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• W2008410014 abstract "Our capacity for tracking how misfolded proteins aggregate inside a cell and how different aggregation states impact cell biology remains enigmatic. To address this, we built a new toolkit that enabled the high throughput tracking of individual cells enriched with polyglutamine-expanded Htt exon 1 (Httex1) monomers, oligomers, and inclusions using biosensors of aggregation state and flow cytometry pulse shape analysis. Supplemented with gel filtration chromatography and fluorescence-adapted sedimentation velocity analysis of cell lysates, we collated a multidimensional view of Httex1 aggregation in cells with respect to time, polyglutamine length, expression levels, cell survival, and overexpression of protein quality control chaperones hsp40 (DNAJB1) and hsp70 (HSPA1A). Cell death rates trended higher for Neuro2a cells containing Httex1 in inclusions than with Httex1 dispersed through the cytosol at time points of expression over 2 days. hsp40 stabilized monomers and suppressed inclusion formation but did not otherwise change Httex1 toxicity. hsp70, however, had no major effect on aggregation of Httex1 but increased the survival rate of cells with inclusions. hsp40 and hsp70 also increased levels of a second bicistronic reporter of Httex1 expression, mKate2, and increased total numbers of cells in culture, suggesting these chaperones partly rectify Httex1-induced deficiencies in quality control and growth rates. Collectively, these data suggest that Httex1 overstretches the protein quality control resources and that the defects can be partly rescued by overexpression of hsp40 and hsp70. Importantly, these effects occurred in a pronounced manner for soluble Httex1, which points to Httex1 aggregation occurring subsequently to more acute impacts on the cell. Our capacity for tracking how misfolded proteins aggregate inside a cell and how different aggregation states impact cell biology remains enigmatic. To address this, we built a new toolkit that enabled the high throughput tracking of individual cells enriched with polyglutamine-expanded Htt exon 1 (Httex1) monomers, oligomers, and inclusions using biosensors of aggregation state and flow cytometry pulse shape analysis. Supplemented with gel filtration chromatography and fluorescence-adapted sedimentation velocity analysis of cell lysates, we collated a multidimensional view of Httex1 aggregation in cells with respect to time, polyglutamine length, expression levels, cell survival, and overexpression of protein quality control chaperones hsp40 (DNAJB1) and hsp70 (HSPA1A). Cell death rates trended higher for Neuro2a cells containing Httex1 in inclusions than with Httex1 dispersed through the cytosol at time points of expression over 2 days. hsp40 stabilized monomers and suppressed inclusion formation but did not otherwise change Httex1 toxicity. hsp70, however, had no major effect on aggregation of Httex1 but increased the survival rate of cells with inclusions. hsp40 and hsp70 also increased levels of a second bicistronic reporter of Httex1 expression, mKate2, and increased total numbers of cells in culture, suggesting these chaperones partly rectify Httex1-induced deficiencies in quality control and growth rates. Collectively, these data suggest that Httex1 overstretches the protein quality control resources and that the defects can be partly rescued by overexpression of hsp40 and hsp70. Importantly, these effects occurred in a pronounced manner for soluble Httex1, which points to Httex1 aggregation occurring subsequently to more acute impacts on the cell. Protein misfolding and aggregation into β-sheet rich amyloid fibrils is a hallmark and possible cause of at least 36 human diseases (1Sipe J.D. Benson M.D. Buxbaum J.N. Ikeda S. Merlini G. Saraiva M.J. Westermark P. Amyloid fibril protein nomenclature: 2010 recommendations from the nomenclature committee of the International Society of Amyloidosis.Amyloid. 