Abstract
Autophagosome formation is governed by sequential functions of autophagy-related (ATG) proteins. Although their genetic hierarchy in terms of localization to the autophagosome formation site has been determined, their temporal relationships remain largely unknown. In this study, we comprehensively analyzed the recruitment of mammalian ATG proteins to the autophagosome formation site by live-cell imaging, and determined their temporal relationships. Although ULK1 and ATG5 are separated in the genetic hierarchy, they synchronously accumulate at pre-existing VMP1-positive punctate structures, followed by recruitment of ATG14, ZFYVE1, and WIPI1. Only a small number of ATG9 vesicles appear to be associated with these structures. Finally, LC3 and SQSTM1/p62 accumulate synchronously, while the other ATG proteins dissociate from the autophagic structures. These results suggest that autophagosome formation takes place on the VMP1-containing domain of the endoplasmic reticulum or a closely related structure, where ULK1 and ATG5 complexes are synchronously recruited.
Keywords: :
Introduction
Macroautophagy plays a major role in intracellular degradation through the delivery of cytoplasmic materials to the lysosome via the autophagosome.Citation1 Autophagosome formation is mediated by ~20 autophagy-related (ATG) proteins.Citation2-Citation4 These proteins are classified into at least six different functional groups both in yeast and mammals, and their genetic hierarchy in terms of localization to the yeast phagophore assembly site or mammalian autophagosome formation site has been determined.Citation5-Citation8 In mammalian cells, the first of the six groups, the UNC-51-like kinase 1 (ULK1) complex [containing ULK1, RB1CC1 (also known as FIP200), ATG13, and C12orf44/ATG101], and the second, transmembrane protein ATG9A, are independently recruited to the autophagosome formation site, and both are required for further recruitment of the third group, class III phosphatidylinositol 3-kinase (PtdIns3K) complex (containing BECN1, ATG14, PIK3C3/VPS34, and PIK3R4/VPS15). This class III PtdIns3K complex is essential for the recruitment of phosphatidylinositol (3)-phosphate (PtdIns3P)-binding proteins such as the fourth group, WD-repeat protein-interacting phosphoinositide (WIPI) proteins and zinc finger FYVE domain-containing protein 1 (ZFYVE1, also known as DFCP1), and the fifth group, the ATG12–ATG5-ATG16L1 complex (– indicates a covalent attachment). The sixth and last group, phosphatidylethanolamine (PE)-conjugated microtubule-associated protein 1 light chain 3 (LC3), is recruited in an ATG12–ATG5-dependent manner. Additionally, vacuole membrane protein 1 (VMP1), which is specific to higher eukaryotes, is essential for autophagosome formation, but recruitment of VMP1 and that of other ATG proteins are not interdependent.Citation6,Citation9
Although the genetic hierarchy indicates functional interdependency among the ATG proteins, it does not necessarily indicate the temporal order of recruitment of these proteins to the autophagosome formation site. To better understand the complicated recruitment mechanism of the ATG proteins, it is essential to determine the temporal relationships among them during autophagosome formation; to date, this has been only partially investigated.Citation10-Citation12 In this study, the recruitment of mammalian ATG proteins to the autophagosome formation site was comprehensively analyzed in live cells. The findings revealed that the ULK1 and ATG5 complexes were synchronously recruited, although they hold different places in the genetic hierarchy. Moreover, these complexes were recruited to the pre-existing VMP1-positive structure, which is potentially the autophagosome formation site.
Results
Validation of the method for monitoring the temporal relationship among ATG proteins in targeting to the autophagic structures
To determine the temporal order of the accumulation of mammalian ATG proteins at the autophagosome formation site, we performed live-cell imaging of fluorescent protein-tagged ATG proteins. We chose ULK1, ATG14, WIPI1, ZFYVE1, ATG5, LC3, ATG9A, and VMP1 as representatives of each functional group. In addition, we included SQSTM1/p62 as a typical selective substrate.Citation13 While ATG9A and VMP1 constitutively form punctate structures, puncta formation of the other ATG proteins is induced by starvation.Citation6-Citation8 We found that ULK1, ATG5, ATG14, ZFYVE1, and WIPI1 accumulated at and dissociated from the autophagosome formation sites within 10 min ( and ). To determine temporal relationship among ATG proteins, the difference of peak time of each fluorescence signal between pairs of two ATG proteins was evaluated.
