Correspondence to Terje Johansen: terjej{at}fagmed.uit.no
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Introduction |
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The p62 protein level increases after oxygen radical stress. Both mRNA and protein levels increase, suggesting an induced transcription of the gene (Ishii et al., 1997). The transcription factor Nrf2 is activated after oxidative stress, and induction of p62 is severely inhibited in cells from Nrf2 knockout mice (Ishii et al., 2000). Inhibition of proteasomal activity also causes induction of p62 (Ishii et al., 1997; Kuusisto et al., 2001b; Thompson et al., 2003). Interestingly, p62 was recently identified as a protein that is induced as a response to the expression of mutant huntingtin (Nagaoka et al., 2004). Huntington's disease is a late onset progressive autosomal dominant neurodegenerative disorder caused by the expression of mutant forms of the huntingtin (Htt) protein containing a polyglutamine expansion encoded by CAG repeats in exon 1 of the huntingtin gene (Vonsattel and DiFiglia, 1998). The disease causes selective neuronal cell death in the striatum. Cells expressing the mutant form of huntingtin display both diffuse and aggregated localization of the protein. The mutant protein has cytotoxic properties, and aggregation seems to be a mechanism for cell survival (Arrasate et al., 2004). Protein inclusions formed by aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by macroautophagy (hereafter referred to as autophagy; Kegel et al., 2000; Ravikumar et al., 2002, 2004), which is a bulk degradation pathway in which a double or multimembrane-bound structure called the autophagosome forms to sequester cytoplasm. Subsequently, the autophagosome fuses with the lysosome, and its content and internal membranes are degraded as it recycles the macromolecules (Levine and Klionsky, 2004; Yoshimori, 2004). Most long-lived proteins and some organelles are degraded by autophagy, and autophagy, in addition to cellular homeostasis, has also been implicated in cellular differentiation, tissue remodelling, growth control, bacterial and viral infections, cell defense, adaptation to adverse environments, neurodegenerative diseases, cardiomyopathies, apoptosis, and cancer (Cuervo, 2004). Among the autophagosomal marker proteins are Atg8 in yeast and light chain 3 (LC3) in mammals (Kabeya et al., 2000). After synthesis, LC3 is cleaved at its COOH terminus to produce the cytosolic LC3-I form. LC3-I is converted to LC3-II, which is tightly associated with the autophagosomal membrane probably via conjugation to phosphatidylethanolamine (Kabeya et al., 2000, 2004).
In this study, we report that the polyubiquitin-binding and homopolymerizing p62 protein may, via LC3, be involved in linking polyubiquitinated protein aggregates to the autophagic machinery, facilitating the clearance of such aggregates and, thereby, contributing to reduced toxicity of mutant huntingtin expression.
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Results |
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Both the PB1 and UBA domains are needed for p62 to form cytoplasmic bodies
To map the domains of p62 involved in the formation of cytoplasmic bodies, different GFP-p62 cDNA constructs were transiently transfected into NIH3T3 cells, and the fusion proteins were analyzed by confocal fluorescence microscopy (Fig. 2 A). NIH3T3 cells were used because they have a low level of endogenous p62, but similar data were obtained with HeLa cells (not depicted). All deletion constructs lacking the PB1 domain (i.e., p62(124440), p62(256440), and p62(385440)) have a completely diffuse distribution in NIH3T3 cells (Fig. 2 A and not depicted). This is also the case with the D69A mutant, which abrogates PB1 domainmediated polymerization of p62 (Fig. 2 A; Lamark et al., 2003). This strongly indicates that the PB1 domainmediated polymerization of p62 is essential for the formation of cytoplasmic bodies.
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In vivo, the length of p62 polymers may be reduced as a result of the binding of p62 to other PB1 domaininteracting partners. In line with this, overexpression of p62 D69A (Fig. 2 B) or p62 R21A (not depicted) counteracted the formation of GFP-p62 bodies in SGFP-p62 cells. These PB1 domain mutants presumably compete with wild-type p62 for binding to a growing chain of p62 molecules acting as chain terminators. However, overexpression of the p62UBA construct myc-p62(1385) also prevented the formation of cytoplasmic dots in SGFP-p62 cells (Fig. 2 B). In contrast to PB1 mutants, deletion of the UBA domain had no effect on the ability of p62 to interact with itself or to form polymers in vitro (unpublished data). Only the PB1 domain is needed for polymerization of p62 in vitro. Therefore, the role of the UBA domain may be to cross-link p62 polymers, presumably by interacting with polyubiquitinated proteins.
