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MINIREVIEW Amyloid oligomers: dynamics and toxicity in the cytosol and nucleus Akira Kitamura 1 and Hiroshi Kubota 2 1 Department of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Kita-ku, Sapporo, Japan 2 Department of Life Science, Faculty of Engineering and Resource Science, Akita University, Akita, Japan Introduction The accumulation of misfolded proteins in the cytosol and nucleus causes neurodegenerative disease [1–3]. For example, proteins harboring expanded polyglutamine (polyQ) tracts cause polyQ diseases, which include Huntington’s disease and several spinocerebellar ataxias [4,5], and mutations in superoxide dismutase 1 (SOD1) lead to familial amyotrophic lateral sclerosis (ALS) [6,7]. In these diseases, inclusions of the mutant proteins are found in the neuronal cells of patients and the accumulation of misfolded proteins is considered to be a primary cause of neuronal dysfunction and death. The aggregation-prone nature of the mutant proteins suggests that misfolded proteins dis- turb neuronal cell functions via unnecessary interactions with normal proteins. However, the mechanism by which mutant proteins exert their cytotoxicity is largely unknown. Although these diseases have a late onset, as symptoms appear in adulthood, the molecular mechanisms underlying the age-dependent onset are poorly understood. Moreover, little is known about Keywords live cell imaging; misfolded protein; molecular chaperone; neurodegenerative disease; neuronal cell death; oligomer; protein aggregation; protein degradation; protein interaction; spectroscopic analysis Correspondence H. Kubota, Department of Life Science, Faculty of Engineering and Resource Science, Akita University, 1-1 Tegatagakuen- cho, Akita 010-8502, Japan Fax: +81 18 75 3053 Tel: +81 18 75 3053 E-mail: hkubota@ipc.akita-u.ac.jp (Received 4 September 2009, revised 29 November 2009, accepted 1 December 2010) doi:10.1111/j.1742-4658.2010.07570.x The accumulation of misfolded proteins in the cytosol and nucleus of neuronal cells leads to neurodegenerative disorders. Polyglutamine diseases are caused by polyglutamine-expanded proteins, whereas mutations in superoxide dismutase 1 lead to amyotrophic lateral sclerosis. These structurally unstable mutant species perturb essential interactions between normal proteins and tend to aggregate because of the presence of exposed hydrophobic surfaces. Accumulating evidence suggests that soluble species, including misfolded monomers and oligomers, are more toxic than large insoluble aggregates or inclusions. Spectroscopic analysis, including fluorescence recovery after photobleaching and fluorescence loss in photobleaching, in living cells revealed that protein aggregates of misfolded proteins are dynamic structures that interact with other proteins, such as molecular chaperones. Fluorescence correlation spectroscopy analysis detected soluble oligomers ⁄ aggregates of misfolded proteins in cell extracts. Fluorescence resonance energy transfer analysis supported the notion that soluble oligomers ⁄ aggregates are formed before the formation of inclusions in vivo. Here, we reviewed the characteristics of oligomers and aggregates of misfolded proteins, with a particular focus on those revealed by spectroscopic analysis, and discussed how these oligomers may be toxic to cells. Abbreviations ALS, amyotrophic lateral sclerosis; AR, androgen receptor; CCT, chaperonin containing t-complex polypeptide 1; CFP, cyan fluorescent protein; FCCS, fluorescence cross-correlation spectroscopy; FCS, fluorescence correlation spectroscopy; FRAP, fluorescence recovery after photobleaching; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; HDAC6, histone deacetylase 6; HSP, heat shock protein; polyQ, polyglutamine; RFP, red fluorescent protein; SCA, spinocerebellar ataxia; SOD1, superoxide dismutase 1; YFP, yellow fluorescent protein. FEBS Journal 277 (2010) 1369–1379 ª 2010 The Authors Journal compilation ª 2010 FEBS 1369 how the mutant proteins specifically damage particular neuronal cells. Recent progress in spectroscopic imaging analysis, using proteins tagged with green fluorescent protein (GFP) and related cyan, yellow and red fluorescent proteins (e.g. CFP, YFP, RFP respectively) allowed us to trace the tagged proteins in living cells [8]. Mutant proteins that cause polyQ disease and ALS have been tagged with these fluorescent proteins and analyzed by fluorescence microscopy-based spectroscopic analysis, as well as by conventional biochemical experiments. The spectroscopic techniques used for living cells include fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP) and fluorescence ⁄ Fo ¨ rster resonance energy transfer (FRET) (Fig. 1). These techniques reveal real-time movements and interactions of misfolded proteins in living cells. The recent application of fluorescence correlation spectroscopy (FCS), which is a microscopy-based technique used for the analysis of fluorescent molecules at the single-molecule sensitivity [9,10], to misfolded mutant proteins succeeded in detecting their soluble oligomers ⁄ aggregates in cell extracts. Together with evidence from other cell biological and biochemical analyses, we discussed the role of soluble oligomers of toxic species in protein-misfolding diseases, including polyQ disease and ALS. Soluble oligomers of misfolded proteins as potentially toxic species PolyQ-expanded proteins and ALS-linked mutant SOD1 are structurally unstable [5,7]. These proteins thus tend to aggregate and interact with other proteins via exposed hydrophobic surfaces, leading to the pertur- bation of cellular activities (Fig. 2). The hydrophobic surfaces of misfolded proteins can be masked by molecular chaperones, and the aggregation of misfolded proteins is inhibited by chaperones through this activity [11,12]. However, the concentration of chaperones is limited in living cells, and these chaperones are required for the folding of newly synthesized normal proteins. Thus, the overloading of cellular chaperoning capacity by misfolded mutant proteins results in increased misfolding of normal proteins and further enhancement of co-aggregation. In this state, the degradation of misfolded proteins is diminished by their insolubility, and cellular functions are severely damaged by a negative chain reaction. This situation can be explained by escape from (or collapse of) the protein homeostasis net- work [13,14], and the accumulating incapacitation of protein homeostasis may explain, in part, the late onset of neurodegenerative disorders associated with protein misfolding. It should be noted that chaperone functions and substrate proteins differ among chaperones, to a certain extent, and their expression levels vary according to cell type. These differences may affect the protein species whose functions are inhibited and the cell types that are damaged under disease conditions. The decrease in the amount of functional proteins (e.g. transcription factors) as a result of becoming trapped in aggregates may explain the toxicity of misfolded proteins. However, a number of studies suggest that sequestration of misfolded proteins into inclusions is protective [15]. The total amount of exposed hydrophobic surfaces in misfolded proteins is much greater Detection of conformational change Dynamics of correctly folded monomer Detection of small oligomer Detection of soluble aggregate or amyloid fibril FRAP/FLIP FCS FCCS FRET Unsuitable Good Not applicable Not applicable Unsuitable Not sensitive* Less sensitive**** Good**** Unsuitable Less sensitive** Good**** Less sensitive*** Unsuitable Good Good**** Less quantitative Dynamics of inclusion body Good Not applicable Not applicable Less quantitative Fig. 1. Suitability of spectroscopic methods for the analysis of protein aggregation. Asterisks indicate the following: *, FCS can be applied only when the size of molecule is greatly altered by conformational change; **, FCS can detect most oligomers but this method is less sensitive for the detection of very small oliomers such as dimers or trimers; ***, FRET detects dimers and larger oligomers ⁄ aggregates as complexes but cannot determine their sizes; and ****, these methods have not been used in in vivo studies for the indicated purposes despite the availability. FCCS, fluorescence cross-correlation spectroscopy; FLIP, fluorescence loss in photobleaching; FRAP, fluorescence recovery after photobleaching. Dynamics and toxicity of cytosolic amyloid oligomers A. Kitamura and H. Kubota 1370 FEBS Journal 277 (2010) 1369–1379 ª 2010 The Authors Journal compilation ª 2010 FEBS in the monomeric and oligomeric states than in the inclusion state under conditions where the number of misfolded proteins per cell is identical. Although misfolded monomers can be trapped and refolded by molecular chaperones and degraded by the ubiquitin– proteasome system [16], oligomers are more resistant to refolding by chaperones and to degradation by the proteasome. Thus, soluble oligomers are considered as highly toxic to cells. Inhibition of oligomer formation is probably useful to protect cells against the toxicity of misfolded proteins. Sequestration of soluble oligomers ⁄ aggregates into inclusions or aggresomes by