Figure 1. Evaluation of the experimental error of the time-course measurement of ATG protein accumulation into punctate structures. MEFs stably coexpressing CFP–ATG5 and Venus–ATG5 (A), CFP-ATG16L1 and Venus–ATG5 (B), CFP–ATG5 and Venus–ZFYVE1 (C), or CFP–ZFYVE1 and Venus–ATG5 (D) were cultured in starvation medium. Time-lapse imaging was performed at 1 frame per 10 s, and selected images (0.5 min interval) are shown. The time course of the fluorescence intensity of the punctate signals shown in the images (left) was plotted in the graphs (right; percent maximum intensity). Dashed-line boxes indicate the video frames at the maximum (peak) intensities. Scale bar: 5 μm.
Figure 2. Time-course analysis of accumulation and disappearance of ATG proteins. MEFs stably coexpressing CFP–ATG5 and Venus–ULK1 (A), CFP–ATG5 and Venus–ATG14 (B), CFP–WIPI1 and Venus–ATG5 (C), CFP–WIPI1 and Venus–ZFYVE1 (D), CFP–LC3 and Venus–DCFP1 (E), or CFP–SQSTM1 and Venus–LC3 (F) were cultured in starvation medium. Time-lapse imaging was performed and analyzed as in . Selected images (1.0 min interval) are shown. Dashed-line boxes indicate the video frames at the maximum (peak) intensities. Scale bar: 5 μm.
We first measured an artificial temporal difference caused by using two different fluorescent proteins, superenhanced cyan fluorescent protein (CFP) and Venus. CFP–ATG5 and Venus–ATG5 appeared to accumulate at and dissociate from punctate structures in a similar manner in starved mouse embryonic fibroblasts (MEFs) ( and , a; Vid. S1). The mean and median time lags between the peak of the CFP–ATG5 and Venus–ATG5 intensities were 13 and 10 s, respectively.
Figure 3. Lag of the peak time of accumulation between ATG proteins. (A) Average fluorescence intensity profiles of all data sets are plotted. The number of samples is indicated in each panel. (B) Histograms show the distribution for the time lag of the maximum peaks of fluorescence intensity profiles between the indicated pairs of CFP- and Venus-fused ATG proteins in (A). Black horizontal, and blue and red vertical bars indicate the standard deviation (sd), mean, and median, respectively. P values were obtained using the unpaired Student t-test between indicated pairs of proteins.
Next, we validated this method by using ATG5 and ATG16L1, both of which are included in the ATG12–ATG5-ATG16L1 complex. The behavior of CFP–ATG16L1 and Venus–ATG5 was quite similar ( and , b), and the mean and median time lags between the peak of the CFP–ATG16L1 and Venus–ATG5 intensities were 14 and 10 s, respectively (, b).
We further validated this method by using two different proteins, ATG5 and ZFYVE1. The peak time of accumulation of CFP–ATG5 was approximately 30 s earlier than that of Venus-ZFYVE1 (; Vid. S2). In cells expressing ATG5 and ZFYVE1 with the opposite tags (CFP–ZFYVE1 and Venus–ATG5), the time lag between their accumulation peaks was very similar (; Vid. S2). The average time-course profiles showed that accumulation of ATG5 was earlier than that of ZFYVE1 for both combinations (, e and f). The peak time lag between CFP-ATG5 and Venus-ZFYVE1 (, e) and that between Venus-ATG5 and CFP-ZFYVE1 (, f) was similar and there was no significant difference between these pairs (P = 0.47). From these results, the measurement error in this study caused by the difference between CFP and Venus was estimated to be approximately 10 to 15 s.
ULK1 and ATG5 synchronously accumulate at and dissociate from the autophagosome formation sites
We used this method for a systematic analysis of the temporal relationship between ATG proteins in targeting to the autophagosome formation site. First, we analyzed the relationship between Venus–ULK1 and CFP–ATG5. The fluorescence signals of Venus–ULK1 puncta gradually appeared and then disappeared within a few minutes (). Although ATG5 is placed downstream of ULK1 in the genetic hierarchy,Citation6 the CFP–ATG5 and Venus–ULK1 signals accumulated, reached a peak and disappeared almost synchronously (; Vid. S3). The fluorescence intensity profiles of 15 examples showed that the patterns of the averaged profiles of CFP–ATG5 and Venus–ULK1 were also very similar (, c). The mean and median time lags between the peak of CFP–ATG5 and Venus–ULK1 were 21 and 10 s, respectively (, c); these values were close to the range of measurement error, as mentioned above. Indeed, statistical analysis demonstrated no significant difference in the peak time lag between the pair of CFP–ATG5 and Venus–ATG5 and that of CFP–ATG5 and Venus–ULK1 (P = 0.35). These results indicate that ULK1 and ATG5 are recruited to the autophagosome formation sites in a similar manner, suggesting that they are included in the same complex or structure.
