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  Cytoplasmic Dynein/Dynactin Mediates the Assembly of Aggresomes Jennifer A. Johnston, *  Michelle E. Illing, and Ron R. Kopito Department of Biological Sciences, Stanford University, Stanford, California Aggresomes are pericentrosomal cytoplasmic structures into which aggregated,ubiquitinated, misfolded proteins are sequestered. Misfolded proteins accumulatein aggresomes when the capacity of the intracellular protein degradation machin-ery is exceeded. Previously, we demonstrated that an intact microtubule cytoskel-eton is required for the aggresome formation [Johnston et al., 1998:  J. Cell Biol. 143:1883–1898]. In this study, we have investigated the involvement of micro-tubules (MT) and MT motors in this process. Induction of aggresomes containingmisfolded   F508 CFTR is accompanied by a redistribution of the retrogrademotor cytoplasmic dynein that colocalizes with aggresomal markers. Coexpres-sion of the p50 (dynamitin) subunit of the dynein/dynactin complex prevents theformation of aggresomes, even in the presence of proteasome inhibitors. Using invitro microtubule binding assays in conjunction with immunogold electron mi-croscopy, our data demonstrate that misfolded   F508 CFTR associate withmicrotubules. We conclude that cytoplasmic dynein/dynactin is responsible for thedirected transport of misfolded protein into aggresomes. The implications of thesefindings with respect to the pathogenesis of neurodegenerative disease are dis-cussed. Cell Motil. Cytoskeleton 53:26–38, 2002. ©  2002 Wiley-Liss, Inc. Key words: aggresome; dynein; proteolysis; centrosome; microtubule INTRODUCTION Aggresomes are pericentriolar cytoplasmic struc-tures in which aggregated, multiubiquitinated misfoldedproteins are sequestered [Johnston et al., 1998]. Aggre-somes, which appear to be a general response to condi-tions where a cell’s capacity to degrade malfolded pro-teins is exceeded, closely resemble ubiquitin-richcytoplasmic “inclusion bodies” that are pathognomonicfor degenerating neurons in Parkinson’s disease, amyo-trophic lateral sclerosis and other neurodegenerative dis-eases [Mayer et al., 1989]. To study the mechanism bywhich aggresomes form in cells may help illuminate thecellular events which underlie these neurological disor-ders. In particular, the restriction of aggresomes to thevicinity of the microtubule organizing center (MTOC)and the dependence of aggresome formation on micro-tubules strongly suggested a central role for the micro-tubule (MT) cytoskeleton in the cellular response toprotein aggregation [Johnston et al., 1998].Microtubules are linear polymers of tubulin thatform polarized tracks extending from the centrosome andradiate outward toward the periphery of eukaryotic cells.A wealth of literature establishes that the MT cytoskel-eton is responsible for most of the directed movement of material inside of cells including: the dynamic assemblyof membrane-bounded organelles like the Golgi appara-tus [Lippincott-Schwartz, 1998], the endoplasmic retic-ulum [Terasaki et al., 1986], the intracellular movementof mitochondria [Gotoh et al., 1985], lysosomes and Abbreviations used: ALLN, N-Acetyl-L-leucyl-L-leucyl-L-norleuci-nal; CFTR, cystic fibrosis transmembrane conductance regulator;MTOC, microtubule organizing center; MT, microtubule.Contract grant sponsor: National Institutes of Health; Contract grantnumber: DK43994.