p62 bodies are found both as membrane-free protein aggregates (sequestosomes) and as membrane-confined autophagosomal and lysosomal structures
To characterize the two populations of p62 bodies, we performed a series of colocalization experiments in both fixed and live cells. We looked at both endogenous p62 and ectopically expressed GFP-p62 or epitope-tagged p62. There was no colocalization of early endosomal markers with p62 bodies. Early endosome antigen 1 (EEA1) did not colocalize with endogenous p62 or ectopically expressed GFP-p62 (Fig. 3 A). In line with this, there was no colocalization between p62 and EGF receptor (fixed cells) or fluorescent-labeled EGF (live cells) at time points up to 15 min of internalization (unpublished data). However, at 30 and 60 min, there was a minor fraction of the smaller p62 bodies that colocalized with EGF receptor or its ligand (unpublished data). Moreover, there was no association of GFP-p62 with the recycling compartment that was visualized by internalization of fluorescently labeled transferrin (unpublished data). Thus, these data suggested that the weakly stained smaller p62 bodies could be late endosomes or lysosomes. CD63 is often used as a marker for the late endosomes/lysosomes and is highly enriched in multivesicular endosomes (Escola et al., 1998). In SGFP-p62 cells, immunostaining with an anti-CD63 antibody revealed colocalization with a fraction of the smaller GFP-p62 structures (Fig. 3 B). The large p62 bodies were consistently negative for CD63. By performing immuno-EM with antibodies against CD63 and GFP, vesicles containing both CD63 and GFP-p62 were identified, although CD63 and GFP-p62 were also found in separate compartments within the same vesicular structures (Fig. 3 C).
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We were not able to detect endogenous p62 by immuno-EM. However, by performing immuno-EM on SGFP-p62 cells, we observed both membrane-free protein aggregates (sequestosomes) and membrane-surrounded p62 bodies (Fig. 4 A). To further characterize the sequestosomes on the ultrastructural level, we performed correlative immunofluorescence and EM. Proteasomal inhibitors such as PSI induce a prominent increase in the amount of p62 protein in cells (Kuusisto et al., 2001b; Thompson et al., 2003). We took advantage of this to increase the size of endogenous p62 bodies in HeLa cells to facilitate EM/immunofluorescence studies. The results show that the large and intensely fluorescent p62 structures are membrane-free protein aggregates that are not related to endocytic vesicles (Fig. 4 B). The aggregates had a filamentous appearance and seemed to exclude any cytosolic material. Both in the stably transfected cell line and in transiently transfected HeLa cells, p62 was found within double membrane structures that are indicative of autophagosomes (Fig. 4 C). Autophagic structures and autolysosomes represented the dominant fraction of p62 bodies in SGFP-p62 cells.
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Presently, there are no antibodies available that allow immunostaining of endogenous LC3 in mammalian cells, and overexpressed myc-LC3 could only be detected using anti-myc antibodies. After transient transfection, myc-LC3 was present in a large fraction of the cytoplasmic bodies formed by endogenous p62 in HeLa cells (not depicted), and it also strongly colocalized with GFP-p62 in SGFP-p62 cells (Fig. 5 E). Generally, it was difficult to find myc-LC3positive dots that did not also contain p62. It should be noted that in HeLa cells, autophagosomes are small and are commonly visualized as dots by fluorescence microscopy (Mizushima, 2004). In cells treated with bafilomycin A1, there was an extensive accumulation of cytoplasmic bodies containing both p62 and myc-LC3 (unpublished data). Coexpression of GFP-LC3 with p62 fused to a novel, very bright, red fluorescent protein, tdTomato (Shaner et al., 2004), enabled the visualization of p62-LC3positive bodies in living cells. Video confocal microscopy of HeLa cells coexpressing tdTomato-p62 and GFP-LC3 showed that many of the punctuate structures containing p62 and LC3 had a high mobility (Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200507002/DC1).
