5 DFCP1-MAPLC3, and 6 MAPLC3-ER, in that order.
Also see Video 3. B Dynamic relation of omegasomes and the ER using TIRFM imaging. Note that omegasomes form in regions containing ER and collapse there. Also see Video 4.
C Thawed cryosection of clone cells 45 min after starvation and double-labeled for anti—GFP-DFCPI arrowheads, 10 nm gold and anti-PDI arrows, 5 nm gold. Note the putative autophagic-like vacuoles asterisks labeled for DFCPI-GFP adjacent to PDI-labeled ER membranes.
This is one panel of a multipanel supplemental figure Fig. D Selected frames from live imaging of GFP-DFCP1 and mRFP-MAP-LC3 during starvation, whereby an intermediate is formed in which MAP-LC3 appears to bud from the omegasome while still being outlined with a DFCP1-staining membrane.
At the bottom of each panel are line drawings of these structures using magnified photographs of the relevant frames. Also see Video 6. E Colocalization of omegasomes with a PI 3 P-binding protein applied exogenously. Clone cells were starved for 60 min, perforated using nitrocellulose, and stained with purified GST-PX domain from p40 phox.
The majority of GSP-PX domain stains early endosomes not depicted but a substantial amount of the protein also binds to DFCP1 omegasomes F and G. F and G Relationship of omegasomes to autophagy proteins and the ER.
Clone cells counterstained with antibodies to endogenous ER and endogenous MAP-LC3 or Atg5 as indicated. Selected examples from such cells in addition to the one shown where the ER was in a single layer and well resolved from cytosol, to allow evaluation of colocalizations, are shown in magnified panels 1—5.
Bars: A, B, and D 1 μm; E—G 20 μm. Double imaging of GFP-DFCP1 green and dsRed-ER red during starvation using TIRF showing omegasome formation and collapse in close association with the ER. Clone cells were transfected with dsRed-ER and imaged in starvation medium at 1 frame per 10 s.
Video showing a 3D reconstruction of GFP-DFCP1 green and mRFP-MAP-LC3 red at different planes of rotation. Images were collected from clone cells transfected with mRFC-MAP-LC3, starved, and fixed. Deconvolution was performed using Autodeblur AutoQuant and 3D reconstructions made using Volocity software Improvision.
Also see the Results section. The display is arbitrarily set at 10 frames per second. Double imaging of GFP-DFCP1 green and mRFP-MAP-LC3 red during starvation showing several examples of the last step in autophagosome formation that involves omegasome exit.
Clone cells were transfected with mRFP-MAP-LC3 and imaged in starvation medium at 1 frame per 10 s. Several panels from different Videos are shown. Autophagosome maturation after omegasome exit. Cells expressing GFP-DFCP1 and mRFP-MAP-LC3 were starved and imaged for the indicated time interval.
At 30 min after starvation, the cells were also incubated with 2 μM MDC. Note that an autophagosome emerges first from an omegasome panels labeled autophagosome formation; arrow indicates the omegasome and then begins to stain with MDC panels labeled autophagosome maturation without appearing to change its appearance or to fuse with another MDC-positive vesicle.
Triple imaging of GFP-DFCP1 green , mRFP-Vps34 red , and CFP-ER blue during starvation showing formation of an omegasome in tight association with a Vps34 particle.
Clone cells expressing mRFP-Vps34 stably were also transfected with CFP-ER and imaged in starvation medium at 1 frame per 10 s. Vps34 dynamics during omegasome formation. A Clone cells were transfected with mRFP-Vps34, and a stable population expressing both proteins was selected last three lanes.
Note that exogenous mRFP-Vps34 was comparable to endogenous Vps34 and stable during amino acid starvation. B Cells as in A were starved for 60 min, fixed, and stained for Lamp C Selected frames from live imaging of mRFP-Vps34 in cells also expressing CFP-ER.
Note that the Vps34 vesicle is in constant proximity to the ER and frequently appears to use the ER strands to move long distances arrows on the bottom. The times refer to the period after amino acid starvation, but similar types of movement are evident without starvation.
D and E Selected frames from live imaging of mRFP-Vps34, GFP-DFCP1, and CFP-ER during amino-acid starvation. D An omegasome being formed in constant and close proximity to a Vps34 particle. Bar, 1 μm. For selected frames of this video, as indicated 10—12 and 35—37 , E shows the relationship of Vps34 to DFCP1 top , Vps34 to ER middle , and all three bottom.
Also see Video 7. F An additional example from live imaging experiments showing an omegasome forming in close proximity to a Vps34 particle.
Potential role of omegasomes in autophagosome biogenesis. Our hypothesis is that during amino acid starvation, Vpscontaining vesicles interact with the ER and form PI 3 P on a membrane connected to the ER step 1, green.
This membrane domain associates with autophagosomal proteins red to create a mixed membrane domain that continues to expand but maintains its spatial separation with PI 3 P on the outside, and autophagosomal membranes inside steps 1 and 2. Note that the connection of this membrane to the ER is not fully shown, thus the question marks at the junction.
Once omegasomes reach their maximum size, the autophagosomal membranes bud inwards steps 2 and 3 , giving rise to a fully formed double-membrane autophagosome step 4. The PI 3 P outer membrane in the intermediate of step 3 may aid the fusion reaction.
At the bottom of steps 2, 3, and 4, we have drawn cut-outs of the relevant structures to indicate the geometry of the bilayer. Sign In or Create an Account. Search Dropdown Menu. header search search input Search input auto suggest.
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Article August 25 Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum In Special Collection: JCB Autophagy.
Axe , Elizabeth L. This Site. Google Scholar. Simon A. Walker , Simon A. Maria Manifava , Maria Manifava. Priya Chandra , Priya Chandra. Llewelyn Roderick , H. Llewelyn Roderick. Anja Habermann , Anja Habermann. Gareth Griffiths , Gareth Griffiths.
Nicholas T. Ktistakis Nicholas T. Author and Article Information. Maria Manifava. Priya Chandra. Anja Habermann. Gareth Griffiths. Correspondence to Nicholas T. Ktistakis: nicholas. ktistakis bbsrc. Axe and S. Walker contributed equally to this paper.
Received: March 26 Accepted: July 28 Online ISSN: The Rockefeller University Press. J Cell Biol 4 : — Article history Received:. Connected Content. How a cell puts itself on the menu. Related Self-eating from an ER-associated cup. Cite Icon Cite.
toolbar search Search Dropdown Menu. toolbar search search input Search input auto suggest. All chemicals were obtained from Sigma-Aldrich unless otherwise stated. Ano, Y. Hattori, M. Oku, H. Mukaiyama, M. Baba, Y. Ohsumi, N. Kato, and Y.
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This Autopphagy was financially supported autophagosomr the National Institutes Boost energy levels Health grant no. and by the Organic Detox Products State University Walter E. Address correspondence to bassham iastate. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors www. org is: Diane C.
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