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Fibrillar Structures of Yeast Prion Sup35 In Vivo
Chapter 25
Fibrillar Structures of Yeast Prion Sup35 In Vivo Hideki Taguchi and Shigeko Kawai-Noma Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
Chapter Outline Introduction271 Yeast Prion Sup35 as a Model Amyloid-Forming Protein in Cells 271 In Vitro Fibril Formation of Sup35 272 Structures of Sup35 Amyloids in Yeast Cells 272 Transmission Electron Microscopy (TEM) 273 Correlative Light Electron Microscopy (CLEM) 274 Cryo-Electron Tomography 274
INTRODUCTION Proteins often form aggregates. Protein aggregates can be categorized into two groups: amorphous (disordered) aggregates and ordered aggregates called amyloids. Amyloids are highly ordered fibrillar protein aggregates with a cross β-sheet structure [1], which binds Congo Red and thioflavin dyes [2,3]. Amyloids were originally defined as extracellular deposits in several human diseases but the nomenclature now also includes many protein aggregates with n on-pathologic origins, raising the possibility that amyloid formation is an inherent property of numerous different proteins with various biologic functions [1]. Representatives of such nonpathologic amyloids are budding yeast Saccharomyces cerevisiae prion proteins, such as Sup35 and Ure2 [4]. Scrapie in sheep, bovine spongiform encephalopathy (also called ‘mad-cow’ disease) in cattle, and Creutzfeldt– Jakob disease and kuru in humans, are transmissible spongiform encephalopathies (TSEs), which are also called prion diseases. A prion is a proteinaceous infectious particle that lacks nucleic acids; this means that it is an infectious protein [5]. In the prion, the altered conformations of a protein auto-catalytically convert the normal structure to the amyloid (Fig. 25.1A). This prion concept was developed Bio-nanoimaging. http://dx.doi.org/10.1016/B978-0-12-394431-3.00025-0 Copyright © 2014 Elsevier Inc. All rights reserved.
Dynamic Properties of Sup35 Amyloids in Living Yeast Cells Single-Cell Imaging System for Monitoring the Fate of Sup35-GFP Foci Fluorescence Correlation Spectroscopy (FCS) Fluorescence Recovery after Photobleaching (FRAP) Concluding Remarks
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for the mammalian neurodegenerative diseases, in which the PrP protein participates. However, the concept has been spread to several non-Mendelian genetic elements in budding yeast, such as [PSI+] and [URE3] in S. cerevisiae [6]. Because yeast is a quite tractable model eukaryote, yeast prions can provide many important insights into prion biology that are usually difficult to access with mammalian prions [7–12]. In particular, the mechanisms by which the prion proteins are propagated and transmitted have been unraveled in the yeast prion model. This chapter provides an overview of the in vivo structure and dynamics of yeast prion protein Sup35.
YEAST PRION Sup35 AS A MODEL AMYLOID-FORMING PROTEIN IN CELLS Although several dozen yeast proteins are known to behave as prions in vivo [13–16], the prion state of the Sup35 protein, the [PSI+] determinant, is the best-characterized prion. Sup35 is an essential protein, which normally functions as a translation termination factor (eRF3) in cooperation with its partner Sup45 (eRF1) [17], but aggregated forms of the Sup35 are the determinant of a prion phenotype [PSI+].
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Sup35, which consists of 685 amino acid residues, has three domains (Fig. 25.1B). The N-terminal portion of Sup35 is a glutamine/asparagine (Q/N)-rich domain, which is responsible for the [PSI+] determinant, and has a high propensity to form amyloid fibrils in vitro [see e.g. references 18–22; (Fig. 25.1)]. The M (middle)-domain is a highly charged domain which has an important role for the stable inheritance of [PSI+] in vivo [23]. However, it is still not understood how the M-domain is involved in the formation of fibrils. The C-terminal domain is sufficient to function as the termination factor and interacts with Sup35[17]. Both the N- and M-domains are dispensable for the essential function of Sup35 in translation termination. In addition, N and M-domains are known as an intrinsically disordered domain [24,25]. The [PSI+] phenotype is nonsense suppression caused by the amyloid-like aggregates of Sup35 [17]. In addition to the amyloid-forming Sup35 protein, the stable maintenance of yeast prions requires several transacting factors. The [PSI+] propagation is strictly dependent on an appropriate amount of Hsp104 [26]. Impairment of Hsp104 function, by either the deletion of Hsp104 [26] or the addition of mM concentrations of guanidine hydrochloride (GuHCl), cures [PSI+] [27].
many of the criteria for typical amyloids, such as binding of Congo Red and thioflavin dyes, seed-dependent self-propagation, and cross-β sheet structures, as revealed by X-ray fiber diffraction [18,22,28].
STRUCTURES OF Sup35 AMYLOIDS IN YEAST CELLS Sup35 fused with GFP in [PSI+] cells gives rise to the formation of visible dot-like aggregates, called ‘foci’, in the cytosol (Fig. 25.3A) (see e.g. references [29–32]), which are one of the hallmarks of the [PSI+] phenotype [8]. Besides the dot-shaped foci, rod- or ring-shaped aggregates may also be formed when Sup35NM-GFP is overexpressed in non-prion [psi–] cells [33,34] and in [PSI+] cells treated with 3∼5 mM guanidine hydrochloride (GuHCl) (Fig. 25.3B), which is known to cure [PSI+] by perturbing Hsp104 [33,35].