LC3 is associated with the isolation membrane during its formation and remains on the membrane after a spherical autophagosome has formed. Transiently overexpressed GFP-LC3 has, therefore, been shown to be a very good marker for autophagy, as its localization changes from diffuse to a punctuate or dotted pattern when autophagy is induced (Mizushima, 2004). GFP-LC3 dots represent isolation membranes and autophagosomes (Mizushima, 2004). Amino acid starvation induces autophagy and results in a transient increase in the number of autophagosomes. In line with this, we found that the fraction of HeLa cells with GFP-LC3 in punctuated structures increased from 27 to 53% after amino acid starvation in Hanks medium for 60 min. Almost all of the LC3-positive bodies stained positive for endogenous p62 (Fig. 6 A). Interestingly, the redistribution of overexpressed LC3 into punctuated structures appeared to depend on the presence of p62. In cells transfected twice with small interfering RNA (siRNA) to deplete endogenous p62, very few cells contained punctuated GFP-LC3 structures (Fig. 6 A). In contrast, cooverexpression of HA-p62 strongly increased the frequency of cells with punctuated GFP-LC3 (Fig. 6 A). Cooverexpression of the D69A mutant, which inhibits PB1 domainmediated polymerization, resulting in a diffuse localization of p62 (Lamark et al., 2003), also resulted in a diffuse localization of GFP-LC3 (Fig. 6 A). Consistent with a direct or indirect association between GFP-LC3 and p62, both endogenous p62 and overexpressed HA-p62 coimmunoprecipitated with GFP-LC3 from HeLa cell extracts (Fig. 6 B). When the D69A mutant was overexpressed, less p62 was coimmunoprecipitated because polymers of p62 were not formed. However, when a p62 mutant lacking the UBA domain was coexpressed with GFP-LC3, both endogenous p62 and p62UBA were efficiently coimmunoprecipitated (Fig. 6 B). Together, the aforementioned results suggest a close association between LC3 and p62 bodies and that a large fraction of p62 bodies are degraded by autophagy.
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Discussion |
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Our data suggest that p62 may link polyubiquitinated proteins to the autophagic machinery. This function seems dependent on both the polymerization of p62 via the NH2-terminal PB1 domain and polyubiquitin binding via the COOH-terminal UBA domain of p62. Both endogenous and ectopically expressed p62 could be copurified with the autophagy marker LC3. Both p62 and LC3 colocalized with mutant huntingtin aggregates. Such aggregates were recently shown to be degraded by autophagy (Ravikumar et al., 2002, 2004). Very recently, studies of conditional knockout mice of Atg7 demonstrated that autophagy is needed for clearance of ubiquitin-positive aggregates (Komatsu et al., 2005). We found that p62 formed a shell surrounding huntingtin aggregates. Cell death induced by the expression of aggregation-prone mutant huntingtin was increased both in HeLa and SHSY-5Y neuroblastoma cells after antisense RNAmediated depletion of p62 levels or by interfering with p62 function by expressing a p62 deletion mutant lacking the UBA domain.
We found that p62 was located in two different types of bodies in the cytosol. The first type of structure appears as large protein aggregates (sequestosomes) that are not surrounded by a membrane and have very low mobility in living cells. However, the majority of p62 bodies in SGFP-p62 cells were generally smaller structures with a much higher mobility that colocalized poorly with early endosomal markers but strongly with coexpressed myc- or GFP-tagged LC3. The finding that a high number of p62 bodies colocalized with LysoTracker and the lysosomal marker CD63 is consistent with p62 being localized to autophagosomes. By detergent extraction of live cells, we observed two populations of p62 bodies. LysoTracker-positive p62 bodies were rapidly lost after extraction, whereas LysoTracker-negative structures were not dissolved by 1% Triton X-100. This is consistent with p62 being partly located to membrane-enclosed autophagosomes and partly in cytosolic sequestosomes. Induction of autophagy by amino acid starvation led to a clear increase in the number of GFP-LC3labeled autophagosomes, and all of these were positive for endogenous p62. The idea that a large fraction of p62 bodies are autophagosomes was also supported by the extensive accumulation of p62-LC3positive structures observed in cells upon treatment with bafilomycin A1. Bafilomycin A1 inhibits the autophagosomelysosome fusion step leading to accumulation of autophagosomal vacuoles. EM experiments confirmed that p62 is found both in autophagosomes and in sequestosomes and that p62 is colocalized with CD63 in cytoplasmic membrane-enclosed autophagosomal structures. Interestingly, we found p62 and LC3 to be components of the same protein complex by coimmunoprecipitation. It should be noted that coexpression of p62 with GFP-LC3 did not increase the total amount of p62 that was copurified with LC3. This suggests that the interaction might not be direct but may depend on a limiting third cellular factor. This notion is consistent with our failure to detect any interaction between GST-p62 and in vitro translated LC3 in a GST pull-down assay (unpublished data). Furthermore, the p62 D69A mutant that inhibits polymerization of p62 was very inefficiently coimmunoprecipitated with GFP-LC3, suggesting that polymeric p62 is important for interaction with LC3. Our data suggest that p62 is needed for the accumulation of GFP-LC3 in dots in HeLa cells in response to amino acid starvation. No GFP-LC3 dots were formed in response to amino acid starvation in cells depleted for endogenous p62 after transfection with siRNA. Similarly, no GFP-LC3 dots were formed in cells coexpressing mutants of p62, resulting in diffuse localization of endogenous p62. These results indicate that p62 polymerization is important for autophagosome formation in HeLa cells.