2010; 17: 101-104Crossref PubMed Scopus (260) Google Scholar). Our basic knowledge of protein misfolding and how the inherent features of proteins and physicochemical environment influence aggregation has largely come from purified peptide and protein model systems (2Lee C.C. Walters R.H. Murphy R.M. Reconsidering the mechanism of polyglutamine peptide aggregation.Biochemistry. 2007; 46: 12810-12820Crossref PubMed Scopus (75) Google Scholar, 3Pappu R.V. Wang X. Vitalis A. Crick S.L. A polymer physics perspective on driving forces and mechanisms for protein aggregation.Arch. Biochem. Biophys. 2008; 469: 132-141Crossref PubMed Scopus (122) Google Scholar, 4Vitalis A. Lyle N. Pappu R.V. Thermodynamics of β-sheet formation in polyglutamine.Biophys. J. 2009; 97: 303-311Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 5Wetzel R. Physical chemistry of polyglutamine: Intriguing tales of a monotonous sequence.J. Mol. Biol. 2012; 421: 466-490Crossref PubMed Scopus (137) Google Scholar). A common theme has emerged that aggregates, notably small oligomeric forms, can incur toxicity to cells (6Bucciantini M. Giannoni E. Chiti F. Baroni F. Formigli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Stefani M. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases.Nature. 2002; 416: 507-511Crossref PubMed Scopus (2153) Google Scholar, 7Campioni S. Mannini B. Zampagni M. Pensalfini A. Parrini C. Evangelisti E. Relini A. Stefani M. Dobson C.M. Cecchi C. Chiti F. A causative link between the structure of aberrant protein oligomers and their toxicity.Nat. Chem. Biol. 2010; 6: 140-147Crossref PubMed Scopus (447) Google Scholar). When aggregates form in a cell, however, they do so in an environment of omnifarious influence from protein quality control mechanisms (8Hartl F.U. Bracher A. Hayer-Hartl M. Molecular chaperones in protein folding and proteostasis.Nature. 2011; 475: 324-332Crossref PubMed Scopus (2189) Google Scholar). Protein quality control processes change the landscape by which proteins spontaneously aggregate by refolding misfolded conformations, disaggregating aggregates, degrading them, and sorting them into different cellular locations such as aggresomes, insoluble protein deposits, and Juxta nuclear quality control compartments for deposition and sequestration (9Johnston J.A. Ward C.L. Kopito R.R. Aggresomes: a cellular response to misfolded proteins.J. Cell Biol. 1998; 143: 1883-1898Crossref PubMed Scopus (1774) Google Scholar, 10Saudou F. Finkbeiner S. Devys D. Greenberg M.E. Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions.Cell. 1998; 95: 55-66Abstract Full Text Full Text PDF PubMed Scopus (1368) Google Scholar, 11Arrasate M. Mitra S. Schweitzer E.S. Segal M.R. Finkbeiner S. Inclusion body formation reduces levels of mutant Huntingtin and the risk of neuronal death.Nature. 2004; 431: 805-810Crossref PubMed Scopus (1606) Google Scholar, 12Kaganovich D. Kopito R. Frydman J. Misfolded proteins partition between two distinct quality control compartments.Nature. 