microtubule-dependent transport is considered to play a role in the removal of the potentially toxic soluble species from the cytosol [17]. Indeed, cells harboring polyQ-expanded Hunting- tin inclusions were reported to be more resistant to the toxicity of misfolded proteins than cells exhibiting dif- fusible patterns [18]. However, an opposing effect was reported using an ALS-linked mutant SOD1 [19], sug- gesting that the cell-protection activity exerted by inclusion ⁄ aggresome formation can be affected by differences in protein characters and other factors (e.g. expression level, time course and cell type). Structural differences between inclusions ⁄ aggresomes may also affect the cell-protection activity; polyQ-expanded Huntingtin proteins are tightly associated and immobile in the inclusion [20], whereas mutant SOD1 proteins are loosely packed in the aggresome and partly exchangeable with cytosolic SOD1 proteins [19]. In a Drosophila model of spinobulbar muscular atrophy, which is a neurodegenerative disease caused by expansion of a polyglutamine repeat in the androgen receptor (AR), histone deacetylase 6 (HDAC6) was shown to play an essential role in preventing polyglutamine toxicity [21]. HDAC6 is a microtuble-associated protein that interacts with polyubiquitinated misfolded proteins and dynein motors [22]. Through these interactions, HDAC6 mediates inclusion ⁄ aggresome formation of misfolded proteins in a microtubule-dependent manner. In this sequestration system, however, the details of transported species (e.g. monomer, oligomer Non-toxic conformation Toxic monomer Toxic small oligomer Degradation by proteasome Degradation resistant Soluble aggregate Inclusion Degradation by autophagy Chaperone Newly-synthesized polypeptide Aberrant interaction with other proteins Conformational change and further binding Inactivation of other proteins A B C Depletion of chaperones b y oli g omers Increased misfolding b y impaired chaperonin g activit y Further stimulation of a gg re g ation Fig. 2. Possible mechanisms of oligomer toxicity in neurodegenerative disease. (A) Small oligomers are hardly degraded by the proteasome and autophagy. (B) Oligomers inhibit protein functions by aberrant interaction and co-aggregation. (C) Depletion of molecular chaperones by oligomers leads to further stimulation of co-aggregation. A. Kitamura and H. Kubota Dynamics and toxicity of cytosolic amyloid oligomers FEBS Journal 277 (2010) 1369–1379 ª 2010 The Authors Journal compilation ª 2010 FEBS 1371 or soluble aggregate) remain unknown. Thus, these observations are not contradictory with the notion that soluble oligomers are highly toxic to cells. The autophagy–lysosome pathway plays a role in the clearance of misfolded protein aggregates [23,24] and this pathway is a candidate system for the removal of soluble oligomers ⁄ aggregates. For example, beclin 1, an essential component of the autophagy system, is required for the effective removal of polyQ aggregates [25]. In the study by Pandey et al. [21] the amount of polyQ-expanded AR aggregates were significantly decreased by the over-expression of HDAC6, but increased by the knockdown of autophagy components. Interestingly, in the fly that expresses polyQ- expanded AR, the rescue of eye degeneration by HDAC6 was dependent on autophagic activity. A similar role of HDAC6 was shown in mammalian cells [26], and an essential role of autophagy in preventing neurodegeneration was reported using knockout mice [27]. These observations suggest a link between the microtubule-dependent formation of aggresome and the autophagy-dependent clearance of misfolded proteins. Recently, the ubiquitin-binding protein p62 (also known as sequestosome 1) was shown to stimulate the aggregation of ubiquitinated proteins and to interact with LC3, an essential component of autophagy [28]. The p62 protein is required for the prevention of pol- yQ toxicity [29] and interacts with ALS-linked mutant SOD1 [30]. Thus, p62-bound misfolded proteins in a soluble state (e.g. soluble aggregates) may be removed by an autophagy-mediated degradation system. How- ever, the autophagy-mediated pathway is probably inefficient for the removal of small oligomers (e.g. trimer, tetramer, etc.), even though proteins such as p62 assist specific recognition, because this system uses bulk sequestration of cytosolic regions via the formation of double membranes. Further investigations are required to understand what size of aggregated species is effectively removed by the autophagy-mediated degradation system in a selective manner in living cells. Two types of neurodegenerative diseases