ATG14 is recruited to the autophagosome formation sites in a manner different from that of the ATG5 and ULK1 complexes
Live-cell imaging of MEFs expressing CFP–ATG5 and Venus–ATG14 showed that accumulation and disappearance of ATG14 were slightly delayed compared with ATG5 ( and , d; Vid. S4). The mean and median time lags between the peak of CFP–ATG5 and Venus–ATG14 were about 41 and 30 s, respectively (, d). These data suggest that the majority of ATG14 was recruited to the autophagosome formation sites slightly later than ULK1 and ATG5, and that the mechanism of recruitment of ATG14 is different from that of ULK1 and ATG5.
PtdIns3K is important for stable accumulation of the ULK1 and ATG5
The synchronous accumulation of ULK1 and ATG5, which is followed by ATG14 is rather contradictory to our previous observation that the ATG14 complex is genetically positioned between the ULK1 and ATG5 complexes.Citation6 We, therefore, reassessed the sensitivity of the ULK1 and ATG5 punctate structures to wortmannin, a PtdIns3K inhibitor. Consistent with previous observation, when starved cells were treated with 0.2 μM wortmannin, almost all preformed CFP–ATG5 puncta disappeared. However, we observed that 67% of preformed ULK1 puncta also disappeared, although 27% of the Venus–ULK1 puncta remained (; Vid. S5). Moreover, formation of new Venus–ULK1 and CFP–ATG5 puncta was severely inhibited; only a very small number of new ULK1 puncta, which were negative for ATG5, were generated. These data suggest that both ULK1 and ATG5 can be stabilized by PtdIns3P at an early phase, and the ULK1 complex but not the ATG5 complex may become PtdIns3P-independent at a later phase of autophagosome formation.
Figure 4. PtdIns3K is important for stable accumulation of ULK1 and ATG5. MEFs stably coexpressing CFP–ATG5 and Venus-ULK1 were cultured in starvation medium for 30 min (upper panels) and then 0.2 μM wortmannin (WM) was added (lower panels). The ULK1+ ATG5+ punctate structures were tracked for 5 min after the addition of wortmannin by time-lapse microscopy and were classified into the four groups: ULK1− ATG5− (arrows), ULK1+ ATG5− (arrowheads), ULK1− ATG5+ and ULK1+ ATG5+. Relative population of each group was shown in the table (right). Scale bar: 10 μm.
The PtdIns3P-binding proteins ZFYVE1 and WIPI1 are synchronously recruited to the omegasome and autophagosome formation site after ATG5 translocation
The ATG14-containing PtdIns3K generates PtdIns3P and recruits the PtdIns3P-binding proteins ZFYVE1 and WIPI1. As shown above, ZFYVE1 was accumulated about 40 to 60 s after ATG5 (; , e and f). Similarly, the CFP–WIPI1 signals also reached peak intensity slightly after those of Venus–ATG5 (; , g; Vid. S6). These results suggest similar temporal behavior of WIPI1 and ZFYVE1. Therefore, we directly compared the kinetics of CFP–WIPI1 and Venus–ZFYVE1 puncta, and confirmed that CFP–WIPI1 and Venus–ZFYVE1 accumulated and disappeared in a similar fashion, even though their structures were not spatially the same (; , h; Vid. S7). Venus–ZFYVE1 signals, which represent endoplasmic reticulum (ER)-derived omegasomes,Citation11 often surrounded the CFP–WIPI1 signals as previously observed.Citation6 These results suggest that WIPI1 and ZFYVE1 are recruited synchronously to the autophagosome formation site and the omegasome, respectively, after ULK1 and ATG5.
SQSTM1 is recruited with LC3
In contrast to the above-mentioned ATG proteins, the LC3 signals appeared and reached a plateau rather than a peak because LC3 remains on completed autophagosomes.Citation10,Citation14 Formation of ATG5 puncta and ZFYVE1-positive omegasomes is known to be followed by the recruitment of LC3.Citation10-Citation12 We confirmed that the CFP–LC3 fluorescence intensity reached a plateau about 2 min after the peak of Venus–ZFYVE1 fluorescence intensity ().