2008; 454: 1088-1095Crossref PubMed Scopus (690) Google Scholar, 13Cohen E. Paulsson J.F. Blinder P. Burstyn-Cohen T. Du D. Estepa G. Adame A. Pham H.M. Holzenberger M. Kelly J.W. Masliah E. Dillin A. Reduced IGF-1 signaling delays age-associated proteotoxicity in mice.Cell. 2009; 139: 1157-1169Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar, 14García-Mata R. Bebök Z. Sorscher E.J. Sztul E.S. Characterization and dynamics of aggresome formation by a cytosolic GFP-chimera.J. Cell Biol. 1999; 146: 1239-1254Crossref PubMed Scopus (503) Google Scholar). The active movement of misfolded proteins into deposits such as aggresomes presents a paradox to the study of the effect of protein misfolding and aggregation in situ. On the one hand, organized aggregation by protein quality control machinery has net benefits to cell health, and on the other hand, spontaneous inappropriate aggregation is capricious (15Hatters D.M. Putting huntingtin “aggregation” in view with windows into the cellular milieu.Curr. Top. Med. Chem. 2012; 12: 2611-2622Crossref PubMed Scopus (15) Google Scholar). It remains plausible that both phenomena occur at the same time especially when protein quality control loses its capacity to control aggregation in an organized fashion (16Morimoto R.I. The heat shock response: Systems biology of proteotoxic stress in aging and disease.Cold Spring Harb. Symp. Quant. Biol. 2011; 76: 91-99Crossref PubMed Scopus (277) Google Scholar). The contradictory nature of these processes and their relevance to disease necessitates more sophisticated approaches to decipher the molecular process of aggregation inside the cell and how this may impact cell health (15Hatters D.M. Putting huntingtin “aggregation” in view with windows into the cellular milieu.Curr. Top. Med. Chem. 2012; 12: 2611-2622Crossref PubMed Scopus (15) Google Scholar). With this in mind, we developed new toolkits to more precisely probe the intracellular conformation and aggregation state of the exon 1 fragment of mutant huntingtin protein (Httex1) 2The abbreviations used are: Httex1Htt exon 1polyQpolyglutamineTCtetracysteinePulSApulse shape analysisSVAsedimentation velocity analysisANOVAanalysis of variancePFRpoor FlAsH-reactiveIRESinternal ribosome entry site. in cells, which has a number of attractive features for this problem (17Ramdzan Y.M. Nisbet R.M. Miller J. Finkbeiner S. Hill A.F. Hatters D.M. Conformation sensors that distinguish monomeric proteins from oligomers in live cells.Chem. Biol. 2010; 17: 371-379Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 18Olshina M.A. Angley L.M. Ramdzan Y.M. Tang J. Bailey M.F. Hill A.F. Hatters D.M. Tracking mutant huntingtin aggregation kinetics in cells reveals three major populations that include an invariant oligomer pool.J. Biol. Chem. 2010; 285: 21807-21816Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 19Ramdzan Y.M. Polling S. Chia C.P. Ng I.H. Ormsby A.R. Croft N.P. Purcell A.W. Bogoyevitch M.A. Ng D.C. Gleeson P.A. Hatters D.M. Tracking protein aggregation and mislocalization in cells with flow cytometry.Nat. Methods. 2012; 9: 467-470Crossref PubMed Scopus (82) Google Scholar). Mutant Httex1 accumulates as intracellular inclusion bodies in Huntington disease, which is caused by mutations that result in an expansion of a polyglutamine (polyQ) sequence within Httex1 to beyond a threshold of 36 glutamines (20MacDonald M.E. Ambrose C.M. Duyao M.P. Myers R.H. Lin C. Srinidhi L. Barnes G. Taylor S.A. James M. Groot N. MacFarlane H. Jenkins B. Anderson M.A. Wexler N.S. Gusella J.F. Bates G.P. Baxendale S. Hummerich H. Kirby S. North M. Youngman S. Mott R. Zehetner G. Sedlacek Z. Poustka A. Frischauf A.-M. Lehrach H. Buckler A.J. Church D. Doucette-Stamm L. O'Donovan M.C. Riba-Ramirez L. Shah M. Stanton V.P. Strobel S.A. Draths K.M. Wales J.L. Dervan P. Housman D.E. Altherr M. Shiang R. Thompson L. Fielder T. Wasmuth J.J. Tagle D. Valdes J. Elmer L. Allard M. Castilla L. Swaroop M. Blanchard K. Collins F.S. Snell R. Holloway T. Gillespie K. Datson N. Shaw D. Harper P.S. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington disease chromosomes.Cell. 1993; 72: 971-983Abstract Full Text PDF PubMed Scopus (7049) Google Scholar, 21Duyao M. Ambrose C. Myers R. Novelletto A. Persichetti F. Frontali M. Folstein S. Ross C. Franz M. Abbott M. Gray J. Conneally P. Young A. Penney J. Hollingsworth Z. Shoulson I. Lazzarini A. Falek A. Koroshetz W. Sax D. Bird E. Vonsattel J. Bonilla E. Alvir J. Bickham Conde J. Cha J.H. Dure L. Gomez F. Ramos M. Sanchez-Ramos J. Snodgrass S. de Young M. Wexler N. Moscowitz C. Penchaszadeh G. MacFarlane H. Anderson M. Jenkins B. Srinidhi J. Barnes G. Gusella J. MacDonald M. Trinucleotide repeat length instability and age of onset in Huntington disease.Nat. Genet. 1993; 4: 387-392Crossref PubMed Scopus (896) Google Scholar). PolyQ expansions in the pathological range lead to Httex1 spontaneously assembling into amyloid-like fibrils in a manner that is faster for longer polyQ lengths (22Perutz M.F. Johnson T. Suzuki M. Finch J.T. Glutamine repeats as polar zippers: Their possible role in inherited neurodegenerative diseases.Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 5355-5358Crossref PubMed Scopus (948) Google Scholar, 23Scherzinger E. Sittler A. Schweiger K. Heiser V. Lurz R. Hasenbank R. Bates G.P. Lehrach H. Wanker E.E. Self-assembly of polyglutamine-containing Huntingtin fragments into amyloid-like fibrils: Implications for Huntington disease pathology.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 4604-4609Crossref PubMed Scopus (577) Google Scholar). Expression of polyQ-expanded proteins in animals and cells recapitulates aggregation and pathology in a polyQ length-dependent manner, demonstrating clear links between the intrinsic biophysical attributes of Httex1, aggregation, and pathology (24Brignull H.R. Morley J.F. Garcia S.M. Morimoto R.I. Modeling polyglutamine pathogenesis in C. elegans.Methods Enzymol. 2006; 412: 256-282Crossref PubMed Scopus (71) Google Scholar, 25Davies S.W. Turmaine M. Cozens B.A. DiFiglia M. Sharp A.H. Ross C.A. Scherzinger E. Wanker E.E. Mangiarini L. Bates G.P. Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation.Cell. 1997; 90: 537-548Abstract Full Text Full Text PDF PubMed Scopus (1908) Google Scholar, 26Warrick J.M. Paulson H.L. Gray-Board G.L. Bui Q.T. Fischbeck K.H. Pittman R.N. Bonini N.M. Expanded polyglutamine protein forms nuclear inclusions and causes neural degeneration in Drosophila.Cell. 1998; 93: 939-949Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar). Htt exon 1 polyglutamine tetracysteine pulse shape analysis sedimentation velocity analysis analysis of variance poor FlAsH-reactive internal ribosome entry site. Our first toolkit involved the development of tetracysteine-based biosensors for detecting the earliest oligomerization steps of the Httex1 in live cells (17Ramdzan Y.M. Nisbet R.M. Miller J. Finkbeiner S. Hill A.F. Hatters D.M. Conformation sensors that distinguish monomeric proteins from oligomers in live cells.Chem. Biol. 2010; 17: 371-379Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The TC9 sensor is a derivative of Httex1 with an engineered tetracysteine (TC) tag embedded with the Httex1 sequence that is masked from binding to biarsenical fluorescent dyes upon self-assembly in vitro (17Ramdzan Y.M. Nisbet R.M. Miller J. Finkbeiner S. Hill A.F. Hatters D.M. Conformation sensors that distinguish monomeric proteins from oligomers in live cells.Chem. Biol. 2010; 17: 371-379Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Httex1TC9 is also tagged C-terminally with a fluorescent protein (e.g. cyan fluorescent protein derivative Cerulean) that independently reports the presence of the protein. Hence, two-color imaging enables readouts of the balance of monomers and aggregates inside live cells, independently to cellular localization (17Ramdzan Y.M. Nisbet R.M. Miller