*Correspondence to: Dr. Jennifer A. Johnston, 800 Gateway Blvd.,South San Francisco, CA 94980.E-mail: jjohnston@elanpharma.comReceived 12 November 2001; Accepted 8 April 2002 Publishedonline22July2002inWileyInterScience www.interscience.wiley.com .DOI:10.1002/cm.10057 Cell Motility and the Cytoskeleton 53:26–38 (2002) ©  2002 Wiley-Liss, Inc.  endosomes [Matteoni and Kreis, 1987], and the move-ment of chromosomes at mitosis [Barton and Goldstein,1996]. MTs function as a dynamic cellular scaffold uponwhich force-generating motor proteins move direction-ally to transport their cargo. Motor proteins fall into twocategories: anterograde motors (mainly kinesins), whichmove cargo from the centrosome to the cell periphery,and retrograde motors such as cytoplasmic dynein, whichmove cargo in the opposite direction. Motor proteins of both types traditionally have been considered to carrymembrane bounded cargo (such as organelles and vesi-cles) [Gotoh et al., 1985]. However, the demonstrationthat cytoplasmic dynein mediates retrograde transport of membrane-free viral capsids [Sodeik et al., 1997] and thedelivery of MT into axons [Ahmad et al., 1998] estab-lishes that the MT transport apparatus is not limited tomembrane-bounded cargo.Cytoplasmic dynein usually conducts cargo in as-sociation with dynactin, a 20S complex consisting of atleast 9 polypeptides that appear to contribute an essentialrole in linking cargo to the dynein motor [Karki andHolzbaur, 1999; Eckley et al., 1999; King and Schroer,2000]. The p50 subunit of dynactin (dynamitin) has beenshown to be directly involved in the attachment of cargoto dynein in a variety of systems. Overexpression of dynamitin dissociates the dynactin-dynein complex anddisrupts cytoplasmic dynein-dependent events includingchromosome alignment and spindle organization in mi-tosis [Echeverri et al., 1996], localization of membranousorganelles in interphase [Burkhardt et al., 1997], andaxonal transport of MT into axons [Ahmad et al., 1998].The p150 glued protein is another subunit of the dynactincomplex and has been shown to interact with dynein in amanner that is critical for dynein function [Vaughan andVallee, 1995; Boylan et al., 2000]. An active area of investigation concerns the determination of how the dy-nein/dynactin complex interacts with speci fi c cargo.We have previously reported that the pericentriolaraccumulation of ubiquitinated and aggregated proteininto aggresomes requires an intact MT cytoskeleton[Johnston et al., 1998]. In the absence of MTs, multiplesmall foci of aggregated protein are distributed randomlythroughout the cytoplasm, suggesting that aggresomeformation requires directed transport on MT. In thepresent report, we have investigated the involvement of MT motors in aggresome formation. Our data indicatethat aggresome formation is accompanied by a partialredistribution of dynein, p50 dynamitin, and p150 glued protein to aggresomes. The overexpression of dynamitinblocks aggresome formation. Together with data show-ing that misfolded protein aggregates co-sediment withMT in vitro, our data establish a central role for thedynein/dynactin complex in the cellular response to pro-tein aggregation. MATERIALS AND METHODS Antibodies and Reagents C. Echeverri and R. Vallee (Worcester Institute,MA) generously donated p50 dynamitin cDNA and an-tibodies. Polyclonal antibodies to the C-terminus of CFTR [Ward and Kopito, 1994] were af  fi nity puri fi ed byhigh pH elution from a CFTR peptide column. Alpha-mannosidaseII antibodies were the gift of M. Farquhar[Velasco et al., 1993]; pericentrin polyclonal antibodieswere the gift of T. Stearns (Stanford University, CA).Other antibodies used: monoclonal dynein IC 70.1 andmonoclonal kinesin IBII (Sigma, St. Louis, MO); anti-p150, p50 (dynamitin) Chemicon (Temecula, CA);  fl u-orophore conjugated secondary antibodies Alexa 488 andAlexa 546 were purchased from Molecular Probes (Eu-gene, OR). HEK 293 cells were transfected by themethod of calcium phosphate precipitation, or were sta-bly expressing GFP-  F508 CFTR as described [Johnstonet al., 1998]. Puri fi ed tubulin was purchased from Cy-toskeleton, Inc (Denver, CO). The proteasome inhibitorsepoxomicin, clasto-lactacystin-  -lactone, MG-132, andALLN were purchased lyophilized from Boston Bio-chem., Inc. (Cambridge, MA) and were reconstituted inDMSO. Immunoblotting