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BIBTEX RIS

Huntingtin and Mutant SOD1 Form Aggregate Structures with Distinct Molecular Properties in Human Cells

2005; Elsevier BV; Volume: 281; Issue: 7 Linguagem: Inglês

10.1074/jbc.m509201200

ISSN

1083-351X

Autores

Gen Matsumoto, Soojin Kim, Richard I. Morimoto,

Tópico(s)

Mitochondrial Function and Pathology

Resumo

Expression of many proteins associated with neurodegenerative disease results in the appearance of misfolded species that readily adopt alternate folded states. In vivo, these appear as punctated subcellular structures typically referred to as aggregates or inclusion bodies. Whereas groupings of these distinct proteins into a common morphological class have been useful conceptually, there is some suggestion that aggregates are not homogeneous and can exhibit a range of biological properties. In this study, we use dynamic imaging analysis of living cells to compare the aggregation and growth properties of mutant huntingtin with polyglutamine expansions or mutant SOD1 (G85R/G93A) to examine the formation of aggregate structures and interactions with other cellular proteins. Using a dual conditional expression system for sequential expression of fluorescence-tagged proteins, we show that mutant huntingtin forms multiple intracellular cytoplasmic and nuclear structures composed of a dense core inaccessible to nascent polypeptides surrounded by a surface that stably sequesters certain transcription factors and interacts transiently with molecular chaperones. In contrast, mutant SOD1 (G85R/G93A) forms a distinct aggregate structure that is porous, through which nascent proteins diffuse. These results reveal that protein aggregates do not correspond to a single common class of subcellular structures, and rather that there may be a wide range of aggregate structures, perhaps each corresponding to the specific disease-associated protein with distinct consequences on the biochemical state of the cell. Expression of many proteins associated with neurodegenerative disease results in the appearance of misfolded species that readily adopt alternate folded states. In vivo, these appear as punctated subcellular structures typically referred to as aggregates or inclusion bodies. Whereas groupings of these distinct proteins into a common morphological class have been useful conceptually, there is some suggestion that aggregates are not homogeneous and can exhibit a range of biological properties. In this study, we use dynamic imaging analysis of living cells to compare the aggregation and growth properties of mutant huntingtin with polyglutamine expansions or mutant SOD1 (G85R/G93A) to examine the formation of aggregate structures and interactions with other cellular proteins. Using a dual conditional expression system for sequential expression of fluorescence-tagged proteins, we show that mutant huntingtin forms multiple intracellular cytoplasmic and nuclear structures composed of a dense core inaccessible to nascent polypeptides surrounded by a surface that stably sequesters certain transcription factors and interacts transiently with molecular chaperones. In contrast, mutant SOD1 (G85R/G93A) forms a distinct aggregate structure that is porous, through which nascent proteins diffuse. These results reveal that protein aggregates do not correspond to a single common class of subcellular structures, and rather that there may be a wide range of aggregate structures, perhaps each corresponding to the specific disease-associated protein with distinct consequences on the biochemical state of the cell. Accumulation of abnormal protein deposits as aggregates or inclusion bodies is a common cytological feature of a number of disease states as represented by clinically related neurodegenerative diseases. The mutant proteins that initiate protein aggregates in many of these diseases have been identified: Aβ in Alzheimer disease, PrP in prion diseases, α-synuclein in Parkinson disease, huntingtin in Huntington disease, tau in tauopathy, and SOD1 in familial amyotrophic lateral sclerosis (1.Taylor J.P. Hardy J. Fischbeck K.H. Science. 