J. Finkbeiner S. Hill A.F. Hatters D.M. Conformation sensors that distinguish monomeric proteins from oligomers in live cells.Chem. Biol. 2010; 17: 371-379Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). This technology was recently merged with a flow cytometry pulse shape analysis (PulSA) method, which utilizes fluorescent pulse width and height information from a flow cytometer to monitor changes in the intracellular distribution of protein (19Ramdzan Y.M. Polling S. Chia C.P. Ng I.H. Ormsby A.R. Croft N.P. Purcell A.W. Bogoyevitch M.A. Ng D.C. Gleeson P.A. Hatters D.M. Tracking protein aggregation and mislocalization in cells with flow cytometry.Nat. Methods. 2012; 9: 467-470Crossref PubMed Scopus (82) Google Scholar). PulSA in combination with the TC9 sensor system enabled a distinction in detection of biochemical aggregates, which can be as small in theory as a dimer (i.e. nanometer scale), from the condensation into microscopically visible structures (i.e. micrometer scale) such as inclusions, providing a new high throughput capacity to track cells enriched with dispersed oligomers of Httex1 from cells with monomers or the inclusions. A second toolkit was sedimentation velocity analysis (SVA) with analytical ultracentrifugation to quantitate the oligomeric size and heterogeneity of GFP-tagged Httex1 aggregate forms in a cell lysate (18Olshina M.A. Angley L.M. Ramdzan Y.M. Tang J. Bailey M.F. Hill A.F. Hatters D.M. Tracking mutant huntingtin aggregation kinetics in cells reveals three major populations that include an invariant oligomer pool.J. Biol. Chem. 2010; 285: 21807-21816Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). For the aggregation prone 46Gln form of Httex1, this approach yielded a heterogeneous mixture of oligomers, most abundantly about 30 nm in diameter. The nonaggregation 25Gln isoform of Httex1 in contrast only yielded monomers. The combination of the single cell approaches with biochemical approaches (e.g. SVA) in principle provides an enabling platform to define the kinetic process of aggregation approaching a molecular scale of detail. Here, we describe an implementation of an integrated platform for defining Httex1 aggregation in the cell by merging our existing toolkits together and developing new capabilities to follow cell death and protein levels. We used this workflow to first monitor the impact of aggregation state on cell death, and second to examine how elevation of key inducible members of the heat shock protein family (hsp70 protein HSPA1A and its hsp40 cofactor DNAJB1) alters the Httex1 aggregation landscape and cell survival when levels are elevated. hsp70 and its co-chaperone hsp40 are key elements that have canonical functions in assisting proteins to fold correctly, and they potently inhibit toxicity of Httex1 in model systems (27Young J.C. Agashe V.R. Siegers K. Hartl F.U. Pathways of chaperone-mediated protein folding in the cytosol.Nat. Rev. Mol. Cell Biol. 2004; 5: 781-791Crossref PubMed Scopus (938) Google Scholar, 28Behrends C. Langer C.A. Boteva R. Böttcher U.M. Stemp M.J. Schaffar G. Rao B.V. Giese A. Kretzschmar H. Siegers K. Hartl F.U. Chaperonin TRiC promotes the assembly of polyQ expansion proteins into nontoxic oligomers.Mol. Cell. 2006; 23: 887-897Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 29Wacker J.L. Zareie M.H. Fong H. Sarikaya M. Muchowski P.J. Hsp70 and Hsp40 attenuate formation of spherical and annular polyglutamine oligomers by partitioning monomer.Nat. Struct. Mol. Biol. 2004; 11: 1215-1222Crossref PubMed Scopus (248) Google Scholar, 30Warrick J.M. Chan H.Y. Gray-Board G.L. Chai Y. Paulson H.L. Bonini N.M. Suppression of polyglutamine-mediated neurodegeneration in Drosophila by the molecular chaperone HSP70.Nat. Genet. 