IN VITRO FIBRIL FORMATION OF Sup35 In vitro, recombinant Sup35 proteins bearing the priondetermining N-terminal domain form fibrils with a diameter of 10∼20 nm (Fig. 25.2) (for fibrils formed from the recombinant Sup35NM fragment), depending on the Sup35 constructs used in the studies [18,22,28]. The fibrils meet
FIGURE 25.2 Electron micrograph of amyloid fibrils formed by yeast prion Sup35. Negatively stained amyloid fibrils formed by recombinant NM fragment of Sup35. Bar indicates 100 nm.
FIGURE 25.1 Prion concept and yeast prion Sup35. (A) Concept of the prion. (B) Domain structures of yeast prion Sup35. Amino acid sequence of Q/N-rich N-terminal domain is shown.
Chapter | 25 Fibrillar Structures of Yeast Prion Sup35 In Vivo
The ability of recombinant Sup35 proteins to form typical amyloid fibrils with cross β-sheet structures in vitro led to an assumption about the amyloid structures of Sup35 in vivo. However, simple observations of Sup35-GFP aggregates by conventional fluorescence microscopy are
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insufficient to investigate the fine structures of Sup35 in cells. Recent results using several techniques to investigate the structures of Sup35 in cells, as shown below, have provided novel insights into the molecular entities of yeast prion Sup35 in [PSI+] cells.
Transmission Electron Microscopy (TEM)
FIGURE 25.3 Sup35-GFP aggregates in [PSI+] cells. (A) Expression of a Sup35-GFP fusion protein in a [PSI+] cell leads to the appearance of visible spherical aggregates (foci). Phase contrast (top) and fluorescent images (bottom) are shown. (B) Rod- or ring-shaped visible aggregates are formed under the conditions where Sup35NM-GFP was overexpressed in a [PSI+] cell that were treated with 3∼5 mM guanidine hydrochloride (GuHCl). (C) Expression of Sup35NM-GFP in a prion-free [psi-] cell results in a diffuse fluorescence in the cytosol. Bars indicate 2 μm. Reprinted with permission from reference 11.
Several TEM analyses have revealed many spherical particles with ordered fibrillar structures in sliced cells [36,37]. Using rapid-freeze EM, several types of ordered structures were observed in [PSI+] cells expressing Sup35NM-GFP. In thin-section EM images, perfectly aligned fibrils were randomly assembled (Fig. 25.4, A and B). Honeycomb structures, which likely correspond to sectioned Sup35 fibrils generated by thin slicing, were also visualized (Fig. 25.4, A and B). The diameter of each fibril in these cells was around 20 nm, which is similar to the diameter of the full-length Sup35 fibrils formed in vitro [18,28]. Immunogold labeling of the thin sections has been conducted to determine whether the fibrillar structures observed in the TEM images contained Sup35 [36,37]. As shown in the images (Fig. 25.5, A and B), the antibody detected
FIGURE 25.4 Rapid-freeze EM images of Sup35NM-GFP amyloids in [PSI+] cells. (A,B) The boxed areas in the left images are magnified on the right. Adapted with permission from reference 36.
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FIGURE 25.5 Immunogold labeling of Sup35NM-GFP in [PSI+] cells. (A,B) Immunogold labeling of the thin-section EM images of aggregates in [PSI+] cells. An anti-GFP antibody was used to attach the gold. Adapted with permission from reference 36.
Sup35NM-GFP within the fibrillar structures, indicating that the fibrillar structures found using rapid-freeze EM contain Sup35NM-GFP in [PSI+] cells.
Correlative Light Electron Microscopy (CLEM) By comparing light and electron miscroscopic images, CLEM provides high-resolution imaging of fluorescently labeled subcellular structures [38], and is therefore wellsuited for detecting the ordered structure of the aggregated form of prion proteins within a cell. Fluorescence imaging of cells expressing Sup35NMGFP showed that Sup35NM-GFP formed spherical dot-like aggregates in [PSI+] cells (Fig. 25.6A, left). Thin-section EM images for the same cell revealed the structure of the same aggregate identified by fluorescence microscopy (Fig. 25.6A, right). The EM image includes some dense structures in the magnified image (Fig. 25.6B). Taken together, CLEM analysis directly demonstrated that fluorescent foci of Sup35-GFP in [PSI+] cells contain fibrillar structures.
Because the shape of the aggregate can be switched from a spherical form to a rod-like form by treatment with GuHCl, an inhibitor of Hsp104[33,35], we also analyzed the structure of rod-like aggregates by CLEM. We treated [PSI+] cells with GuHCl, and about half of the cells contained the rod-shaped aggregates upon Sup35NM-GFP expression [35], as shown in Figures 25.3B and Fig. 25.7A. CLEM analysis revealed that the rod-shaped fluorescent aggregates were composed of bundled fibrils that were longer than those observed in [PSI+] cells without GuHCl treatment (Fig. 25.7, B and C). The fibrils were aligned laterally, and the lengths of the bundled structures ranged from ∼0.5 to ∼2 μm.