2002; 296: 1991-1995Crossref PubMed Scopus (981) Google Scholar, 2.Caughey B. Lansbury Jr., P.T. Annu. Rev. Neurosci. 2003; 2003: 267-298Crossref Scopus (1423) Google Scholar, 3.Bossy-Wetzel E. Schwarzenbacher R. Lipton S.A. Nat. Med. 2004; 10: 2-9Crossref PubMed Scopus (619) Google Scholar). Although these proteins do not share distinctive common features in their respective primary sequences, they have all been shown to adopt alternate conformational states and form misfolded protein structures that appear visually as aggregates and inclusion bodies that correlate with disease pathology. There is increasing evidence to support a "toxic gain-of-function" mechanism by which misfolded protein and aggregate structures lead to a dominant pathological phenotype (2.Caughey B. Lansbury Jr., P.T. Annu. Rev. Neurosci. 2003; 2003: 267-298Crossref Scopus (1423) Google Scholar, 4.Zoghbi H.Y. Orr H.T. Annu. Rev. Neurosci. 2000; 23: 217-247Crossref PubMed Scopus (1082) Google Scholar). A molecular basis for proteotoxicity is the aberrant interactions between aggregation-prone proteins and other cellular proteins. 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The recruitment and sequestration of these cellular proteins has been proposed to lead to functional depletion as described for the transcription factors TATA-binding protein (TBP), 4The abbreviations used are: TBP, TATA-binding protein; CBP, CREB-binding protein; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; FRAP, fluorescent recovery after photobleaching; FLIP, fluorescence loss in photobleaching; RFI, relative fluorescence intensity; TRE, tetracycline response element; FRET, fluorescence resonance energy transfer.4The abbreviations used are: TBP, TATA-binding protein; CBP, CREB-binding protein; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; FRAP, fluorescent recovery after photobleaching; FLIP, fluorescence loss in photobleaching; RFI, relative fluorescence intensity; TRE, tetracycline response element; FRET, fluorescence resonance energy transfer. CREB-binding protein (CBP), Sp1 (specificity protein 1), and TBP-associated factor (TAFII130) (15.Huang C.C. Faber P.W. Persichetti F. Mittal V. Vonsattel J.P. MacDonald M.E. Gusella J.F. Somat. Cell Mol. Genet. 1998; 24: 217-233Crossref PubMed Scopus (227) Google Scholar, 16.Schaffar G. Breuer P. Boteva R. Behrends C. Tzvetkov N. Strippel N. Sakahira H. Siegers K. Hayer-Hartl M. Hartl F.U. Mol. Cell. 2004; 15: 95-105Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 17.Steffan J.S. Kazantsev A. Spasic-Boskovic O. Greenwald M. Zhu Y.-Z. Gohler H. Wanker E.E. Bates G.P. Housman D.E. Thompson L.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6763-6768Crossref PubMed Scopus (861) Google Scholar, 18.Nucifora Jr., F.C. Sasaki M. Peters M.F. Huang H. Cooper J.K. Yamada M. Takahashi H. Tsuji S. Troncoso J. Dawson V.L. Dawson T.M. Ross C.A. Science. 2001; 291: 2423-2428Crossref PubMed Scopus (926) Google Scholar, 19.Dunah A.W. Jeong H. Griffin A. Kim Y. Standaert D.G. 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Consequently, the properties of aggregates will be influenced by multiple factors such as intrinsic rate of self-association, small molecule ligands, post-translational modifications, association with other cellular proteins that share related structural motifs, interactions with molecular chaperones, and association with degradation machinery (11.Muchowski P.J. Neuron. 2002; 35: 9-12Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 15.Huang C.C. Faber P.W. Persichetti F. Mittal V. Vonsattel J.P. MacDonald M.E. Gusella J.F. Somat. Cell Mol. Genet. 1998; 24: 217-233Crossref PubMed Scopus (227) Google Scholar, 17.Steffan J.S. Kazantsev A. Spasic-Boskovic O. Greenwald M. Zhu Y.-Z. Gohler H. Wanker E.E. Bates G.P. Housman D.E. Thompson L.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6763-6768Crossref PubMed Scopus (861) Google Scholar, 18.Nucifora Jr., F.C. Sasaki M. Peters M.F. Huang H. Cooper J.K. Yamada M. Takahashi H. Tsuji S. Troncoso J. Dawson V.L. Dawson T.M. 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Kaytor M.D. Duvick L.A. Zu T. Tousey S.K. Zoghbi H.Y. Clark H.B. Orr H.T. Neuron. 