1999; 23: 425-428Crossref PubMed Scopus (727) Google Scholar). How they do this remains enigmatic because protection does not always occur with reducing inclusions, which seems counterintuitive to their canonical role in assisting proteins to fold (30Warrick J.M. Chan H.Y. Gray-Board G.L. Chai Y. Paulson H.L. Bonini N.M. Suppression of polyglutamine-mediated neurodegeneration in Drosophila by the molecular chaperone HSP70.Nat. Genet. 1999; 23: 425-428Crossref PubMed Scopus (727) Google Scholar, 31Jana N.R. Tanaka M. Wang G.-h. Nukina N. Polyglutamine length-dependent interaction of Hsp40 and Hsp70 family chaperones with truncated N-terminal Huntingtin: their role in suppression of aggregation and cellular toxicity.Hum. Mol. Genet. 2000; 9: 2009-2018Crossref PubMed Scopus (363) Google Scholar, 32Zhou H. Li S.-H. Li X.-J. Chaperone suppression of cellular toxicity of Huntingtin is independent of polyglutamine aggregation.J. Biol. Chem. 2001; 276: 48417-48424Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 33Rujano M.A. Kampinga H.H. Salomons F.A. Modulation of polyglutamine inclusion formation by the Hsp70 chaperone machine.Exp. Cell Res. 2007; 313: 3568-3578Crossref PubMed Scopus (44) Google Scholar, 34Wacker J.L. Huang S.-Y. Steele A.D. Aron R. Lotz G.P. Nguyen Q. Giorgini F. Roberson E.D. Lindquist S. Masliah E. Muchowski P.J. Loss of Hsp70 exacerbates pathogenesis but not levels of fibrillar aggregates in a mouse model of Huntington disease.J. Neurosci. 2009; 29: 9104-9114Crossref PubMed Scopus (95) Google Scholar, 35Cummings C.J. Sun Y. Opal P. Antalffy B. Mestril R. Orr H.T. Dillmann W.H. Zoghbi H.Y. Over-expression of inducible HSP70 chaperone suppresses neuropathology and improves motor function in SCA1 mice.Hum. Mol. Genet. 2001; 10: 1511-1518Crossref PubMed Scopus (423) Google Scholar, 36Muchowski P.J. Schaffar G. Sittler A. Wanker E.E. Hayer-Hartl M.K. Hartl F.U. Hsp70 and Hsp40 chaperones can inhibit self-assembly of polyglutamine proteins into amyloid-like fibrils.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 7841-7846Crossref PubMed Scopus (542) Google Scholar). The TC9 variant of Httex1 was generated as described (17Ramdzan Y.M. Nisbet R.M. Miller J. Finkbeiner S. Hill A.F. Hatters D.M. Conformation sensors that distinguish monomeric proteins from oligomers in live cells.Chem. Biol. 2010; 17: 371-379Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The Httex1-Emerald constructs were produced as described (18Olshina M.A. Angley L.M. Ramdzan Y.M. Tang J. Bailey M.F. Hill A.F. Hatters D.M. Tracking mutant huntingtin aggregation kinetics in cells reveals three major populations that include an invariant oligomer pool.J. Biol. Chem. 2010; 285: 21807-21816Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) to produce Httex1 with a C-terminal Emerald fusion in the pT-Rex vector backbone (Invitrogen). The IRES vectors were made by inserting an IRES sequence C-terminally to the Httex1TC9-Cerulean moiety in the pT-Rex backbone. Specifically, we ligated the following synthetic gene (Geneart, Invitrogen) cut from the cloning vector with MfeI and EcoRI into a unique EcoRI site at the 3′ of the stop codon of Httex1TC9-Cerulean (Sequence 1), where key features are annotated as follows: CAATTG, MfeI restriction site; SEQUENCE, IRES sequence; SEQUENCE, mKate2; shaded SEQUENCE (37Shcherbo D. Murphy C.S. Ermakova G.V. Solovieva E.A. Chepurnykh T.V. Shcheglov A.S. Verkhusha V.V. Pletnev V.Z. Hazelwood K.L. Roche P.M. Lukyanov S. Zaraisky A.G. Davidson M.W. Chudakov D.M. Far-red fluorescent tags for protein imaging in living tissues.Biochem. J. 2009; 418: 567-574Crossref PubMed Scopus (390) Google Scholar), farnesylation tag; CGTACG with dots below, BsiW1 restriction site; GAATTC with rule and dots below, EcoRI restriction site. The nonfarnesylated mKate2 version of the IRES vector was created by excision of the farnesylation tag with BsiW1 digestion and vector religation. Hsp40 and Hsp70 chaperones were provided from Paul Muchowski (Gladstone Institutes) and verified by DNA sequencing. Neuro-2a cells were maintained in OptiMEM (Invitrogen) supplemented with 10% fetal calf serum, 1 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin in a humidified incubator with 5% atmospheric CO2. 