Cryo-Electron Tomography Cryo-electron tomography has also revealed that sections of [PSI+] cells contain aligned bundles of regularly spaced Sup35 fibrils [39]. In addition, the subtomogram-averaging method has suggested additional structures intercalated between the Sup35 fibrils [39].
Chapter | 25 Fibrillar Structures of Yeast Prion Sup35 In Vivo
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FIGURE 25.6 CLEM images of spherical aggregates in a [PSI+] cell. (A) Fluorescence (left) and EM (right) images of a Sup35NM-GFP aggregate in a [PSI+] cell. Cells expressing Sup35NM-GFP were fixed after fluorescence imaging (left), and the thinsection containing the same spherical aggregate identified by the fluorescence microscopy was subjected to EM analysis (right). CW, cell wall; V, vacuole; N, nucleus; ER, endoplasmic reticulum; M, mitochondrion. (B) The area within the red box in (A) is magnified. Adapted with permission from reference 36.
DYNAMIC PROPERTIES OF Sup35 AMYLOIDS IN LIVING YEAST CELLS Prion phenomena are intrinsically dynamic processes because prion aggregates propagate, remodel, and transmit during the protein-based inheritance in yeast prions [7,40]. Simple static observations of Sup35-GFP aggregates by conventional fluorescence microscopy are insufficient to investigate the dynamic aspects of the prions in the cells. Recent advances using several techniques to investigate the dynamics of protein molecules in living cells have provided novel insights into the molecular mechanism by which the Sup35 prion aggregates are propagated and transmitted in [PSI+] cells.
Single-Cell Imaging System for Monitoring the Fate of Sup35-GFP Foci Several genetic analyses combined with fluorescent microscopic observations of visible Sup35-GFP foci have suggested that the foci do not directly represent [PSI+]. However, such an ensemble method does not provide direct evidence for the significance of the visible foci in the transmission of [PSI+]. To gain insight into the dynamics of
Sup35-GFP foci in [PSI+] cells, an on-chip single-cell cultivation system was developed to investigate the dynamic properties of prion aggregates directly [32,41]. Individual live-cell imaging showed that the diameter of the foci gradually decreased, and the foci eventually disappeared (Fig. 25.8, see also supplemental movie) [32]. The disappearance of the foci was not due to GFP photobleaching or degradation [32]. The disappearance of visible foci of Sup35-GFP was also reported by analyses of a microcolony assay system in which preformed Sup35-GFP foci became undetectable as the cells grew [42]. The dot-like foci re-appeared when Sup35-GFP was re-induced, indicating that the seeds of the foci were not lost in the cells after the foci disappeared. The live-cell imaging showed the appearance of the foci in the daughter cell at almost the same time as when the foci re-appeared in the mother cell, indicating that the seeds are transmitted from the mother to the daughter cell. In addition, several lines of evidence showed that the [PSI+] phenotype was maintained in the cells after the foci dispersed [32]. Taken together, the single-cell imaging of Sup35-GFP foci clearly revealed that the foci dynamically dispersed into a state that functions as the seeds of the foci.
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FIGURE 25.7 CLEM images of rod-like aggregates in [PSI+] cells treated with GuHCl. (A–C) Fluorescence (A) and EM (B,C) images of a rod-like Sup35NM-GFP aggregate in a [PSI+] cell treated with GuHCl for 15 hours. Cells expressing Sup35NM-GFP were fixed after fluorescence imaging (A), and the thin-section containing the same rod-like aggregate identified by the fluorescence microscopy was subjected to EM analysis (B). (C) Magnified image of the boxed area in (B). Adapted with permission from reference 36.