2003; 38: 375-387Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 34.Urushitani M. Kurisu J. Tateno M. Hatakeyama S. Nakayama K. Kato S. Takahashi R. J. Neurochem. 2004; 90: 231-244Crossref PubMed Scopus (150) Google Scholar, 35.Shimizu, N., Asakawa, S., Minoshima, S., Kitada, T., Hattori, N., Matsumine, H., Yokochi, M., Yamamura, Y., and Mizuno, Y. (2000) J. Neural Transm. Suppl., 19–30Google Scholar, 36.Goldberg A.L. Nature. 2003; 426: 895-899Crossref PubMed Scopus (1635) Google Scholar). We have shown previously that heat shock protein 70 (Hsp70) associates transiently with the surface of polyglutamine aggregates and suggested that the activity of this chaperone reflects the presence of non-native substrates to which Hsp70 binds on the aggregate surface (14.Kim S. Nollen E.A. Kitagawa K. Bindokas V.P. Morimoto R.I. Nat. Cell Biol. 2002; 4: 826-831Crossref PubMed Scopus (247) Google Scholar). Recent in vitro studies show that the interaction of TBP with huntingtin is, indeed, prevented by Hsp70 (16.Schaffar G. Breuer P. Boteva R. Behrends C. Tzvetkov N. Strippel N. Sakahira H. Siegers K. Hayer-Hartl M. Hartl F.U. Mol. Cell. 2004; 15: 95-105Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar). This also suggests that protein aggregates in vivo may contain specific sites and surfaces to which nascent proteins become associated, whether transiently or irreversibly. Here, we show that the intracellular structures of mutant huntingtin aggregates consist of distinct layers with an inner dense core and a recruitment surface to which nascent proteins become associated in transfected cultured cells. Since these layers are not exchangeable with each other, mutant huntingtin proteins in the core are completely separated from cellular proteins. In contrast, mutant SOD1 (G85R/G93A) forms porous structures into which nascent proteins can diffuse and associate. These results demonstrate, for the first time, in a comparative analysis that different disease-associated proteins form distinct classes of aggregate structures and consequently associate differentially with other cellular proteins. Constructs—The pEYFP-N1-TBP and pEYFP-N1-HSP70 constructs were previously described (14.Kim S. Nollen E.A. Kitagawa K. Bindokas V.P. Morimoto R.I. Nat. Cell Biol. 2002; 4: 826-831Crossref PubMed Scopus (247) Google Scholar). pEYFP-C1-CBP was generated by subcloning BamHI-digested CBP fragment from pRc/RSV-mCBP-HA-RK (37.Kwok R.P. Laurance M.E. Lundblad J.R. Goldman P.S. Shih H. Connor L.M. Marriott S.J. Goodman R.H. Nature. 1996; 380: 642-646Crossref PubMed Scopus (308) Google Scholar) into the BglII site of pEYFP-C1. The pTRE-YFP or pTRE-CFP vectors were generated by PCR amplification of YFP from pEYFP-N1 or CFP from pECFP-N1 (Clontech, BD Biosciences) using the forward primer 5′-TTTCAGCTGCAGGCTAGCGCTAGCAAGGGCGAGG-3′ and reverse primer 5′-TTAGCTAGCACGCGTTTACTTGTACAGCTCG-3′ and subcloning into the PvuII/MluI sites of pTRE2hyg (Clontech, BD Biosciences, CA). To construct pTRE-httQ78-YFP, pTRE-httQ78-CFP, and pTRE-httQ23-YFP, the respective BamHI/SphI-digested httQ150 or httQ23 fragments from pcDNA3-Q150 or pcDNA3-Q23 (gift from Dr. M. Macdonald (Harvard University)) were subcloned into the BamHI/PvuII sites of pTRE-YFP or pTRE-CFP vector. DNA sequence analysis revealed a deletion of the httQ150 construct from 150 CAG repeats to 78 repeats, resulting in pTRE-httQ78-YFP and pTRE-httQ78-CFP. pTRE-SOD1-wt-YFP, pTRE-SOD1-wt-CFP, pTRE-SOD1-G85R-YFP, and pTRE-SOD1-G85R-CFP were generated by PCR amplification of wild type SOD1 and G85R mutant SOD1 from plQL01 or plQL03 (gift from Dr. Q. Liu, Harvard Medical School), respectively, using the forward primer 5′-CTCCACCGCGGATCCATGGCGACGAAGGCCGTGTG-3′ and reverse primer 5′-TTTCAGCTGCAGTTGGGCGATCCCAATTACAC-3′ and inserting into BamHI/PvuII sites of pTRE-YFP or pTRE-CFP. pTRE-SOD1-G93A-YFP and pTRE-SOD1-G93A-CFP were generated by PCR-based site-directed mutagenesis using the forward primer 5′-GTGACTGCTGACAAAGATGCTGTGGCCGATGTGTCTATTG-3′ and the reverse complement primer 5′-CAATAGACACATCGGCCACAGCATCTTTGTCAGCAGTCAC-3′ to change glycine at amino acid residue 93 to alanine of pTRE-SOD1-wt-YFP or pTRE-SOD1-wt-CFP. The