caused by misfolded proteins in the cytosol and nucleus PolyQ diseases Expansion of the polyQ tract in at least nine proteins causes neurodegenerative disorders [4,5,31,32]. PolyQ expansion in the Huntingtin protein causes Hunting- ton’s disease, and polyQ-expanded ataxin-1 leads to spinocerebellar ataxia type 1 (SCA1; also known as olivopontocerebellar atrophy type 1). PolyQ expansion in ataxin-3 is the cause of the most common dominant ataxia, spinocerebellar ataxia type 3 (SCA3, also known as Machado–Joseph disease). SCA2, SCA6, SCA7 and SCA17 are also caused by polyQ expansion, and expansion of the polyQ repeat in the AR is responsible for spinal and bulbar muscular atrophy. The expanded polyQ tract is encoded by a CAG repeat in the causative genes, and polyQ diseases are inherited dominantly. PolyQ repeats contain approximately 10–30 glutamine residues in healthy individuals, whereas they are expanded to more than 40 repeats in patients. In the polyQ diseases, inclusions containing polyQ- expanded proteins are found in the nucleus and ⁄ or cytoplasm of neuronal cells in the brain of patients. The function of neuronal cells is progressively dis- turbed, leading to cell death. PolyQ-expanded proteins aggregate easily in vivo and in vitro, and these aggregates are very difficult to dissolve, even in the presence of strong detergents, such as SDS [33]. PolyQ- expanded proteins trap normal functional proteins in the aggregation process, which suggests a possible mechanism of toxicity. PolyQ aggregates have been shown to be rich in b-sheets [34], and Nagai et al. [35] demonstrated that b-sheet-containing monomers and oligomers produced in vitro are toxic to cultured neuronal cells when introduced by microinjection. A recent study of the conformation and toxicity of polyQ-expanded Huntingtin indicated that fragile amyloid, which is rich in exposed and flexible regions, is significantly more toxic than rigid amyloid, which comprises buried and fixed regions [36]. As the former is considered to break into oligomers and to be accessible to other proteins more easily than the latter, these observations are consistent with the notion that oligomers with specific conformations are toxic to cells. Molecular chaperones, including heat shock protein (HSP)70, cognate of HSP70 (HSC70), chaperonin containing t-complex polypeptide 1 (CCT) (also called TRiC), HSP40 (DnaJ) and small HSPs (e.g. HSP27 and crystalins), play crucial roles in the protection of cells against the toxicity of polyQ-expanded proteins [11,12]. For example, HSP70, which is a cytosolic molecular chaperone that interacts with the hydrophobic surfaces of denatured and misfolded proteins, prevents aggregation of polyQ-expanded proteins and inhibits their toxicity [37–39]. The cytosolic chaperonin CCT is a molecular chaperone that prevents b-sheet aggregation by recognizing hydrophobic b-strands [40]; this chaperone prevents aggregate formation and toxicity of polyQ-expanded proteins [41–44]. CCT weakly recognizes monomeric and oligomeric forms (2–5 mers) Dynamics and toxicity of cytosolic amyloid oligomers A. Kitamura and H. Kubota 1372 FEBS Journal 277 (2010) 1369–1379 ª 2010 The Authors Journal compilation ª 2010 FEBS of Huntingin-Q53, but not fibril forms of the protein [43]. Depletion of CCT activity by RNA interference in polyQ-expressing cells results in increased amounts of soluble aggregates and cell death [42], which suggests that CCT interferes with polyQ aggregation by trapping monomers or small oligomers, thus inhibiting their toxicity. These observations support the notion that b-sheet-rich oligomers of polyQ-expanded proteins are toxic to cells and that inhibition of oligomer formation is an effective strategy for the inhibition of polyQ toxicity. Familial ALS caused by mutant SOD1 ALS is a neurodegenerative disorder characterized by the progressive loss of motor neurons. Although 90% of ALS cases are sporadic, the remaining 10% are caused by genetic mutations that are inherited dominantly. Dominant inheritance of familial ALS suggests a toxic gain of function, similarly to other protein- misfolding diseases, such as the polyQ diseases. However, the molecular mechanisms of ALS toxicity are largely unknown. Moreover, little is known about how the mutant gene products specifically damage and kill motor neurons. Several causative genes have been identified for familial ALS, including SOD1, TDP-43 and FUS ⁄ TLS [45]. Mutations in SOD1 are the most common cause of familial ALS, and SOD1 mutants aggregate in the cytosol of neuronal cells [6,46,47]. More than 100 ALS-linked mutations have been identified in SOD1 