Previous studies have established p62 as a stress response protein induced by oxidative stress (Ishii et al., 1997, 2000). The protein has also been identified as a common component in protein aggregates that was found in a wide range of protein aggregation diseases (Zatloukal et al., 2002). Recently, expression of mutant huntingtin was shown to induce p62 (Nagaoka et al., 2004). Interestingly, reactive oxygen species are produced in response to proteasomal inhibition (Ling et al., 2003), and aggregation-prone mutant proteins with expanded polyglutamine stretches inhibit proteasomal activity (Bence et al., 2001). Thus, the induction of p62 in aggregation diseases might also be caused by reactive oxygen species. Prostaglandin J2 is known to induce oxidative stress by causing decreases in glutathione, glutathione peroxidase, and mitochondrial membrane potential as well as increases in the production of protein-bound lipid peroxidation products (Kondo et al., 2001). In human neuroblastoma cells, p62 is needed for the sequestration of ubiquitinated proteins into bodies in response to treatment with the inflammatory agent prostaglandin J2 (Wang and Figueiredo-Pereira, 2005). Our present results provide a molecular mechanism of how p62 might recognize ubiquitinated protein bodies and present these to the autophagic machinery.
Mutant huntingtin is found to be both diffuse in some cells and aggregated in others. It seems clear that aggregation is a mechanism for cell survival (Arrasate et al., 2004). In line with this, Steffan et al. (2004) found that ubiquitination of mutant huntingtin is an important way to detoxify the protein, whereas sumoylation of the same residues prevents aggregation and leads to cell death. Autophagy is important for the clearance of huntingtin aggregates, and induced autophagy leads to increased cell survival of cells expressing mutant huntingtin (Ravikumar et al., 2002, 2004). Similar to Nagaoka et al. (2004), we found that mutant huntingtin could also form aggregates in the absence of p62. Thus, we believe that the protective role of p62 may be to recruit autophagosomal components to the polyubiquitinylated protein aggregates rather than to help or facilitate the formation of these aggregates. Given the essential role of autophagy in preventing protein aggregateinduced neurodegeneration, p62 and proteins with related functions could be attractive targets for the development of neuroprotective drugs.
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Materials and methods |
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Antibodies and reagents
The following antibodies were used: anti-p62 and EEA1 mAbs (BD Transduction Laboratories); p62 pAb (Progen Biotechnik); polyubiquitin mAb (clone FK1; Affinity BioReagents, Inc.); CD63 mAbs (Developmental Studies Hybridoma Bank, University of Iowa); anti-GFP antibody (Ab290; Abcam Ltd.); anti-EGF receptor and myc-tag antibodies (9E10; Santa Cruz Biotechnology, Inc.); antiactin antibody (Sigma-Aldrich); and LC3 antibody (gift from T. Yoshimori, National Institute of Genetics, Shizuoka, Japan; Kabeya et al., 2000). All fluorescent Alexa-labeled secondary antibodies, LysoTracker, fluorescent-labeled dextran, and EGF as well as a Zenon kit for direct Alexa labeling of mAbs were obtained from Invitrogen. Bafilomycin A1, epoximicin, 3-methyladenine, and rapamycin were all purchased from Sigma-Aldrich. Proteasome inhibitor I (PSI) was obtained from Calbiochem. Draq5 was obtained from Biostatus Ltd. Redivue Pro-mix 35S-methionine was obtained from GE Healthcare.