Fluorescence Correlation Spectroscopy (FCS) The single-cell imaging described above revealed that the [PSI+] cells – after the dispersion of the foci – have an entity that behaves as prions. The next question is, what is the prion entity left in the cytoplasm after the foci have dispersed? Conventional fluorescence microscopic observation can hardly distinguish the difference between [PSI+] cells without the foci and [psi−] cells in their appearance – because both cells have diffuse GFP fluorescence in the cytoplasm. To elucidate the physical properties of Sup35-GFP in living [PSI+] cells without the foci, fluorescence correlation spectroscopy (FCS) has been applied [32]. FCS is a technique for analyzing the diffusion properties of fluorescent molecules by calculating the fluorescence autocorrelation function (FAF) in a microscopic detection volume at the femtoliter level [43,44]. FCS conveniently
allows the determination of diffusion constants, which are directly correlated with the size of the molecules, of fluctuating fluorescent molecules under equilibrium conditions. Because FCS is usually combined with confocal laser scanning microscopy, we can define the detection volume at any position of interest inside a living cell, in a non-invasive manner. Because the dynamics of prions are basically dependent on the conversion from monomers to aggregates, and vice versa, FCS is ideally suited to estimate the size of Sup35-GFP in living yeast cells. Fluorescence fluctuations of Sup35-GFP in [psi−] and [PSI+] cells, with or without the foci, were measured by FCS (Fig. 25.9) (32). The FAFs of Sup35-GFP in [psi−] cells were almost the same as those in cells expressing the GFP monomer alone, indicating that [psi−] cells contain mostly monomers of Sup35-GFP. In contrast, the FAF profiles in [PSI+] cells, irrespective of the presence of foci, were shifted to the right, as compared to those
Chapter | 25 Fibrillar Structures of Yeast Prion Sup35 In Vivo
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FIGURE 25.8 Dynamics of the Sup35NM-GFP foci in individual living cells detected using the on-chip cultivation system. (A,B) Time-lapse imaging of living [PSI+] cells cultured in rich SC medium (A, see also supplemental movie) and in an isotonic nutrient-free buffer (B). Phase contrast (Ph) and fluorescent images are shown. After the formation of Sup35NM-GFP foci induced by SC containing galactose, the medium was exchanged to SC or the isotonic buffer. (C) Foci size measurements in individual [PSI+] cells cultured in SC (closed symbols) or isotonic buffer (open symbols). Cross-sectional areas of the fluorescent foci were digitally determined. Sizes of the foci at 0 minutes from the medium exchange were set to 100%. Adapted with permission from reference 32.
FIGURE 25.9 Fluorescence correlation spectroscopy (FCS) measurement of yeast prion aggregates in living yeast cells. Normalized autocorrelation curves of Sup35NM-GFP in living yeast cells. Diffusion profiles of Sup35-GFP in the cytoplasm of [psi−], [PSI+(+foci)], and [PSI+(−foci)] cells were measured. The averages ± standard deviations of six independent measurements are shown. Adapted with permission from reference 32.
in [psi−] cells, indicating that the Sup35-GFP species in [PSI+] cells were much slower, and thus larger, than those in [psi−] cells. These results indicate that the larger species, referred to here as diffuse oligomers, are dispersed in the cytoplasm of [PSI+] cells, regardless of the presence of foci [32]. The combination of FCS with the on-chip single cultivation system (time-lapse FCS system) allows measurements of the size of Sup35-GFP in the daughter cells immediately after the transmission from the mother [PSI+] cells [32]. Time-lapse FCS experiments of both mother and daughter cells have demonstrated that the oligomeric species dispersed in the mother cells are directly transmitted to their daughter cells [32]. The single mother–daughter pair analysis using FCS was extended to investigate the effect of Hsp104 on the transmission of Sup35-GFP [35]. An FCS analysis of GuHCl-treated [PSI+] cells revealed that Sup35-GFP diffusion in the daughter cells was faster; that is, the Sup35-GFP
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particle was smaller than that in the mother cells under the Hsp104-inactivated conditions [35]. To clarify the shape of diffuse oligomers, we conducted a simulation based on the experimental results of FCS and semi-denaturing detergent agarose gel electrophoresis [45]. The simulation has revealed that the Sup35NM-GFP oligomers in [PSI+] lysates are fibrillar in shape, suggesting that the oligomers are fragmented forms of the fibrils that were observed in the EM images [36].
Fluorescence Recovery after Photobleaching (FRAP) As an alternative approach to analyze the protein dynamics in living cells, the diffusion of a fluorescent protein can be measured by using a photobleaching technique called fluorescence recovery after photobleaching (FRAP) [43]. In this technique, fluorescent molecules in a small region of the cell are irreversibly photobleached by transient exposure to a laser beam, and the subsequent recovery of fluorescence in the photobleached region is recorded [43]. The technique has been applied to the Sup35-GFP fusion proteins in living yeast cells [30,35,42,46–49]. The FRAP analysis showed that the fluorescence recovery was slower in [PSI+] cells than that in [psi−] cells, indicating that the Sup35-GFP was in an aggregated form in [PSI+] cells [30]. In addition to the conventional FRAP analysis, a modified FRAP technique has been developed to directly measure the flux of Sup35 aggregates between mother and daughter cells [35], because conventional FRAP, as well as FCS, cannot address the flux of Sup35 between mother and daughter. In the modified FRAP technique, the GFP fluorescence in the whole daughter cell is photobleached, to assess the flux rate from the mother to the daughter cell. When the modified FRAP, called MD-FRAP (mother to daughter), was conducted with the [PSI+] cells, the flux of Sup35NM-GFP in the [psi−] cells was faster than that in the [PSI+] cells, reflecting the existence of diffuse oligomers of Sup35GFP in the [PSI+] cells.
CONCLUDING REMARKS To understand the amyloids, previous efforts have focused on in vitro amyloid fibril formation or in vivo phonotype analyses of amyloid-related diseases or yeast prions. So far, there has been not so much connection between the in vitro and in vivo studies. This chapter introduced several attempts to elucidate the amyloid fibrils inside cells in regard to in vivo structures and dynamics of yeast prion Sup35. The techniques shown here can be applied and extended to other prions and amyloid fibrils inside cells, providing important insights into prion/amyloid biology in the cell.