Cell pellets from washed and transfected HEK cellswere lysed in 250   l of ice-cold IPB buffer (10 mMTris-HCl [pH    7.5], 5 mM EDTA, 1% NP-40, 0.5%deoxycholate, 150 mM NaCl) plus protease inhibitors(100   M TLCK, 100   M TPCK, 1 mM PMSF) for 30min on ice. Insoluble material was recovered by centrif-ugation at 13,000 g  for 15 min and solubilized in 50  l 10mM Tris-HCl, 1% SDS for 10 min at room temperature.After addition of 200  l IPB, samples were sonicated for20 s with a tip sonicator. Cell fractions, normalized fortotal protein, were separated on 7.5% SDS-PAGE andelectroblotted. Chemiluminescent detection was carriedout with the Renaissance detection kit (New EnglandNuclear). Microtubule Assembly Assays Microtubules were assembled from 0.25 – 0.5mg/ml puri fi ed tubulin in G-PEM Buffer (80 mM Pipes,1 mM MgCl 2 , 1 mM EGTA) at room temperature by theaddition of 10  M paclitaxel (Taxol) in DMSO (Sigma)and 25   M GTP. After 20 min at room temperature,GFP-  F508 CFTR cell extract was added (1/3 volume of MT assembly reaction) and incubated at 16 ° C for 1 – 12 h(routinely 1-h incubations were used, with occasional12-h overnight incubations; no observable difference inassociated CFTR after 1 h). GFP-  F508 CFTR cell ex-tracts were prepared as follows: 90% con fl uent T75 Microtubule Motors and Aggresome Formation 27  fl asks of cell lines stable expressing GFP-  F508 CFTRwere treated with 25  M MG-132 for 5 – 7 h. Cells wereharvested by release into 1 ml PBS/4mM EDTA, 3,000 g spin and then resuspended in 500   l of G-PEM   1%NP-40. Extraction was allowed to proceed on ice for 20min, then centrifuged at 4 ° C for 5 min at 3,000 g . Thesupernatant from the 3,000 g  spin was then centrifugedfor 20 min at 15,000 g . The resulting material is extractthat was added to MT assembly assays. Protein not usedimmediately was quick frozen in liquid nitrogen andstored at  80 ° C. After 16 ° C incubation for MT af  fi nityreactions, mixtures were underlain with a 10% sucrosecushion made in G-PEM containing taxol and GTP (ex-cept for the control reactions, which do not contain anytaxol or GTP), and centrifuged at 10,000 g  for 15 min atroom temperature. Collected MTs were resuspended inequal volume of G-PEM, 10   l from both Supernatantand Pellet fractions were removed to which equal vol-umes of 2   sample buffer was added for analysis bySDS-PAGE. Electron Microscopy  Microtubule assembly assays were performed asdescribed above with the exception that assay mixtureswere  fi xed with 2% glutaraldehyde (EMS, Fort Wash-ington, PA) for 20 min at room temperature prior tosedimentation through 10% sucrose. Microtubule pelletsfrom in vitro MT assembly assays were resuspended in100   l of G-PEM buffer. Formvar-Ni 200mesh grids(EMS, Inc.) were  fl oated on 20  l of   fi xed MTs for 5 minat room temperature. Nonspeci fi c binding was blockedby 5% BSA/PBS for 10 min, followed by 25 min inprimary antibody in PBS/1%BSA. Following  fi ve PBS/ 1%BSA washes, grids were  fl oated for 20 min on pre-cleared 15-nm gold conjugated secondary antibodies(EMS, Inc.) diluted 1:10 in PBS/BSA. Subsequently,  fi vePBS washes and  fi ve water washes, a 5-min post  fi x in2% glutaraldehyde followed by  fi ve more water washeswere carried out. Washed grids were stained with 1%uranyl acetate for 10 min at room temperature, air dried,and observed with a JOEL transmission electron micro-scope at 60 kV. Immunofluorescence Microscopy  In all experiments, HEK 293 cells grown on no. 1coverslips were  fi xed in  20 methanol for 6 min. After fi xation, cells were washed 5   in PBS, followed by 10min in 5% BSA. Primary antibodies were added to eachcoverslip in 1% BSA/PBS and incubated for 30 min to2 h at room temperature. Cells were washed in PBS andstained for 3 min with 10  g/ml bisbenzimide. Followingone  fi nal wash in PBS, secondary antibodies conjugatedto  fl uorophore were added for 