pLac/MCS vector was generated by introducing a multiple cloning site into the NotI site of pOPRSVCAT (Stratagene) using the synthesized oligonucleotides, 5′-GGCCGGTACCAGATCTCATATGGATATCCTCGAGACGCGTTCTAGAGC-3′ and 5′-GGCCGCTCTAGAACGCGTCTCGAGGATATCCATATGAGATCTGGTACC-3′. The pLac-httQ78-YFP, pLac-httQ78-CFP, pLac-SOD1-G85R-YFP, and pLac-SOD1-G93A-YFP were generated by insertion of BamHI/BglII-digested httQ78-YFP, httQ78-CFP, SOD1-G85R-YFP, or SOD1-G93A-YFP fragments from the respective pTRE-constructs into the BglII site of pLacO/MCS. The BamHI fragment containing the lac operator-2 was subsequently removed from each construct, since we observed an enhanced level of induction with this deletion. The pLac-TBP-YFP and pLac-Hsp70-YFP were generated by subcloning a BglII/NotI-digested TBP-YFP fragment from pEYFP-N1-TBP or Hsp70-YFP fragment from pEYFP-N1-Hsp70 into the BglII/NotI sites of pLac/MCS. To construct pLac-CBP-YFP, pLac-EYFP-C1 was first generated by cloning the NheI/BclI-digested YFP fragment from pEYFP-C1 (Clontech, BD Biosciences) into blunt-ended HindIII/BglII sites of pLacO/MCS. The pLac-CBP-YFP was then constructed by subcloning a BamHI-digested CBP fragment from pRc/RSV-mCBP-HA-RK into the BglII site of pLac-EYFP-C1. All constructs were verified by sequencing. Cell Culture and Sequential Expression by the Dual Conditional Protein Expression System—HeTOFLI cells were generated by transfecting HeLa Tet-Off cells (Clontech, BD Biosciences) with the pCMV-LacI-NLS construct (Stratagene) using Lipofectamine PLUS reagent (Invitrogen) and selecting with 0.5 mg/ml hygromycin. The HeTOFLI cell line was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin, 0.2 mg/ml G418, and 0.5 mg/ml hygromycin at 37 °C in an atmosphere of 5% CO2, 95% air. For co-localization studies, cells were grown in 2-well glass slide chambers (Lab-Tek). For live cell analysis, cells were grown in 35-mm glass bottom cell culture dishes (MatTek Corp.). Transient transfections were performed using Lipofectamine-PLUS reagent, as described in the protocol provided by the manufacturer. pTRE- and pLacO-constructs were co-transfected into HeTOFLI cells at ratios of 2:3. pTRE- and pEYFP-constructs were co-transfected into HeTOFLI cells at ratio of 2:1. For sequential expression, the transfected HeTOFLI cells were incubated for 24 h in the absence of doxycycline, and then the protein expression from the tetracycline response element (TRE) promoter was down-regulated by adding 1 μg/ml doxycycline, and expression from the Lac promoter was simultaneously induced by adding 30 mm isopropyl-β-d-thiogalactopyranoside. Samples were analyzed 12 h after the isopropyl-β-d-thiogalactopyranoside induction. Visualization of YFP- and CFP-tagged Protein and Live Cell Imaging—Transfected HeTOFLI cells were fixed in 4% formaldehyde in 1× phosphate-buffered saline for 10 min, quenched in 0.1 m Tris-HCl, pH 8.0, for 5 min, washed in 1× phosphate-buffered saline at room temperature, and mounted in Vectashield anti-fading solution (Vector Laboratories, Inc.). Fixed samples were examined using a Leica TCS SP2/Leica DM-IRE2 inverted confocal microscope equipped with a × 63 oil objective lens (Leica Microsystems Inc.). For live cell imaging, cells were maintained at 37 °C for the duration of the experiment. Fluorescent recovery after photobleaching (FRAP) analysis was performed on a Zeiss LSM510 Axiovert confocal microscope (Carl Zeiss MicroImaging Inc.) as described previously with the following modifications: an area of 12.5 μm2 was photobleached for 3 s (20 iterations) with 514-nm laser wavelength at 100% laser power, and single scan images were collected before and every 3 s after photobleaching at 5× zoom power (14.Kim S. Nollen E.A. Kitagawa K. Bindokas V.P. Morimoto R.I. Nat. Cell Biol. 2002; 4: 826-831Crossref PubMed Scopus (247) Google Scholar). Fluorescence loss in photobleaching (FLIP) analysis was performed using a Leica TCS SP2/Leica DM-IRE2 inverted confocal microscope equipped with × 63 oil objective lens, and images were taken at 4× zoom before photobleaching and every 30 s while photobleaching an 8.4-μm2 region continuously with 514-nm laser wavelength