Next we determined the temporal relationship between LC3 and the selective autophagy substrate SQSTM1. Live imaging of LC3 and SQSTM1 showed that they accumulated synchronously, suggesting that SQSTM1 molecules are incorporated into the autophagosome together with LC3 during membrane elongation (; Vid. S8). This is consistent with the proposal that SQSTM1 is incorporated into the autophagosome via a direct association with LC3.Citation15-Citation17 Although SQSTM1 has the ability to directly localize to the autophagosome formation site in an LC3-independent manner,Citation18 this pathway does not seem to be major in autophagy-competent cells.
The ATG9A structure does not accumulate and is transiently recruited to the autophagosome formation sites
ATG9A is one of the most upstream ATG proteins in the genetic hierarchy and can localize to the autophagosome formation site independently of the ULK1 complex.Citation7,Citation8 ATG9A is a transmembrane protein and is present on vesicle structures as well as on the trans-Golgi network and endosomes.Citation19 Indeed, we detected a large number of small ATG9A puncta constitutively (). To track these ATG9A puncta, movies were taken at 160 ms/frame, which was more than 60 times higher than the speed required for the observation of other ATG proteins. We detected very faint mRFP–ATG9A signals around the GFP–ULK1 structures (; Vid. S9). The GFP-ULK1 and mRFP–ATG9A puncta moved together for a short period but mRFP–ATG9A signals did not further accumulate. These data suggest that only a small number of rapidly moving ATG9A vesicles are recruited to the autophagosome formation site. Recently, Orsi et al. have reported the dynamic and transient interaction of mammalian ATG9 with autophagosomes by live-cell imaging,Citation8 which is consistent with our observations.
Figure 5. ATG9 vesicles are transiently recruited to the autophagosome formation site. (A) Structured illumination microscopy (SIM) imaging of MEFs stably expressing GFP–ATG9A cultured in starvation medium. Scale bar: 10 μm. (B) Time-lapse images of GFP–ULK1 and mRFP–ATG9A in starved MEFs were simultaneously produced at a rate of 160 ms/frame by conventional fluorescence microscopy using a fluorescence-split system. Selected images (1.6 s interval) are shown. Arrowheads indicate mRFP–ATG9A structures that are associated with a GFP–ULK1 punctate structure. Scale bar: 2 μm.
ATG proteins are recruited to VMP1-positive structures
As we reported previously,Citation6 VMP1–GFP constitutively localizes to the ER network and Golgi apparatus as well as to punctate structures (). Previously, using fixed cells, we showed that colocalization between ULK1 and VMP1 was very limited in starved cells and was only apparent in wortmannin-treated cells, in which autophagosome formation was blocked at a PtdIns3K-dependent step.Citation6 Thus, we thought that VMP1 only transiently associated with the autophagosome formation site. However, live imaging showed that approximately 70% of the Venus–ULK1 puncta emerged on the pre-existing VMP1–CFP structures. The Venus–ULK1 and VMP1–CFP puncta moved together for a few minutes. Finally, the Venus–ULK1 signals disappeared, whereas VMP1–CFP puncta remained (; Vid. S10). In our previous study, weak VMP1–GFP signals might have been undetected or destroyed during the fixation process if they were not accumulated by wortmannin treatment.Citation6 These results suggest that ULK1 and other ATG proteins are recruited to the pre-existing VMP1-positive region of the ER or a closely related structure, which is likely to represent the autophagosome formation site in mammalian cells.
Figure 6. ULK1 is recruited to pre-existing VMP1-positive punctate structures. (A) SIM imaging of MEFs stably expressing VMP1–GFP cultured in normal medium. Scale bar: 10 μm. (B) Time-lapse imaging of VMP1–CFP and Venus–ULK1 in starved MEFs was performed at 1 frame per 10 s, and selected images (0.5 min interval) are shown. Arrows indicate VMP1–CFP and Venus–ULK1 puncta, which colocalize with each other for certain times. Scale bar: 5 μm.
Discussion
We determined the temporal-relationships among mammalian ATG proteins in recruitment to the autophagosome formation site by comprehensive live imaging. In this study, we compared the timing of accumulation peaks among mammalian ATG proteins. Because the peak time indicates the time when the dissociation rate becomes greater than the association rate, the peak time order may not necessarily represent the order in which they function. Actual timing that each protein plays a substantial role may be prior to the peak timing if recruitment of only a small number of ATG molecules is sufficient to fulfill its function. The temporal relationship rather represents the recruitment/dissociation mechanisms of each complex. For example, if two factors show a similar pattern, it suggests that they are part of the same complex or share the same targeting mechanism. Therefore, these temporal analyses do not necessarily indicate the time order of function.