20 – 45 min at room tem-perature. Cells were washed again 5  in PBS, and thenmounted in 50%glycerol/50% PBS on microscope slidesand viewed with a Zeiss Axiovert, using Metamorphsoftware (Universal Imaging) and a Princeton Instru-ments CCD to collect and analyze images. Adobe Pho-toshop v6.0 (San Jose, CA) was used to prepare  fi nalimages for publication. RESULTS To examine the role of molecular motors in aggre-some formation, we used indirect immuno fl uorescencemicroscopy to assess the distribution of the anterogrademotor kinesin (Fig. 1) and the retrograde motor cytoplas-mic dynein (Fig. 2) in HEK cells stably expressing GFP-  F508 CFTR and exposed to proteasome inhibitors. This  F508 mutant membrane protein has previously beenshown to be quantitatively incapable of folding and isnormally rapidly degraded in a proteasome- and ubiq-uitin-dependent fashion [Jensen et al., 1995; Ward et al.,1995]. We previously reported that overexpression of   F508 CFTR leads spontaneously to the formation of aggresomes containing ubiquitinated misfolded   F508CFTR molecules that have been extracted or  “ dislocated ” from the ER membrane [Johnston et al., 1998]. Exposureof these cells to proteasome inhibitor (epoxomicin [Sin,1999], lactacystin [Fenteany and Schreiber, 1998], MG-132 [Bogyo et al., 1997], or ALLN [Vinitsky et al.,1992]) leads to formation of massive aggresomes within8 – 16 h [Johnston et al., 1998]. Epoxomicin is currentlythe most speci fi c inhibitor available [Kim, 1999; Sin,1999]. Large CFTR immunopositive aggresomes, evi-dent in epoxomicin treated GFP-  F508 CFTR express-ing cells (Figs. 1C, 2D), were also strongly stained withantibody to cytoplasmic dynein (Fig. 2C) but not withantibody to kinesin (Fig. 1D). By contrast, cytoplasmicdynein exhibited a diffuse, somewhat punctate distribu-tion throughout the cytoplasm of untreated untransfectedHEK cells (Fig. 2G) as well as HEK cells stably express-ing GFP-  F508 CFTR (Fig. 2A). Pericentrin staining inFigure 2H demonstrates the cellular location of the cen-trosome, and a comparison to Figure 2G suggests thatdynein is not normally concentrated at the centrosomearea (merged image in Fig. 2I). When untransfected HEKcells were exposed overnight to epoxomicin, a signi fi cantportion of the cell ’ s cytoplasmic dynein was found to bestrongly colocalized with pericentrin staining (Fig. 2J – L). This redistribution of dynein was accompanied withthe distortion of the nuclear envelope that is highlycharacteristic of aggresome formation [Johnston et al.,1998]. These data suggest that proteasome inhibitionredistributed dynein, but not kinesin, to the area occupiedby aggresomes, where dynein colocalizes both with cen-trosomal markers and with misfolded aggregated protein. 28 Johnston et al.  Moreover, the data in Figure 2J – L indicates this redistri-bution is not dependent on CFTR expressing cells.Although GFP-  F508 molecules that accumulatein aggresomes are misfolded, and largely insoluble innon-denaturing detergent [Johnston et al., 1998] (Fig.3B, compare lanes 3,4 with 5,6), neither the mobility northe detergent solubility of dynein was affected by pro-teasome inhibition (Fig. 3A compare lanes 1, 2 with 7, 8).These data suggest that dynein is not a substrate for theaggresome pathway.Retrograde transport of cargo by cytoplasmic dy-nein usually occurs in the context of a dynein/dynactincomplex, which, in addition to the force-generating dy-nein heavy chain, contains components like dynamitin Fig. 1. Kinesin does not redistribute to aggresomes. HEK cells stably expressing GFP CFTR (A – D) andimaged for GFP  fl uorescence ( A,C ) or indirect immuno fl uorescence using kinesin antibodies ( B,D ).Vehicle treated cells are shown in A,B; C,D demonstrate cells treated with 3  M epoxomicin. Bar  15  m. Microtubule Motors and Aggresome Formation 29
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