at 100% laser power. Average fluorescence intensity of the aggregates in FRAP and FLIP analysis was determined using Metamorph software (Universal Imaging Corp.). Relative fluorescence intensity (RFI) for FRAP and FLIP was determined using the equation, RFI = ((Net/N1t)/(Ne0/N10)) × 100, where Net is the average intensity of an aggregate at a given time point and N1t is the average intensity of a nonphotobleached area of the aggregate at the corresponding time points as a control for general photobleaching (14.Kim S. Nollen E.A. Kitagawa K. Bindokas V.P. Morimoto R.I. Nat. Cell Biol. 2002; 4: 826-831Crossref PubMed Scopus (247) Google Scholar, 38.Lippincott-Schwartz J. Snapp E. Kenworthy A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 444-456Crossref PubMed Scopus (962) Google Scholar). Ne0 and N10 represent the average intensity before photobleaching of the bleached or nonbleached area, respectively. RFI values are the average of at least three data points. All images were processed by Adobe Photoshop software (Adobe Systems Inc.). Fluorescence Resonance Energy Transfer (FRET) Analysis—FRET analysis was carried out with Leica inverted microscope (DM-IRE2) with a × 63 objective. CFP (430-nm excitation/470-nm emission), YFP (500-nm excitation/535-nm emission) and FRET (430-nm excitation/535-nm emission) channel images were taken with the beam splitter 86002v2 JP4 for CFP (excitation 430/25 nm and emission 470/30 nm) and YFP (excitation 500/20 nm and emission 535/30 nm) (Chroma Technology Corp.). The acquired images were then analyzed using Metamorph imaging software with the equation, FRETC = (FRET - 95) - 0.46(CFP - 95) - 0.016(YFP - 100) - 8 (14.Kim S. Nollen E.A. Kitagawa K. Bindokas V.P. Morimoto R.I. Nat. Cell Biol. 2002; 4: 826-831Crossref PubMed Scopus (247) Google Scholar, 39.Gordon G.W. Berry G. Liang X.H. Levine B. Herman B. Biophys. J. 1998; 74: 2702-2713Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar). The FRET ratio image was then generated by calculating the ratio between FRETC (corrected FRET) and CFP images, ranging from 0 to 3. Detection of "Ring" Structures Formed by Co-expression of TBP, CBP, and Hsp70 with httQ78—To monitor the properties of huntingtin aggregates and their growth in vivo, we expressed the amino-terminal fragment of huntingtin shown to be associated with the appearance of aggregates and inclusions in Huntington disease (40.Goldberg Y.P. Nicholson D.W. Rasper D.M. Kalchman M.A. Koide H.B. Graham R.K. Bromm M. Kazemi-Esfarjani P. Thornberry N.A. Vaillancourt J.P. Hayden M.R. Nat. Genet. 1996; 13: 442-449Crossref PubMed Scopus (498) Google Scholar, 41.DiFiglia M. Sapp E. Chase K.O. Davies S.W. Bates G.P. Vonsattel J.P. Aronin N. Science. 1997; 277: 1990-1993Crossref PubMed Scopus (2256) Google Scholar). These huntingtin constructs containing 78 glutamine repeats and tagged with either YFP or CFP (httQ78-YFP, httQ78-CFP) or 23 glutamine repeats (httQ23-YFP) were used to examine interactions with the polyglutamine aggregate-associated proteins, TBP (TBP-YFP), CBP (YFP-CBP), and Hsp70 (Hsp70-YFP). These chimera proteins are functional as previously described (14.Kim S. Nollen E.A. Kitagawa K. Bindokas V.P. Morimoto R.I. Nat. Cell Biol. 2002; 4: 826-831Crossref PubMed Scopus (247) Google Scholar, 42.Patterson G.H. Schroeder S.C. Bai Y. Weil A. Piston D.W. Yeast. 1998; 14: 813-825Crossref PubMed Scopus (18) Google Scholar, 43.Chen D. Hinkley C.S. Henry R.W. Huang S. Mol. Biol. Cell. 2002; 13: 276-284Crossref PubMed Scopus (123) Google Scholar, 44.Chong J.A. Moran M.M. Teichmann M. Kaczmarek J.S. Roeder R. Clapham D.E. Mol. Cell. Biol. 2005; 25: 2632-2643Crossref PubMed Scopus (37) Google Scholar, 45.Boisvert F.M. Kruhlak M.J. Box A.K. Hendzel M.J. Bazett-Jones D.P. J. Cell Biol. 2001; 152: 1099-1106Crossref PubMed Scopus (128) Google Scholar). Co-expression of httQ78-YFP and httQ78-CFP resulted in the appearance of aggregates with both proteins uniformly distributed throughout (Fig. 1A), whereas HttQ23-YFP and Hsp70-YFP were diffuse in the cytosol, and TBP-YFP and YFP-CBP were localized to the nucleus (Fig. 1, F-I). The subcellular distribution of httQ23-YFP, TBP-YFP, YFP-CBP, and