Our temporal analysis highlighted two significant differences compared with genetic analyses ().Citation6 First, the recruitment and dissociation of ATG5 occurred together with ULK1, which is earlier than those of the majority of ATG14, although the ATG5 complex is genetically placed downstream of the ULK1 and ATG14 complexes.Citation6 This is consistent with the recent findings that the ULK1 and ATG5 complexes interact with each other via a direct binding between RB1CC1 and ATG16L1.Citation20,Citation21 This interaction is independent of PtdIns3K.Citation21 It is puzzling why localization of ATG5 and ATG16L1 to the phagophore appears to be dependent on the PtdIns3K activity.Citation6,Citation10 We reassessed this issue and found that not only ATG5 and ATG16L1 puncta, but also many of the ULK1 puncta, are sensitive to wortmannin (). This is consistent with a recent observation by Nicholas Ktistakis’ lab (personal communication). Whereas almost all ATG5 and ATG16L1 puncta disappeared following wortmannin treatment, approximately one fourth of the ULK1 puncta remained. This could explain the previous hierarchical model. However, our data suggest that ULK1 is not simply upstream of PtdIns3K. We hypothesize that translocation of the ULK1 and ATG12–ATG5-ATG16L1 complexes to the autophagosome formation site could be independent of PtdIns3P, but PtdIns3P is required for stable accumulation of these complexes. In this case, a small number of the ATG14 complex may be sufficient to produce enough amount of PtdIns3P at the autophagosome formation site even before the amount of ATG14 reaches the peak level. At a later step, the ULK1 complex, but not the ATG5 complex, would become independent of PtdIns3P.
Figure 7. Schematic models of the genetic hierarchy and temporal relationship among mammalian ATG proteins. Genetic hierarchy of mammalian ATG proteins based on previous reportsCitation6-Citation8 (left) and their temporal relationship revealed by the current study (right) are shown.
Second, previous studies show that the silencing of VMP1 causes accumulation of almost all ATG proteins at the autophagosome formation site, suggesting that VMP1 functions at a late step of autophagosome formation.Citation6,Citation9 However, the present live-imaging study showed that VMP1 could be the most “temporally” upstream factor among currently known autophagy-related proteins, suggesting that it could be a resident protein at the autophagosome formation site. We do not know whether all the VMP1-positive structures are involved in autophagosome formation or some of them are specialized for it. The VMP1-positive structure or domain should be further characterized structurally and biochemically.
Our results also showed that the timing of dissociation from the phagophore and autophagosome varies among ATG proteins. For example, ATG5 dissociates faster than ATG14 and WIPI1 ( and ). This suggests that dissociation of ATG proteins is not regulated by a single mechanism. One possible mechanism of the dissociation is dephosphorylation of PtdIns3P by PtdIns3-phosphatases such as MTMR14/Jumpy and MTMR3.Citation22,Citation23 However, as recruitment of upstream factors such as ULK1 and ATG14 is independent of PtdIns3P,Citation6 several different mechanisms would regulate dissociation of ATG proteins.
Materials and Methods
Plasmids
cDNAs encoding proteins for rat LC3B, mouse ATG5, human ATG9A, human ATG14, mouse ZFYVE1, human WIPI1, mouse ULK1, human VMP1, mouse SQSTM1, and human ATG16L1 were obtained as previously reported.Citation6,Citation7,Citation21 Full-length cDNAs encoding these ATG proteins were subcloned into pMRX-IP (provided by Shoji Yamaoka, Tokyo Medical and Dental University) together with super-enhanced cyan fluorescent protein (CFP) or Venus (provided by Atsushi Miyawaki, Riken). cDNA encoding ATG9A was subcloned into pMXs-puro (provided by Toshio Kitamura, University of Tokyo) with mRFP (provided by RY Tsien, University of California, San Diego).Citation24 pMXs-IP-GFP–ULK1 has been described previously.Citation25
Cell culture and transfection
Mouse embryonic fibroblasts (MEFs) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, D6546) supplemented with 10% fetal bovine serum, 50 μg/ml penicillin and streptomycin (GIBCO, 1570) in a 5% CO2 incubator (regular medium). For starvation treatment, cells were washed with phosphate-buffered saline and incubated in amino acid (AA)-free DMEM without serum (starvation medium) (GIBCO, custom-made). FuGENE 6 reagent (Roche Applied Science, 1814443) was used for transfection. Wortmannin (W1628) and puromycin (P8333) were purchased from Sigma-Aldrich.