Plasmids
The following vectors have been described previously (Lamark et al., 2003): pCW7-mycUbi (a gift from J. Lukas, Danish Cancer Society, Copenhagen, Denmark; Thullberg et al., 2000); pcDNA3-myc-LC3 and pEGFP-C1-LC3 (Simonsen et al., 2004); the Gateway entry clones pENTR-p62 and pENTR-p40phox; the Gateway destination vectors pDestEGFP-C1 and pDestmyc; and the Gateway expression vectors pDestHA-p62, pDestmyc-p62, pDestHA-p62 D69A, pDestmyc-p62 D69A, pDestEGFP-p62 D69A, and pEGFP-p62. pENTR-p62 I431A was constructed by the mutagenesis of pENTR-p62 using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The p62 deletion constructs pENTR-p62123386, pENTR-p62(387440), pENTR-p62(1122), pENTR-p62(124440), and pENTR-p62(1385) were made by subcloning of the indicated p62 fragments from pENTR-p62 into Gateway entry vectors (pENTR1A or pENTR3C; Invitrogen). pENTR-Htt(1171)68Q-Flag was made by the subcloning of human huntingtin(1171)68Q-FLAG cDNA from plasmid pcDNA1-Htt(1171)68Q-Flag (obtained from U. Moens, University of Tromsø, Tromsø, Norway; Saudou et al., 1998) into pENTR3C. The mammalian tdTomato fusion expression vector ptdTomato-C1 was made by exchanging the EGFP gene of pEGFP-C1 (CLONTECH Laboratories, Inc.) with tdTomato from the bacterial expression vector pRSET-B-tdTomato (obtained from R. Tsien, University of California, San Diego, San Diego, CA; Shaner et al., 2004). ptdTomato-LC3 was made by inserting a 400-bp EcoRIBamHI fragment from pEGFP-C1-LC3 into ptdTomato-C1 cut with the same enzymes. The Gateway destination vector pDest-tdTomato-C1 was made by exchanging the EGFP gene of pDestEGFP-C1 with tdTomato, and Gateway destination vector pDestDsRED2-C1 was constructed by the insertion of Gateway cassette B (Gateway vector conversion system; Invitrogen) into pDsRED2-C1 (CLONTECH Laboratories, Inc.). The Gateway expression vectors pDestDsRED2-p62, pDest-tdTomato-p62, pDestEGFP-p62(1122), pDestEGFP-p62(387440), pDestEGFP-p62
123386, pDestEGFP-p62(1385), pDestmyc-p62(1385), pDestEGFP-p62 (124440), pDestEGFP-p62 I431A, pDestEGFP-Htt(1171)68Q-Flag, and pDestEGFP-p40phox were made using Gateway recombination reactions (Invitrogen). The antisense p62 construct pAS-p62-c was made by religation of pAS-p62 (Lamark et al., 2003) after digestion with EcoRV. All constructs were verified by DNA sequencing (BigDye; Applied Biosystems). Oligonucleotides for mutagenesis, PCR, and DNA sequencing reactions were obtained from Eurogentec.
Confocal microscopy analyses
The cell cultures were directly examined under the microscope or fixed in 4% PFA and stained as previously described (Lamark et al., 2003). Live cells were imaged at 37°C in Hanks medium containing 10% serum, whereas fixed cells were imaged at RT in PBS. Images were collected using a microscope (Axiovert 200; Carl Zeiss MicroImaging, Inc.) equipped with a 40x 1.2W C-Apochroma objective and a confocal module (LSM510 META; Carl Zeiss MicroImaging, Inc.) and using the LSM5 software version 3.2 (Carl Zeiss MicroImaging, Inc.). Images were processed using Photoshop (Adobe). Z-stack video images were acquired using an imager (Ultraview RS Live Cell; PerkinElmer) on the Axiovert microscope with a 63x NA 1.4 plan-Apochromat objective. In total, 810 confocal 1-µm planes covering the whole height of the cells were superimposed for each time point. The video was acquired with Imaging Suite software (PerkinElmer) and compressed using Premiere Pro software (Adobe). Two-color live cell videos were obtained using the LSM510 META confocal module imaging a single confocal plane. Videos were compressed using QuickTime Pro.