Hsp70-YFP are all strikingly altered, however, when co-expressed with httQ78-CFP. For TBP, CBP, and Hsp70, their subcellular localization is visualized by confocal microscopy in nearly all cells (81, 100, and 94%, respectively, for TBP, CBP, and Hsp70) as a "ring" surrounding a core structure composed of the huntingtin aggregate (Fig. 1, C-E). Co-localization of these transcription factors is not due to the presence of YFP, since YFP alone does not associate with the huntingtin aggregate but rather is excluded from the aggregates (supplemental Fig. S1A). Moreover, endogenous TBP (supplemental Fig. S1B) and CBP (18.Nucifora Jr., F.C. Sasaki M. Peters M.F. Huang H. Cooper J.K. Yamada M. Takahashi H. Tsuji S. Troncoso J. Dawson V.L. Dawson T.M. Ross C.A. Science. 2001; 291: 2423-2428Crossref PubMed Scopus (926) Google Scholar) also colocalize with the "ring" structure of huntingtin aggregates, demonstrating that recruitment of the transcription factors is due to the intrinsic properties of these transcription factors rather than a consequence of chimeras with YFP or CFP. In contrast, httQ23-YFP co-associated with httQ78-CFP only in the core and did not form ring structures similar to the pattern of co-localization observed for httQ78 self-association (Fig. 1B). Taken together, these results suggest that the structure of the aggregate core is composed preferentially of huntingtin protein with polyglutamine expansions. TBP and CBP appear to be excluded from this core despite both transcription factors containing polyglutamine expansions. This suggests that the process in which cellular proteins are recruited to a polyglutamine aggregate must be influenced strongly by other properties of cellular proteins, such as the sequences adjacent to the polyglutamine expansion or other structural features. Establishing a Dual Conditional System for the Sequential Expression of Proteins Recruited to the Surface of Huntingtin Aggregates—To test directly whether huntingtin aggregates have localized surfaces for recruitment of nascent proteins, it was necessary to establish a dual conditional protein expression system to allow for the sequential expression of CFP- or YFP-tagged proteins. The dual conditional protein expression system employed the Tet-off and Lac regulatory systems in which tTA and LacO-NLS stably expressing HeLa cells (HeTOFLI) was used to express two different genes under the control of the TRE or RSV-LacO (Lac) promoters (supplemental Fig. S2). With this system, we reasoned that it would then be possible to address whether proteins either co-expressed or sequentially expressed formed homogenous or heterogeneous aggregate structures. The initial expression of httQ78 would allow formation of a visual seeding structure, and the subsequent expression of an aggregate-associated protein would allow us to address the process of protein recruitment. In vivo sequential imaging analysis was performed on cells cotransfected with pTRE-httQ78-CFP and either pLac-TBP-YFP, pLac-YFP-CBP, or pLac-Hsp70-YFP. HttQ78-CFP was expressed for 24 h, after which its expression was repressed, and the expression of TBP-YFP, YFP-CBP, or Hsp70-YFP was subsequently induced. Newly synthesized TBP-YFP and YFP-CBP were recruited efficiently to the exterior surface of nuclear aggregates and detected as YFP "ring" structures surrounding a CFP huntingtin core (Fig. 2, A-C). The appearance of TBP-YFP "ring" structures was less frequently detected in cytoplasmic or perinuclear aggregates than in nuclear aggregates (Fig. 2B), consistent with the expectation that interactions between huntingtin aggregates and transcription factors is a more frequent event in the nuclear compartment. In contrast, Hsp70-YFP was detected on the surface of both nuclear and cytoplasmic aggregates (Fig. 2D). These results demonstrate that the "ring" structures observed in cells expressing poly(Q)-containing proteins are, indeed, due to recruitment of cellular proteins to the exterior surface of the aggregate and moreover that the huntingtin aggregate continues to recruit other cellular protein even when express

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