2 × 105 cells were plated in individual wells of a 24-well tissue culture plate. The following day, the cells in each well were transfected with 2 μl of Lipofectamine 2000/0.8 μg of vector DNA according to the manufacturer's instructions (Invitrogen). The next day, the media were refreshed (500 μl), and for the time course the media were refreshed daily. Cell suspensions were kept on ice until analysis by flow cytometry. FlAsH staining was performed as described previously (19Ramdzan Y.M. Polling S. Chia C.P. Ng I.H. Ormsby A.R. Croft N.P. Purcell A.W. Bogoyevitch M.A. Ng D.C. Gleeson P.A. Hatters D.M. Tracking protein aggregation and mislocalization in cells with flow cytometry.Nat. Methods. 2012; 9: 467-470Crossref PubMed Scopus (82) Google Scholar) with the exception of using a 12-well tissue culture plate setup, and all volumes used during the transfection and FlAsH staining protocols were doubled. For SYTOX staining, the media from the cultured cells were removed 24 h after transfection and kept aside in parallel format in 24-well plates and retained in the cell culture incubator. Fresh media were added to the cells, and at further time points of analysis, the media were collected and added to the initial media collections (so as to collect any detached cells). Remaining adherent cells were detached by gentle agitation and pipetting in 500 μl of phosphate-buffered saline (PBS). The cell suspension was added to the set-aside media and then pelleted (1600 × g; 3 min; room temperature). The supernatant was discarded and the pellet resuspended in 500 μl of PBS followed by 0.5 μl of 5 μm SYTOX Red Dead stain (Invitrogen). Cell suspensions were kept on ice until analysis by flow cytometry (which was all completed within 1 h after labeling). Cells were analyzed at high flow rate in an LSRFortessa flow cytometer, equipped with 405- and 488-nm lasers (BD Biosciences). 50,000–100,000 events were collected, using a forward scatter threshold of 5,000. Data were collected in pulse height, area, and width parameters for each channel. For Cerulean fluorescence, data were collected with the 405-nm laser and Pacific blue filter. For FlAsH and GFP, data were collected with the 488-nm laser and FITC filter. mKate2 fluorescence was collected in the PE-Texas Red filter. SYTOX Red Dead stain was collected using 640-nm laser and the APC filter. All flow cytometry data were analyzed with FACSDiva software (BD Biosciences), FlowJo (Tree Star Inc.), or manually in Excel (Microsoft). 1 × 106 cells were plated in individual wells of a 6-well tissue culture plate. The following day, the cells in each well were transfected with 10 μl of Lipofectamine 2000 and 4 μg of vector DNA according to the manufacturer's instructions (Invitrogen). After 24 h, the media were refreshed (2 ml). Cells were harvested at 48 h post-transfection by first rinsing in PBS, followed by resuspension in PBS with a cell scraper, and gentle pipetting. Cells were pelleted (1,600 × g; 3 min) and resuspended in 2 ml of 2% (v/v) paraformaldehyde for 30 min at room temperature. Cells were again pelleted (1,600 × g; 3 min), resuspended in 2 ml of PBS, and filtered through 100-μm nylon mesh before analysis and recovery on a BD FacsAria cell sorter (BD Biosciences). Cells were imaged on a Leica SP2 confocal microscope using a HC PL APO lbd.BL 20.0 × 0.70 IMM objective (TCS SP2 Leica). 