Retroviral infection and generation of stable cell lines
Cells stably expressing fluorescence protein-tagged ATG proteins were generated using a retroviral expression system as previously described.Citation25 Briefly, Plat E cells (provided by Toshio Kitamura) were transiently transfected with the pMXs vectors using FuGENE 6 reagent. After 72 h of culture, the growth medium containing retrovirus was collected. MEFs were incubated with the collected virus-containing medium with 8 μg/ml hexadimethrine bromide (Sigma-Aldrich, H9268) for 24 h. Uninfected cells were removed by puromycin selection.
Live-cell imaging
Live-cell fluorescence imaging was performed with an inverted microscope (Olympus, IX81-ZDC), a 60× PlanAPO oil-immersion objective lens (Olympus, NA 1.42) and a cooled CCD camera (Photometrics, CoolSNAP HQ2). MEFs stably expressing GFP-variant-fused ATG proteins were placed on a glass bottom dish (IWAKI, 11-004-008) 2 d before observation. During live-cell imaging, the culture dish was mounted in a chamber (TOKAI HIT, INUB-ONI-F2) to maintain the culture conditions (37 °C and 5% CO2). GFP-variant proteins were illuminated with a 100 W mercury arc lamp attenuated to 3~12% by neutral-density filters, and two-color time-lapse images were acquired with appropriate exposure times at 5 or 10 s intervals. For high-speed observation of GFP–ULK1 and mRFP–ATG9, in order to obtain two-color images simultaneously, a dual-band pass excitation filter (Chroma, 59004x), dichroic beam splitter (Chroma, 59004bs) and a fluorescence-split system (Olympus, U-SIP) were used instead of filter wheels. The external devices (shutters, filter wheels, X–Y stage and camera) were controlled by MetaMorph (ver 7.0, Molecular Devices Japan).
Measurement of fluorescence intensities of punctate structures
Time series of 16-bit TIFF images were converted into 8-bit AVI images using MetaMorph. The signal intensity for each punctate structure, of which fluorescence intensity was found to increase, was measured frame by frame in a 1.6 × 1.6-μm area (5 × 5 pixels on the image), and the background intensity of an adjacent 1.6 × 1.6-μm area was always subtracted. All graph creations and unpaired t-tests between the histograms were performed using OriginPro (ver.8, Microcal Software).
Analysis of temporal relationships for ATG-protein accumulation
All time-course data were analyzed using OriginPro. Time-course curves for accumulation of two different ATG proteins labeled with either CFP or Venus into the same punctate structure were obtained and compared as follows. (1) Sliding window smoothing over six time points was conducted for each time-course fluorescence intensity plot. (2) The fluorescence intensity (arbitrary unit) was normalized to between 0% and 100% by setting the maximum value to 100%. (3) The time-course profiles of CFP and Venus fluorescence intensities were overlaid onto the same graph ( and ).
Next, the time when the fluorescence intensity reached a peak was statistically analyzed as follows. (1) The time of peak fluorescence intensity was determined for the individual time-course profile, and the time lag between the peak times for CFP and Venus was obtained. (2) These time-lag data were assembled for all observations (). (3) Unpaired t-tests were used to determine differences between the data for different ATG protein pairs.
To obtain the average time-course accumulation curves (shown in ), time-course profiles for different observations were processed as follows. (1) Linear interpolation was used to add an extra point midway between two points. (2) The pair of CFP and Venus profiles for each observation was horizontally shifted so that the intermediate value between their peak time points was set to 0. (3) All profiles for different observations were overlaid, and the mean value for CFP and Venus at each time point was calculated; this resulted in the average time-course accumulation curves for two different ATG proteins, labeled with either CFP or Venus.