and these mutant proteins are structurally unstable [7,48]. Because of structural instability, the SOD1 mutants are thought to expose hydrophobic surfaces more easily than the wild-type protein. Thus, these proteins tend to aggregate and potentially exert their toxicity via aberrant interactions with other normal proteins. Recently, Wang et al. [49] used a mouse model of ALS-linked mutant SOD1 (G85R) to show that soluble oligomers of mutant SOD1 are detectable biochemically in spinal cord extracts before the onset of visible motor neuron dysfunction. Similar oligomers were also detected biochemically in Caenorhabditis elegans expressing the G85R mutant [50]. In the mouse model, insoluble aggregates were detected at the onset of symptoms, which suggests that soluble oligomers are further aggregated into inclusions. These observations suggest that soluble oligomers of mutant SOD1 appear when cellular chaperoning and other quality-control pathways are overwhelmed by the accumulation of misfolded proteins. Although the molecular chaperone HSC70 was associated with soluble species of mutant SOD1 at any stage, HSP110, which is a nucleotide exchange factor of HSP70 ⁄ HSC70, was associated with the mutant protein after the initiation of motor neuron dysfunction. The structure and toxicity of soluble oligomers may differ according to the stage of disease progression. Extracellular oligomers have been suggested to be a pathogenic factor of neurodegenerative diseases, including Alzheimer’s disease and prion diseases [51– 53]. For example, amyloid-b peptide (Ab) oligomers induce synaptic disfunction, probably by interfering with receptor-dependent signaling pathways via binding to synaptic plasma membranes. In the case of the ALS-linked mutant, SOD1, Urushitani et al. [54] indicated (using cultured cells) that these mutants are secreted from neuronal cells through a chromogranin- mediated pathway and that extracellular mutant SOD1 triggers microgliosis and neuronal cell death. In a mouse model of ALS, a conditional knockout of mutant SOD1 in astrocytes revealed that these cells affect the disease progression, but not the onset, of ALS in a noncell autonomous manner [55]. In this report, extracellular mutant SOD1 was suggested as a candidate for the mediator. These observations suggest that extracellular mutant SOD1 may play an addi- tional role in the pathogenesis of mutant SOD1-mediated ALS. As in vitro studies for mutant SOD1 indicate that post-translational events (including metal binding and disulfide formation) affect oligomer and fibril formation [56,57], the aggregation state may be altered by extracellular environmental conditions. Thus, like other neurodegenerative diseases, extracellular oligomers of mutant SOD1 might act as a toxic species, although this possibility remains to be investigated. In vivo dynamics of misfolded proteins revealed by spectroscopic imaging analyses FRAP and FLIP analyses of aggregates and interacting proteins Time-lapse observation of fluorescently labeled molecules is often used to trace the movement of cellular structures. However, this method cannot analyze the mobility of molecules distributed uniformly and is unsuitable for the determination of molecular-exchange rates from one structure to another. FRAP is a method that measures the mobility of rapidly moving fluorescent molecules in a living cell [8]. Molecules labeled with a fluorescent protein (i.e. GFP and related proteins) are bleached in a region of interest for a short time-period and the subsequent movement of fluorescent molecules from the unbleached area is quantitatively analyzed by A. Kitamura and H. Kubota Dynamics and toxicity of cytosolic amyloid oligomers FEBS Journal 277 (2010) 1369–1379 ª 2010 The Authors Journal compilation ª 2010 FEBS 1373 the recovery of fluorescence intensity. This method is useful for the quantitative analysis of the mobility of aggregation-prone proteins in a living cell (Fig. 1). In FLIP analysis, fluorescently labeled molecules are con- tinuously bleached in a region and the fluorescence of unbleached areas is measured. FLIP can be used for the analysis of molecular transfers between two or more regions, regardless of the speed of movement, even if this method is less quantitative than FRAP. Thus, FRAP is very useful for determining the locoregional mobility of proteins, whereas FLIP can comprehen- sively analyze protein trafficking. The mobility of polyQ-expanded proteins in inclusions has been analyzed by FRAP and FLIP. FRAP analysis of polyQ-expanded ataxin-3 tagged with GFP (GFP-ataxin-3-Q82) revealed that polyQ-expanded ataxin-3 is immobile