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Chapter | 25 Fibrillar Structures of Yeast Prion Sup35 In Vivo
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Fibrillar Structures of Yeast Prion Sup35 In Vivo Hideki Taguchi and Shigeko Kawai-Noma Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
Chapter Outline Introduction271 Yeast Prion Sup35 as a Model Amyloid-Forming Protein in Cells 271 In Vitro Fibril Formation of Sup35 272 Structures of Sup35 Amyloids in Yeast Cells 272 Transmission Electron Microscopy (TEM) 273 Correlative Light Electron Microscopy (CLEM) 274 Cryo-Electron Tomography 274
INTRODUCTION Proteins often form aggregates. Protein aggregates can be categorized into two groups: amorphous (disordered) aggregates and ordered aggregates called amyloids. Amyloids are highly ordered fibrillar protein aggregates with a cross β-sheet structure [1], which binds Congo Red and thioflavin dyes [2,3]. Amyloids were originally defined as extracellular deposits in several human diseases but the nomenclature now also includes many protein aggregates with n on-pathologic origins, raising the possibility that amyloid formation is an inherent property of numerous different proteins with various biologic functions [1]. Representatives of such nonpathologic amyloids are budding yeast Saccharomyces cerevisiae prion proteins, such as Sup35 and Ure2 [4]. Scrapie in sheep, bovine spongiform encephalopathy (also called ‘mad-cow’ disease) in cattle, and Creutzfeldt– Jakob disease and kuru in humans, are transmissible spongiform encephalopathies (TSEs), which are also called prion diseases. A prion is a proteinaceous infectious particle that lacks nucleic acids; this means that it is an infectious protein [5]. In the prion, the altered conformations of a protein auto-catalytically convert the normal structure to the amyloid (Fig. 25.1A). This prion concept was developed Bio-nanoimaging. http://dx.doi.org/10.1016/B978-0-12-394431-3.00025-0 Copyright © 2014 Elsevier Inc. All rights reserved.
Dynamic Properties of Sup35 Amyloids in Living Yeast Cells Single-Cell Imaging System for Monitoring the Fate of Sup35-GFP Foci Fluorescence Correlation Spectroscopy (FCS) Fluorescence Recovery after Photobleaching (FRAP) Concluding Remarks
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for the mammalian neurodegenerative diseases, in which the PrP protein participates. However, the concept has been spread to several non-Mendelian genetic elements in budding yeast, such as [PSI+] and [URE3] in S. cerevisiae [6]. Because yeast is a quite tractable model eukaryote, yeast prions can provide many important insights into prion biology that are usually difficult to access with mammalian prions [7–12]. In particular, the mechanisms by which the prion proteins are propagated and transmitted have been unraveled in the yeast prion model. This chapter provides an overview of the in vivo structure and dynamics of yeast prion protein Sup35.
YEAST PRION Sup35 AS A MODEL AMYLOID-FORMING PROTEIN IN CELLS Although several dozen yeast proteins are known to behave as prions in vivo [13–16], the prion state of the Sup35 protein, the [PSI+] determinant, is the best-characterized prion. Sup35 is an essential protein, which normally functions as a translation termination factor (eRF3) in cooperation with its partner Sup45 (eRF1) [17], but aggregated forms of the Sup35 are the determinant of a prion phenotype [PSI+].
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Sup35, which consists of 685 amino acid residues, has three domains (Fig. 25.1B). The N-terminal portion of Sup35 is a glutamine/asparagine (Q/N)-rich domain, which is responsible for the [PSI+] determinant, and has a high propensity to form amyloid fibrils in vitro [see e.g. references 18–22; (Fig. 25.1)]. The M (middle)-domain is a highly charged domain which has an important role for the stable inheritance of [PSI+] in vivo [23]. However, it is still not understood how the M-domain is involved in the formation of fibrils. The C-terminal domain is sufficient to function as the termination factor and interacts with Sup35[17]. Both the N- and M-domains are dispensable for the essential function of Sup35 in translation termination. In addition, N and M-domains are known as an intrinsically disordered domain [24,25]. The [PSI+] phenotype is nonsense suppression caused by the amyloid-like aggregates of Sup35 [17]. In addition to the amyloid-forming Sup35 protein, the stable maintenance of yeast prions requires several transacting factors. The [PSI+] propagation is strictly dependent on an appropriate amount of Hsp104 [26]. Impairment of Hsp104 function, by either the deletion of Hsp104 [26] or the addition of mM concentrations of guanidine hydrochloride (GuHCl), cures [PSI+] [27].
many of the criteria for typical amyloids, such as binding of Congo Red and thioflavin dyes, seed-dependent self-propagation, and cross-β sheet structures, as revealed by X-ray fiber diffraction [18,22,28].
STRUCTURES OF Sup35 AMYLOIDS IN YEAST CELLS Sup35 fused with GFP in [PSI+] cells gives rise to the formation of visible dot-like aggregates, called ‘foci’, in the cytosol (Fig. 25.3A) (see e.g. references [29–32]), which are one of the hallmarks of the [PSI+] phenotype [8]. Besides the dot-shaped foci, rod- or ring-shaped aggregates may also be formed when Sup35NM-GFP is overexpressed in non-prion [psi–] cells [33,34] and in [PSI+] cells treated with 3∼5 mM guanidine hydrochloride (GuHCl) (Fig. 25.3B), which is known to cure [PSI+] by perturbing Hsp104 [33,35].