Structured illumination microscopy (SIM) imaging
MEFs stably expressing fluorescent protein-fused ATG proteins were placed on number 1.5 coverslips (Carl Zeiss Microscopy GmbH) 2 d before fixation. Cells were treated with starvation medium for 1 h and fixed with 4% paraformaldehyde for 10 min at room temperature. SIM was performed with ELYRA S1 (Carl Zeiss Microscopy GmbH) and a 63× PlanAPO oil-immersion objective lens (NA 1.40, Carl Zeiss). Images were processed with ZEN 2010 software (Carl Zeiss Microscopy GmbH).
| Abbreviations: | ||
| ATG | = | autophagy-related |
| BECN1 | = | Beclin 1, autophagy-related |
| CCD | = | charge coupled device |
| CFP | = | cyan fluorescent protein |
| C12orf44 | = | chromosome 12 open reading frame 44 |
| DFCP1 | = | double FYVE-containing protein 1 |
| DMEM | = | Dulbecco’s modified Eagle’s medium |
| ER | = | endoplasmic reticulum |
| FIP200 | = | FAK family kinase-interacting protein 200 kDa |
| GFP | = | green fluorescent protein |
| LC3 | = | microtubule-associated protein 1 light chain 3 |
| MEF | = | mouse embryonic fibroblast |
| MTMR | = | myotubularin-related protein |
| mRFP | = | monomeric red fluorescent protein |
| PE | = | phosphatidylethanolamine |
| PIK3C3 | = | phosphatidylinositol 3-kinase, catalytic subunit type 3 |
| PIK3R4 | = | phosphatidylinositol 3-kinase, regulatory subunit 4 |
| PtdIns 3-kinase | = | phosphatidylinositol 3-kinase |
| RB1CC1 | = | RB1-inducible coiled-coil 1 |
| SIM | = | structured illumination microscopy |
| SQSTM1 | = | sequestosome 1 |
| ULK1 | = | unc-51-like kinase 1 (C. elegans) |
| VMP1 | = | vacuole membrane protein 1 |
| VPS | = | vascular protein sorting |
| WIPI | = | WD repeat domain, phosphoinositide interacting |
| WM | = | wortmannin |
| ZFYVE1 | = | zinc finger, FYVE domain containing 1 |
Additional material
Download Zip (5.6 MB)Acknowledgments
We thank Toshio Kitamura and Shoji Yamaoka for the retroviral vectors and Plat-E cells, Atsushi Miyawaki for the plasmid encoding superenhanced CFP and Venus, and Roger Y Tsien for the cDNA coding for mRFP. We also thank Akihiko Kusumi for help with the particle tracking assay. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, by the Funding Program for Next Generation World-Leading Researchers (to NM) and by research fellowships from the Japan Society for the Promotion of Science for Young Scientists (to IK-H and EI).
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Supplemental Materials
Supplemental materials may be found here: www.landesbioscience.com/journals/autophagy/article/25529
References
- Tooze SA, Yoshimori T. The origin of the autophagosomal membrane. Nat Cell Biol 2010; 12:831 - 5; http://dx.doi.org/10.1038/ncb0910-831; PMID: 20811355
- Xie Z, Klionsky DJ. Autophagosome formation: core machinery and adaptations. Nat Cell Biol 2007; 9:1102 - 9; http://dx.doi.org/10.1038/ncb1007-1102; PMID: 17909521
- Nakatogawa H, Suzuki K, Kamada Y, Ohsumi Y. Dynamics and diversity in autophagy mechanisms: lessons from yeast. Nat Rev Mol Cell Biol 2009; 10:458 - 67; http://dx.doi.org/10.1038/nrm2708; PMID: 19491929
- Mizushima N, Yoshimori T, Ohsumi Y. The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol 2011; 27:107 - 32; http://dx.doi.org/10.1146/annurev-cellbio-092910-154005; PMID: 21801009
- Suzuki K, Kubota Y, Sekito T, Ohsumi Y. Hierarchy of Atg proteins in pre-autophagosomal structure organization. Genes Cells 2007; 12:209 - 18; http://dx.doi.org/10.1111/j.1365-2443.2007.01050.x; PMID: 17295840
- Itakura E, Mizushima N. Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins. Autophagy 2010; 6:764 - 76; http://dx.doi.org/10.4161/auto.6.6.12709; PMID: 20639694
- Itakura E, Kishi-Itakura C, Koyama-Honda I, Mizushima N. Structures containing Atg9A and the ULK1 complex independently target depolarized mitochondria at initial stages of Parkin-mediated mitophagy. J Cell Sci 2012; 125:1488 - 99; http://dx.doi.org/10.1242/jcs.094110; PMID: 22275429