in the nuclear inclusion [58]. In addition, FLIP analysis indicated that polyQ-expanded ataxin-3 is unable to shuttle between the inclusions and the nucleoplasm. These results demonstrate that the inclusion body formed by polyQ-expanded ataxin-3 is a structure that is immobilized in the nucleus. By contrast, FRAP analysis of GFP-ataxin-1-Q84 demonstrated that ataxin-1 is mobile in nucleoplasmic inclusions [59]. Interestingly, there are two types of ataxin-1 inclusions: one undergoes fast and complete exchange with a nucleoplasmic pool and the other exhibits slow exchange rates. The slowly exchanging inclusions contain high levels of ubiquitin and low levels of proteasome, which suggests a role that is distinct from that of the rapidly exchanging inclusions. Inverse FRAP analysis of ataxin- 1 indicated that wild-type ataxin-1 shuttles between the nucleus and the cytosol, whereas polyQ-expanded ataxin-1 is not exported from the nucleus [60]. These observations suggest that the ataxin-1 accumulated in the nucleus becomes a species that is unable to pass through nuclear pores. FRAP was also used to analyze the dynamics of ALS-linked mutant SOD1 in cytosolic inclusions [19]. Mutant SOD1 shuttles dynamically between the inclusion body and the cytosol in neuronal cells, which suggests that the inclusion body of mutant SOD1 is not an immobile structure. By contrast, polyQ and polyQ-expanded Huntingtin formed immobile inclusions in the cytosol and in the nucleus [20]. Thus, there are at least two types of inclusions – mobile inclusions and immobile inclusions – which is consistent with a recent study proposing two distinct inclusion-like compartments for protein quality control [61]. FRAP and FLIP are also useful for analyzing the transient association of other proteins with the inclusions. FRAP analysis of HSP70–YFP in Huntingtin- 150Q–CFP inclusions revealed that HSP70 is mobile within the inclusion [20]. As the movement of HSP70 is significantly slower in the inclusion than in the cytosol, HSP70 appears to interact transiently with aggregated mutant proteins in the inclusion. These observations indicate that HSP70 localized in inclusions is not co- aggregated in inclusions and thus may play a role in the modulation of the potentially toxic hydrophobic surfaces of polyQ aggregates. Interaction of HSP70 with the ALS-linked mutant SOD1 was analyzed using FLIP [19]. By continuous photobleaching of YFP–SOD1 in a small cytosolic region, the fluorescence intensity of the nonbleached area was decreased more slowly in aggregate-containing cells than in aggregate-free cells. These observations suggest a dynamic interaction between HSP70 and mobile inclusions of mutant SOD1 in living cells. HSP70 might shuttle with misfolded mutant SOD1 between the inclusions and the cytosol. FCS analysis of oligomers and soluble aggregates The mobility or exchange rate of aggregate-prone proteins in inclusions has been estimated using FRAP, as described above. However, this method is unsuitable for determining the diffusion coefficients of rapidly moving molecules (or particles), because the diffusion rates are faster than the image capture rate on the detector. By contrast, FCS is appropriate for this pur- pose [8–10]. For FCS analysis, a very small fluorescence detection volume (the so-called confocal volume) is cre- ated using optics similar to that of a confocal micro- scope. In the FCS optics, fluorescent molecules are excited by a diffracted narrow laser beam and detected in a pinhole aperture-regulated thin layer. When fluorescent molecules pass through the confocal volume, fluorescence fluctuation is detected using a highly sensitive photodetector. The fluctuation is analyzed as a calculated autocorrelation function, which provides the residence time of diffusing molecules in the confocal volume. As diffusion coefficients correlate with the fric- tion between the molecule and the solvent, the molecular mass of the molecules can be calculated by assuming the molecular shape (e.g. sphere or rod). FCS analysis allows determination of the concentration of fluorescent molecules and of fluorescence intensity per molecule (or counts per particle) thus, the distribution of differently sized oligomeric species can be estimated. As FCS analysis can be performed in living cells as well as in solution [10,62], this technique is becoming a pow- erful tool for the quantitative analysis of protein complexes, including soluble oligomers ⁄ aggregates of misfolded proteins, as described below. The presence of soluble oligomers (or soluble aggregates) has been demonstrated by FCS analysis using Dynamics and