EM
Cells for immuno-EM were fixed and embedded as described previously (Peters et al., 1991). Small blocks were cut and infused with 2.3 M sucrose for 1 h, mounted on silver pins, and frozen in liquid nitrogen. Ultrathin cryosections were cut at 110°C on an ultramicrotome (Ultracut; Leica) and collected with a 1:1 mixture of 2% methyl cellulose and 2.3 M sucrose. Sections were transferred to formvar/carbon-coated grids and labeled with primary antibodies followed by a bridging secondary antibody and protein Agold conjugates essentially as described previously (Slot et al., 1991). For double-labeling experiments, we included a blocking step between the first protein Agold and the second primary antibody (15-min incubation in 0.1% glutaraldehyde and 0.1 M PBS). After embedding in 2% methyl cellulose/0.4% uranyl acetate, we observed sections at 80 kV in an electron microscope (CM10; Philips).
Correlative immunofluorescence/EM was performed on HeLa cells that were grown on gridded coverslips (Eppendorf). Cells were treated with PSI for 3 h, fixed in 3% PFA/PBS (30 min), permeabilized with 0.05% saponin/PBS, and labeled with mouse anti-p62 (1:2,000) followed by donkey antimouse Cy2 (1:500). After embedding in moviol, the samples were observed on a microscope (LSM510 META; Carl Zeiss MicroImaging, Inc.), and the localization of interesting cells was recorded. For EM observation, the same coverslips were then fixed with 2% glutaraldehyde in 0.1 M phosphate buffer and postfixed with 2% OsO4 and 1.5% KFeCN in 0.1 M phosphate buffer. Thereafter, the coverslips were stained en bloc with 4% uranyl acetate for 60 min, dehydrated in ethanol, and embedded in Epon. After polymerization, the coverslips were removed with 40% hydrofluoric acid, and the flat specimens were glued onto Epon stubs. The block was thereafter trimmed down to the regions observed on the gridded coverslips in the fluorescence microscope and sectioned parallel to the substratum at 5070-nm section thickness. The sections were poststained with lead citrate (2 min). Electron micrographs were taken and overlaid with the confocal images in Photoshop.
Immunoblot and immunoprecipitations
All expression constructs were controlled by the immunoblotting of total cell extracts that was made after stable transfection or 24 h after transient transfection as described previously (Lamark et al., 2003). Cells were labeled with 0.14 µCi/ml 35S-methionine by a 30-min pulse in methionine-free medium, washed in PBS, and incubated for different times in normal medium. Endogenous p62 was immunoprecipitated using the p62 mAb from total cell lysates from 35S-methionine cells as described previously (Lamark et al., 2003). GFP-LC3 was immunoprecipitated from total cellular extracts using anti-GFP antibody.
Online supplemental material
Video 1 shows the migration of SGFP-p62 expressed in HeLa cells over 2 min. In total, 130 image stacks were collected with a time interval of 1 s. Video 2 shows the migration of SGFP-p62 expressed in HeLa cells over 1 h. 60 image stacks were collected with a time interval of 60 s. Video 3 shows dynamic movements of pDest-tdTomato-LC3 and GFP-p62 transiently coexpressed in HeLa cells. One confocal plane was imaged for 3 min with a time interval of 4 s. Videos 1, 2, and 3 are displayed as 15, 6, and 10 frames per second, respectively. Fig. S1 shows rapid detergent extraction of both GFPp40phox- and LysoTracker-positive vesicles in HeLa cells. Fig. S2 shows a lack of association between DsRed fluorescent protein and a GFPN-HttQ68 aggregate. Fig. S3 shows the accumulation of endogenous p62 and polyubiquitin in mutant huntingtin aggregates. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200507002/DC1.
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Acknowledgments |
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This work was supported by grants to T. Johansen from the Norwegian Research Council, the Norwegian Cancer Society, the Aakre Foundation, Simon Fougner Hartmanns Familiefond, and the Blix Foundation. A. Brech is the recipient of a career fellowship from The National Programme for Research in Functional Genomics in Norway of the Norwegian Research Council.
Submitted: 1 July 2005
Accepted: 12 October 2005
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