6 × 106 cells were plated in 75-cm2 tissue culture flasks. The following day, cells were transfected with 24 μg of DNA and 60 μl of Lipofectamine 2000. 24 h after transfection, media were either refreshed for the 30-h time point, or cells were harvested by scraping. Cells were then pelleted (1,600 × g; 3 min; room temperature) and snap-frozen in liquid nitrogen. Cells were then lysed as described previously (18Olshina M.A. Angley L.M. Ramdzan Y.M. Tang J. Bailey M.F. Hill A.F. Hatters D.M. Tracking mutant huntingtin aggregation kinetics in cells reveals three major populations that include an invariant oligomer pool.J. Biol. Chem. 2010; 285: 21807-21816Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Running buffer (20 mm Tris, pH 7.4, 150 mm NaCl, 1% Triton X-100) was used to pack and equilibrate Sephacryl S-1000 superfine medium (GE Healthcare) into a 1.0 × 30-cm chromatography Econo-Column (Bio-Rad). 200 μl of cell lysate was run through the column at a flow rate of 1.1 ml/min. 4-Drop fractions were collected into three U-shaped black-bottomed 96-well plates. Fluorescence of fractions was assessed with a Varioskan Flash spectral scanning multimode plate reader. Excitation/emission wavelength of 470/511 nm was used. Cell preparation, lysis, and analysis were all performed as described previously (18Olshina M.A. Angley L.M. Ramdzan Y.M. Tang J. Bailey M.F. Hill A.F. Hatters D.M. Tracking mutant huntingtin aggregation kinetics in cells reveals three major populations that include an invariant oligomer pool.J. Biol. Chem. 2010; 285: 21807-21816Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Data were analyzed for differences by either a one- or three-way ANOVA with the Holm-Sidak Test comparing each chaperone treatment with the Htt alone control. We first developed strategies to examine how the transition of mutant Httex1 from monomers to diffuse oligomers and large inclusions in individual mammalian cells correlate with cell death. Our TC9-based biosensor system of the Htt aggregation state (17Ramdzan Y.M. Nisbet R.M. Miller J. Finkbeiner S. Hill A.F. Hatters D.M. Conformation sensors that distinguish monomeric proteins from oligomers in live cells.Chem. Biol. 2010; 17: 371-379Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) was adapted into a bicistronic expression system (homemade pTIREX vectors) to independently mark cells that had expressed Httex1 with a second membrane-associated red fluorescent protein mKate2 (Fig. 1A). The mKate2 was C-terminally tagged with a farnesylation targeting sequence (mKate2-F), which targets proteins to the plasma membrane (39Hancock J.F. Cadwallader K. Paterson H. Marshall C.J. A CAAX or a CAAL motif and a second signal are sufficient for plasma membrane targeting of Ras proteins.EMBO J. 1991; 10: 4033-4039Crossref PubMed Scopus (378) Google Scholar, 40Aronheim A. Engelberg D. Li N. al-Alawi N. Schlessinger J. Karin M. Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway.Cell. 1994; 78: 949-961Abstract Full Text PDF PubMed Scopus (423) Google Scholar). This was designed to fluorescently detect cells that may have leached their cytosolic contents upon cell death, such as when sma" @default.
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• W2008410014 date "2013-12-01" @default.
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• W2008410014 title "A Platform to View Huntingtin Exon 1 Aggregation Flux in the Cell Reveals Divergent Influences from Chaperones hsp40 and hsp70" @default.
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