toxicity of cytosolic amyloid oligomers A. Kitamura and H. Kubota 1374 FEBS Journal 277 (2010) 1369–1379 ª 2010 The Authors Journal compilation ª 2010 FEBS extracts of cultured cells expressing long polyQ repeats or polyQ-expanded Huntingtin tagged with GFP or YFP [42,63]. The amount of soluble oligomers ⁄ aggregates was significantly increased by the RNA interference-mediated knockdown of the cytosolic chaperonin CCT, which suggests that CCT prevents oligomer formation of polyQ-expanded proteins in an early step of aggregation, under normal conditions [42]. In this study, records of count rate indicated that bright particles of Q82–GFP and Huntingtin-Q143–YFP passed through the confocal volume in the CCT-depleted cell extract. In another study using FCS, fluorescence intensity per particle increased for Q45–GFP and Q81–GFP in a time-dependent manner [63]. Further- more, a polyQ-binding polypeptide (QBP1) significantly inhibited the increase of fluorescence intensity per particle for Q45–GFP. These observations suggest that the soluble oligomers ⁄ aggregates detected by FCS contain multiple polyQ-expanded proteins, which are probably homo-oligomeric, at least in part. Fluorescence cross-correlation spectroscopy (FCCS) detects the direct interaction between two fluorescent molecules at a near single-molecule sensitivity [10,64]. For FCCS measurements, two molecules are labeled with different fluorophores that are distant in wave- length, and a solution of these labeled molecules is analyzed using FCS equipment. An interaction between the denatured proteins and small HSPs was determined using FCCS in vitro [65]. Although this study was carried out in vitro, FCCS can be performed in living cells or in cell lysates. Thus, this method has the potential to analyze the interaction between aggregation-prone protein and binding protein (e.g. chaperones) in living cells. FRET measurements to analyze molecular interactions in aggregates FRET provides a useful tool with which to detect interactions between proteins labeled with a fluorescent tag. FRET analysis is a method that measures energy transfer from a donor fluorophore to a nearby acceptor chromophore. FRET efficiency is highly dependent on the distance between the donor and the acceptor. Sev- eral methods have been used to detect FRET signals, including spectral scanning, ratio imaging, the recovery of donor fluorescence after acceptor photobleaching and fluorescence lifetime measurement of the donor. Molecular interactions between neurodegenerative disease-associated proteins in aggregates have been analyzed by FRET using the ratio imaging method. For example, polyQ-expanded proteins show strong FRET in the inclusions when they are tagged with enhanced (E)CFP as a donor and EYFP as an acceptor [20,66]. PolyQ-expanded proteins (Q82–CFP) co-aggregate with normal-length polyQ (Q19–YFP), as detected by FRET [20]. Because FRAP analysis in Q82–FLAG-based inclusions indicates that the exchange rate of Q19–GFP is faster than that of Q82–GFP, Q19 interacts weakly with Q82. Although TATA-box binding protein, which contains a short polyQ tract, is also sequestered in the Q82 inclusion, this protein exhibited more rapid exchange than Q19–GFP. Thus, protein mobility in inclusions appears to depend on the external polypeptide sequence as well as on the length of the polyQ repeat. In this report, the FRET efficiency was variable among cells, which suggests the presence of cell-to-cell heterogeneity in the molecular interactions within Q82–CFP ⁄ Q19–YFP aggregates. As FRET analysis via fluorescence recovery of the donor after acceptor photobleaching for Q40 in C. elegans also indicates the presence of heterogeneity in living neurons [67], the molecular interactions of polyQ proteins may be affected by unknown cellular conditions. FRET was also used to screen for inhibitors of polyQ aggregation in cultured cells, which indicates that this method has high-through- put potential [68]. Recently, Takahashi et al. [69] reported that soluble FRET-positive species of polyQ- expanded proteins were detected before inclusion-body formation, and FRET signals were significantly decreased by inhibitors of aggregation. These observations suggest that soluble oligomers ⁄ aggregates of pol- yQ-expanded proteins are formed before the formation of inclusion bodies, which is consistent with the results of FCS analysis described above [42,63] and with studies reporting 4-50 nm particles of immunopurified polyQ- expansion proteins by atomic force microscopy [70,71]. Fluorescence lifetime measurement of a donor fluorophore is available for quantitative FRET