- Orsi A, Razi M, Dooley HC, Robinson D, Weston AE, Collinson LM, et al. Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy. Mol Biol Cell 2012; 23:1860 - 73; http://dx.doi.org/10.1091/mbc.E11-09-0746; PMID: 22456507
- Tian Y, Li Z, Hu W, Ren H, Tian E, Zhao Y, et al. C. elegans screen identifies autophagy genes specific to multicellular organisms. Cell 2010; 141:1042 - 55; http://dx.doi.org/10.1016/j.cell.2010.04.034; PMID: 20550938
- Mizushima N, Yamamoto A, Hatano M, Kobayashi Y, Kabeya Y, Suzuki K, et al. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J Cell Biol 2001; 152:657 - 68; http://dx.doi.org/10.1083/jcb.152.4.657; PMID: 11266458
- Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, Habermann A, et al. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J Cell Biol 2008; 182:685 - 701; http://dx.doi.org/10.1083/jcb.200803137; PMID: 18725538
- Hailey DW, Rambold AS, Satpute-Krishnan P, Mitra K, Sougrat R, Kim PK, et al. Mitochondria supply membranes for autophagosome biogenesis during starvation. Cell 2010; 141:656 - 67; http://dx.doi.org/10.1016/j.cell.2010.04.009; PMID: 20478256
- Johansen T, Lamark T. Selective autophagy mediated by autophagic adapter proteins. Autophagy 2011; 7:279 - 96; http://dx.doi.org/10.4161/auto.7.3.14487; PMID: 21189453
- Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 2000; 19:5720 - 8; http://dx.doi.org/10.1093/emboj/19.21.5720; PMID: 11060023
- Bjørkøy G, Lamark T, Brech A, Outzen H, Perander M, Øvervatn A, et al. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J Cell Biol 2005; 171:603 - 14; http://dx.doi.org/10.1083/jcb.200507002; PMID: 16286508
- Komatsu M, Waguri S, Koike M, Sou YS, Ueno T, Hara T, et al. Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. Cell 2007; 131:1149 - 63; http://dx.doi.org/10.1016/j.cell.2007.10.035; PMID: 18083104
- Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen H, et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem 2007; 282:24131 - 45; http://dx.doi.org/10.1074/jbc.M702824200; PMID: 17580304
- Itakura E, Mizushima N. p62 Targeting to the autophagosome formation site requires self-oligomerization but not LC3 binding. J Cell Biol 2011; 192:17 - 27; http://dx.doi.org/10.1083/jcb.201009067; PMID: 21220506
- Young ARJ, Chan EYW, Hu XW, Köchl R, Crawshaw SG, High S, et al. Starvation and ULK1-dependent cycling of mammalian Atg9 between the TGN and endosomes. J Cell Sci 2006; 119:3888 - 900; http://dx.doi.org/10.1242/jcs.03172; PMID: 16940348
- Gammoh N, Florey O, Overholtzer M, Jiang X. Interaction between FIP200 and ATG16L1 distinguishes ULK1 complex-dependent and -independent autophagy. Nat Struct Mol Biol 2013; 20:144 - 9; http://dx.doi.org/10.1038/nsmb.2475; PMID: 23262492
- Nishimura T, Kaizuka T, Cadwell K, Sahani MH, Saitoh T, Akira S, et al. FIP200 regulates targeting of Atg16L1 to the isolation membrane. EMBO Rep 2013; 14:284 - 91; http://dx.doi.org/10.1038/embor.2013.6; PMID: 23392225
- Vergne I, Roberts E, Elmaoued RA, Tosch V, Delgado MA, Proikas-Cezanne T, et al. Control of autophagy initiation by phosphoinositide 3-phosphatase Jumpy. EMBO J 2009; 28:2244 - 58; http://dx.doi.org/10.1038/emboj.2009.159; PMID: 19590496
- Taguchi-Atarashi N, Hamasaki M, Matsunaga K, Omori H, Ktistakis NT, Yoshimori T, et al. Modulation of local PtdIns3P levels by the PI phosphatase MTMR3 regulates constitutive autophagy. Traffic 2010; 11:468 - 78; http://dx.doi.org/10.1111/j.1600-0854.2010.01034.x; PMID: 20059746
- Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, et al. A monomeric red fluorescent protein. Proc Natl Acad Sci U S A 2002; 99:7877 - 82; http://dx.doi.org/10.1073/pnas.082243699; PMID: 12060735
- Hara T, Takamura A, Kishi C, Iemura S, Natsume T, Guan JL, et al. FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells. J Cell Biol 2008; 181:497 - 510; http://dx.doi.org/10.1083/jcb.200712064; PMID: 18443221