IN VITRO FIBRIL FORMATION OF Sup35 In vitro, recombinant Sup35 proteins bearing the priondetermining N-terminal domain form fibrils with a diameter of 10∼20 nm (Fig. 25.2) (for fibrils formed from the recombinant Sup35NM fragment), depending on the Sup35 constructs used in the studies [18,22,28]. The fibrils meet
FIGURE 25.2 Electron micrograph of amyloid fibrils formed by yeast prion Sup35. Negatively stained amyloid fibrils formed by recombinant NM fragment of Sup35. Bar indicates 100 nm.
FIGURE 25.1 Prion concept and yeast prion Sup35. (A) Concept of the prion. (B) Domain structures of yeast prion Sup35. Amino acid sequence of Q/N-rich N-terminal domain is shown.
Chapter | 25 Fibrillar Structures of Yeast Prion Sup35 In Vivo
The ability of recombinant Sup35 proteins to form typical amyloid fibrils with cross β-sheet structures in vitro led to an assumption about the amyloid structures of Sup35 in vivo. However, simple observations of Sup35-GFP aggregates by conventional fluorescence microscopy are
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insufficient to investigate the fine structures of Sup35 in cells. Recent results using several techniques to investigate the structures of Sup35 in cells, as shown below, have provided novel insights into the molecular entities of yeast prion Sup35 in [PSI+] cells.
Transmission Electron Microscopy (TEM)
FIGURE 25.3 Sup35-GFP aggregates in [PSI+] cells. (A) Expression of a Sup35-GFP fusion protein in a [PSI+] cell leads to the appearance of visible spherical aggregates (foci). Phase contrast (top) and fluorescent images (bottom) are shown. (B) Rod- or ring-shaped visible aggregates are formed under the conditions where Sup35NM-GFP was overexpressed in a [PSI+] cell that were treated with 3∼5 mM guanidine hydrochloride (GuHCl). (C) Expression of Sup35NM-GFP in a prion-free [psi-] cell results in a diffuse fluorescence in the cytosol. Bars indicate 2 μm. Reprinted with permission from reference 11.
Several TEM analyses have revealed many spherical particles with ordered fibrillar structures in sliced cells [36,37]. Using rapid-freeze EM, several types of ordered structures were observed in [PSI+] cells expressing Sup35NM-GFP. In thin-section EM images, perfectly aligned fibrils were randomly assembled (Fig. 25.4, A and B). Honeycomb structures, which likely correspond to sectioned Sup35 fibrils generated by thin slicing, were also visualized (Fig. 25.4, A and B). The diameter of each fibril in these cells was around 20 nm, which is similar to the diameter of the full-length Sup35 fibrils formed in vitro [18,28]. Immunogold labeling of the thin sections has been conducted to determine whether the fibrillar structures observed in the TEM images contained Sup35 [36,37]. As shown in the images (Fig. 25.5, A and B), the antibody detected
FIGURE 25.4 Rapid-freeze EM images of Sup35NM-GFP amyloids in [PSI+] cells. (A,B) The boxed areas in the left images are magnified on the right. Adapted with permission from reference 36.
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FIGURE 25.5 Immunogold labeling of Sup35NM-GFP in [PSI+] cells. (A,B) Immunogold labeling of the thin-section EM images of aggregates in [PSI+] cells. An anti-GFP antibody was used to attach the gold. Adapted with permission from reference 36.
Sup35NM-GFP within the fibrillar structures, indicating that the fibrillar structures found using rapid-freeze EM contain Sup35NM-GFP in [PSI+] cells.
Correlative Light Electron Microscopy (CLEM) By comparing light and electron miscroscopic images, CLEM provides high-resolution imaging of fluorescently labeled subcellular structures [38], and is therefore wellsuited for detecting the ordered structure of the aggregated form of prion proteins within a cell. Fluorescence imaging of cells expressing Sup35NMGFP showed that Sup35NM-GFP formed spherical dot-like aggregates in [PSI+] cells (Fig. 25.6A, left). Thin-section EM images for the same cell revealed the structure of the same aggregate identified by fluorescence microscopy (Fig. 25.6A, right). The EM image includes some dense structures in the magnified image (Fig. 25.6B). Taken together, CLEM analysis directly demonstrated that fluorescent foci of Sup35-GFP in [PSI+] cells contain fibrillar structures.
Because the shape of the aggregate can be switched from a spherical form to a rod-like form by treatment with GuHCl, an inhibitor of Hsp104[33,35], we also analyzed the structure of rod-like aggregates by CLEM. We treated [PSI+] cells with GuHCl, and about half of the cells contained the rod-shaped aggregates upon Sup35NM-GFP expression [35], as shown in Figures 25.3B and Fig. 25.7A. CLEM analysis revealed that the rod-shaped fluorescent aggregates were composed of bundled fibrils that were longer than those observed in [PSI+] cells without GuHCl treatment (Fig. 25.7, B and C). The fibrils were aligned laterally, and the lengths of the bundled structures ranged from ∼0.5 to ∼2 μm.