analysis [72]. Fluorescence lifetime imaging microscopy can determine the distribution of fluorescence lifetime and is appropriate for cell-based assays. Fluorescence lifetime imaging microscopy analysis led to the detection of FRET signals for an ubiquitination substrate protein tagged with EGFP as a donor and ubiquitin tagged with REACh (a non-fluorescent variant of yellow fluorescent protein) as an acceptor in cultured cells, which indicates that this method can be used to analyze the distribution of ubiquitin conjugates of specific proteins [73]. The ubiquitination of ALS-linked SOD1 in cultured cells was analyzed using this system, which revealed that the distribution of fluorescence lifetime was different for the G85R and G93A mutants [74]. These observations are consistent with the fact that mutant SOD1 is polyubiquitinated for rapid degradation by the proteasome [75,76] and that the A. Kitamura and H. Kubota Dynamics and toxicity of cytosolic amyloid oligomers FEBS Journal 277 (2010) 1369–1379 ª 2010 The Authors Journal compilation ª 2010 FEBS 1375 G85R mutant is more structurally unstable than the G93A mutant [6,7]. Interestingly, the FRET efficiency of the polyubiquitinated mutant, SOD1–G85R, was stronger in a region near the plasma membrane than in inclusions, which suggests a role for the juxtamem- brane region in protein quality control. As proteaso- mal function is important for eliminating the potential toxicity of mutant proteins, such as SOD1, detailed spatiotemporal examination of the polyubiquitination of mutant SOD1 using FRET may be useful for inves- tigating the details of the role of the ubiquitin–proteasome system in neurodegenerative diseases in vivo.By contrast, polyubiquitinated SOD1–G93A emitted strong FRET signals in perinuclear inclusions. The localization of the polyubiquitinated mutant SOD1 may be affected by the structure of the mutant proteins, because the G93A mutant is more structurally stable than the G85R mutant. The FRET efficiency of SOD1–ubiquitin conjugates in cultured cells was highly correlated with that of SOD1–HSP70 complexes, which suggests that HSP70 plays a role in the polyubiquitination of mutant SOD1, perhaps by maintain- ing mutant SOD1 in a ubiquitination-competent state. Although FRET signals indicate steady-state molecular interactions, the combination of FRET analysis with other spectroscopic methods, including FRAP and FCS, provides important information on the dynamic biophysical properties of protein aggregates. The optical system of FCS can be applied to single- molecule FRET analysis. This method was used to analyze aggregate formation and protein interactions of polyQ-expanded proteins in vitro [43]. In this study, the fluorescence intensity of FRET and non-FRET signals was measured at the single-molecule sensitivity level using a dual-color system, and direct interaction between polyQ-expanded Huntingtin (Huntingtin-Q53) and the molecular chaperone CCT was detected using single-molecule FRET. Similar methods may be applicable to the analysis of the molecular structure of polyQ-expanded proteins or ALS-linked SOD1 in oligomers and aggregates formed in living cells. Inter- molecular FRET analysis has been applied to yeast Sup35 prion proteins and revealed that Sup35 exists as a monomer at low concentrations in vitro and adopts a compact state in yeast [77]. Intermolecular FRET may also be useful for the analysis of the aggregation of polyQ-expanded proteins or ALS-linked SOD1 in vivo. Conclusions and perspectives We reviewed the dynamics and toxicity of misfolded proteins (including polyQ-expanded proteins and ALS- linked SOD1) in living cells, with a particular focus on soluble oligomers ⁄ aggregates. Accumulating evidence strongly suggests that soluble oligomers of misfolded proteins are toxic to cells. However, the exact molecular mechanisms that underlie this cytotoxicity remain unknown. Real-time spatiotemporal observation of misfolded proteins in vivo is essential for the full understanding of these mechanisms, and spectroscopic analyses in living cells will greatly aid the detailed analysis of the processes involved in these diseases. In addition to the techniques described in this review, single-molecule observations in living cells may be required to elucidate how misfolded proteins produce toxic oligomers and interact with other proteins. 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MINIREVIEW Amyloid oligomers: dynamics and toxicity in the cytosol and nucleus Akira Kitamura 1 and Hiroshi Kubota 2 1 Department of Molecular Cell Dynamics,

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