Cryo-Electron Tomography Cryo-electron tomography has also revealed that sections of [PSI+] cells contain aligned bundles of regularly spaced Sup35 fibrils [39]. In addition, the subtomogram-averaging method has suggested additional structures intercalated between the Sup35 fibrils [39].
Chapter | 25 Fibrillar Structures of Yeast Prion Sup35 In Vivo
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FIGURE 25.6 CLEM images of spherical aggregates in a [PSI+] cell. (A) Fluorescence (left) and EM (right) images of a Sup35NM-GFP aggregate in a [PSI+] cell. Cells expressing Sup35NM-GFP were fixed after fluorescence imaging (left), and the thinsection containing the same spherical aggregate identified by the fluorescence microscopy was subjected to EM analysis (right). CW, cell wall; V, vacuole; N, nucleus; ER, endoplasmic reticulum; M, mitochondrion. (B) The area within the red box in (A) is magnified. Adapted with permission from reference 36.
DYNAMIC PROPERTIES OF Sup35 AMYLOIDS IN LIVING YEAST CELLS Prion phenomena are intrinsically dynamic processes because prion aggregates propagate, remodel, and transmit during the protein-based inheritance in yeast prions [7,40]. Simple static observations of Sup35-GFP aggregates by conventional fluorescence microscopy are insufficient to investigate the dynamic aspects of the prions in the cells. Recent advances using several techniques to investigate the dynamics of protein molecules in living cells have provided novel insights into the molecular mechanism by which the Sup35 prion aggregates are propagated and transmitted in [PSI+] cells.
Single-Cell Imaging System for Monitoring the Fate of Sup35-GFP Foci Several genetic analyses combined with fluorescent microscopic observations of visible Sup35-GFP foci have suggested that the foci do not directly represent [PSI+]. However, such an ensemble method does not provide direct evidence for the significance of the visible foci in the transmission of [PSI+]. To gain insight into the dynamics of
Sup35-GFP foci in [PSI+] cells, an on-chip single-cell cultivation system was developed to investigate the dynamic properties of prion aggregates directly [32,41]. Individual live-cell imaging showed that the diameter of the foci gradually decreased, and the foci eventually disappeared (Fig. 25.8, see also supplemental movie) [32]. The disappearance of the foci was not due to GFP photobleaching or degradation [32]. The disappearance of visible foci of Sup35-GFP was also reported by analyses of a microcolony assay system in which preformed Sup35-GFP foci became undetectable as the cells grew [42]. The dot-like foci re-appeared when Sup35-GFP was re-induced, indicating that the seeds of the foci were not lost in the cells after the foci disappeared. The live-cell imaging showed the appearance of the foci in the daughter cell at almost the same time as when the foci re-appeared in the mother cell, indicating that the seeds are transmitted from the mother to the daughter cell. In addition, several lines of evidence showed that the [PSI+] phenotype was maintained in the cells after the foci dispersed [32]. Taken together, the single-cell imaging of Sup35-GFP foci clearly revealed that the foci dynamically dispersed into a state that functions as the seeds of the foci.
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FIGURE 25.7 CLEM images of rod-like aggregates in [PSI+] cells treated with GuHCl. (A–C) Fluorescence (A) and EM (B,C) images of a rod-like Sup35NM-GFP aggregate in a [PSI+] cell treated with GuHCl for 15 hours. Cells expressing Sup35NM-GFP were fixed after fluorescence imaging (A), and the thin-section containing the same rod-like aggregate identified by the fluorescence microscopy was subjected to EM analysis (B). (C) Magnified image of the boxed area in (B). Adapted with permission from reference 36.
Fluorescence Correlation Spectroscopy (FCS) The single-cell imaging described above revealed that the [PSI+] cells – after the dispersion of the foci – have an entity that behaves as prions. The next question is, what is the prion entity left in the cytoplasm after the foci have dispersed? Conventional fluorescence microscopic observation can hardly distinguish the difference between [PSI+] cells without the foci and [psi−] cells in their appearance – because both cells have diffuse GFP fluorescence in the cytoplasm. To elucidate the physical properties of Sup35-GFP in living [PSI+] cells without the foci, fluorescence correlation spectroscopy (FCS) has been applied [32]. FCS is a technique for analyzing the diffusion properties of fluorescent molecules by calculating the fluorescence autocorrelation function (FAF) in a microscopic detection volume at the femtoliter level [43,44]. FCS conveniently
allows the determination of diffusion constants, which are directly correlated with the size of the molecules, of fluctuating fluorescent molecules under equilibrium conditions. Because FCS is usually combined with confocal laser scanning microscopy, we can define the detection volume at any position of interest inside a living cell, in a non-invasive manner. Because the dynamics of prions are basically dependent on the conversion from monomers to aggregates, and vice versa, FCS is ideally suited to estimate the size of Sup35-GFP in living yeast cells. Fluorescence fluctuations of Sup35-GFP in [psi−] and [PSI+] cells, with or without the foci, were measured by FCS (Fig. 25.9) (32). The FAFs of Sup35-GFP in [psi−] cells were almost the same as those in cells expressing the GFP monomer alone, indicating that [psi−] cells contain mostly monomers of Sup35-GFP. In contrast, the FAF profiles in [PSI+] cells, irrespective of the presence of foci, were shifted to the right, as compared to those
Chapter | 25 Fibrillar Structures of Yeast Prion Sup35 In Vivo
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FIGURE 25.8 Dynamics of the Sup35NM-GFP foci in individual living cells detected using the on-chip cultivation system. (A,B) Time-lapse imaging of living [PSI+] cells cultured in rich SC medium (A, see also supplemental movie) and in an isotonic nutrient-free buffer (B). Phase contrast (Ph) and fluorescent images are shown. After the formation of Sup35NM-GFP foci induced by SC containing galactose, the medium was exchanged to SC or the isotonic buffer. (C) Foci size measurements in individual [PSI+] cells cultured in SC (closed symbols) or isotonic buffer (open symbols). Cross-sectional areas of the fluorescent foci were digitally determined. Sizes of the foci at 0 minutes from the medium exchange were set to 100%. Adapted with permission from reference 32.
FIGURE 25.9 Fluorescence correlation spectroscopy (FCS) measurement of yeast prion aggregates in living yeast cells. Normalized autocorrelation curves of Sup35NM-GFP in living yeast cells. Diffusion profiles of Sup35-GFP in the cytoplasm of [psi−], [PSI+(+foci)], and [PSI+(−foci)] cells were measured. The averages ± standard deviations of six independent measurements are shown. Adapted with permission from reference 32.
in [psi−] cells, indicating that the Sup35-GFP species in [PSI+] cells were much slower, and thus larger, than those in [psi−] cells. These results indicate that the larger species, referred to here as diffuse oligomers, are dispersed in the cytoplasm of [PSI+] cells, regardless of the presence of foci [32]. The combination of FCS with the on-chip single cultivation system (time-lapse FCS system) allows measurements of the size of Sup35-GFP in the daughter cells immediately after the transmission from the mother [PSI+] cells [32]. Time-lapse FCS experiments of both mother and daughter cells have demonstrated that the oligomeric species dispersed in the mother cells are directly transmitted to their daughter cells [32]. The single mother–daughter pair analysis using FCS was extended to investigate the effect of Hsp104 on the transmission of Sup35-GFP [35]. An FCS analysis of GuHCl-treated [PSI+] cells revealed that Sup35-GFP diffusion in the daughter cells was faster; that is, the Sup35-GFP
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particle was smaller than that in the mother cells under the Hsp104-inactivated conditions [35]. To clarify the shape of diffuse oligomers, we conducted a simulation based on the experimental results of FCS and semi-denaturing detergent agarose gel electrophoresis [45]. The simulation has revealed that the Sup35NM-GFP oligomers in [PSI+] lysates are fibrillar in shape, suggesting that the oligomers are fragmented forms of the fibrils that were observed in the EM images [36].
Fluorescence Recovery after Photobleaching (FRAP) As an alternative approach to analyze the protein dynamics in living cells, the diffusion of a fluorescent protein can be measured by using a photobleaching technique called fluorescence recovery after photobleaching (FRAP) [43]. In this technique, fluorescent molecules in a small region of the cell are irreversibly photobleached by transient exposure to a laser beam, and the subsequent recovery of fluorescence in the photobleached region is recorded [43]. The technique has been applied to the Sup35-GFP fusion proteins in living yeast cells [30,35,42,46–49]. The FRAP analysis showed that the fluorescence recovery was slower in [PSI+] cells than that in [psi−] cells, indicating that the Sup35-GFP was in an aggregated form in [PSI+] cells [30]. In addition to the conventional FRAP analysis, a modified FRAP technique has been developed to directly measure the flux of Sup35 aggregates between mother and daughter cells [35], because conventional FRAP, as well as FCS, cannot address the flux of Sup35 between mother and daughter. In the modified FRAP technique, the GFP fluorescence in the whole daughter cell is photobleached, to assess the flux rate from the mother to the daughter cell. When the modified FRAP, called MD-FRAP (mother to daughter), was conducted with the [PSI+] cells, the flux of Sup35NM-GFP in the [psi−] cells was faster than that in the [PSI+] cells, reflecting the existence of diffuse oligomers of Sup35GFP in the [PSI+] cells.
CONCLUDING REMARKS To understand the amyloids, previous efforts have focused on in vitro amyloid fibril formation or in vivo phonotype analyses of amyloid-related diseases or yeast prions. So far, there has been not so much connection between the in vitro and in vivo studies. This chapter introduced several attempts to elucidate the amyloid fibrils inside cells in regard to in vivo structures and dynamics of yeast prion Sup35. The techniques shown here can be applied and extended to other prions and amyloid fibrils inside cells, providing important insights into prion/amyloid biology in the cell.
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Chapter | 25 Fibrillar Structures of Yeast Prion Sup35 In Vivo
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