| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | ADVANCED SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, FSB401, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan
2 Laboratory of Molecular Cell Dynamics, Graduate School of Life Sciences, Hokkaido University, Sapporo 001-0021, Japan
3 Cellular Systems Modeling Team, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
As prions are transmissible altered conformations, the elucidation of the molecular mechanism by which they are transmitted to the next generation is central to understanding prions. So far, tremendous efforts utilizing a variety of techniques have been made to address the transmissible entity of the yeast prions. First, sophisticated yeast genetics revealed that [PSI+] cells have so-called propagons to transmit and maintain the prion phenotype (Eaglestone et al. 2000; Cox et al. 2003; Byrne et al. 2007). Second, biochemical analyses, using semi-denaturing agarose gel electrophoresis, have shown that the oligomeric form is the major species in [PSI+] lysates with SDS (Kryndushkin et al. 2003; Bagriantsev & Liebman 2004; Salnikova et al. 2005; Song et al. 2005; Tanaka et al. 2006). In addition, the oligomers (called Sup35 polymers) were recently purified from [PSI+] lysates and characterized (Bagriantsev et al. 2008). Finally, cell biological approaches using Sup35 fused with GFP revealed that the transmissible entity is composed of diffuse oligomers (Song et al. 2005; Wu et al. 2005; Kawai-Noma et al. 2006; Satpute-Krishnan et al. 2007).
The next question to be addressed is how such Sup35 prion oligomers are maintained and then transmitted to daughter cells in the protein-based heritable phenomenon. Hsp104, a molecular chaperone for thermotolerance in yeast (Sanchez & Lindquist 1990; Parsell et al. 1994a,b; Bosl et al. 2006), is known as an essential factor for yeast prion maintenance (Chernoff et al. 1995). Deletion, inactivation, or overexpression of Hsp104 cures [PSI+] (Chernoff et al. 1995). Among these cures, the addition of a mM concentration of guanidine hydrochloride (GuHCl) leads to the elimination of [PSI+] through the inhibition of Hsp104 (Eaglestone et al. 2000; Ferreira et al. 2001; Jung & Masison 2001; Wegrzyn et al. 2001; Jung et al. 2002; Ness et al. 2002; Grimminger et al. 2004). A lag of four to five cell generations is observed before [psi–] cells appear (Eaglestone et al. 2000; Ferreira et al. 2001). Based on the kinetics of the cure, it has been proposed that the GuHCl treatment blocks the generation of new propagons (prion seeds). Several previous studies have described the status of the Sup35–GFP in Hsp104-inactivated [PSI+] cells (Wegrzyn et al. 2001; Zhou et al. 2001; Ness et al. 2002; Wu et al. 2005; Satpute-Krishnan et al. 2007). Satpute-Krishnan et al. clearly showed that the loss of Sup35 aggregate remodelling by Hsp104 creates a segregation bias that limits the transmission of the Sup35 aggregates to daughter cells. Although their conclusion was based on microcolony observations and the fluorescence recovery after photobleaching (FRAP) technique within the cytoplasm (Satpute-Krishnan et al. 2007), the mechanism underlying the segregation bias has yet to be fully elucidated.
In this study, we have developed unique methods to probe the dynamics of Sup35 inside the cytoplasms of mother and daughter cells, using fluorescence correlation spectroscopy (FCS) as well as a modified FRAP technique. FCS is an effective technique to determine the diffusion times of fluorescent molecules. As 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 noninvasive manner (Lippincott-Schwartz et al. 2001; Kawai-Noma et al. 2006; Pack et al. 2006). Therefore, the technique provides information about the size of Sup35–GFP in living yeast cells, as shown in our previous study (Kawai-Noma et al. 2006). In addition, we directly analysed the flux of the Sup35 oligomers from the mother to daughter cells, using the modified FRAP technique. Using these approaches, we investigated the dynamics of Sup35 prion states (oligomers) in the GuHCl-treated [PSI+] cells. The FCS and the modified FRAP techniques are both applicable to other types of protein-based transmission inside living cells.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
In our previous study, we utilized FCS to analyse the size of prion aggregates in living yeast cells and found that the transmissible prion entity is composed of diffuse oligomers (Kawai-Noma et al. 2006). We further extended this approach to investigate the mechanism underlying the transmission bias in GuHCl-treated [PSI+] cells. For this purpose, we modified the 74-D694 strain by integrating a GFP gene within the endogenous SUP35 gene (referred to as G74-D694). The integration of GFP did not change the [PSI+] genetic background in G74-D694, as G74-D694 [PSI+] was viable in medium without adenine, and formed white colonies on YPD medium (data not shown). In addition, like the 74-D694 strain, G74-D694 [PSI+] was cured during growth on YPD containing 5 mM GuHCl (data not shown).
The cytoplasm of G74-D694 [PSI+] and [psi–] cells were analysed by FCS. Typical fluorescence autocorrelation functions [FAFs; G(
)], which reflect the diffusion property of the fluorescent molecules in the confocal volume, revealed clear difference between the [PSI+] and [psi–] cell (Fig. 1a). The autocorrelation decay of the [PSI+] cell generated a slower profile than that of the [psi–] cells, confirming that the Sup35–GFP in the [PSI+] cells was much larger than that in the [psi–] cells.
|
Rod-shaped Sup35NM–GFP aggregates appear in the GuHCl-treated [PSI+] cells
As the GuHCl treatment cures [PSI+], the GuHCl-dependent appearance of another population in the [PSI+] cells (Fig. 1d) seems to reflect the conversion of [PSI+] to [psi–]. Actually, the newly appearing distribution in the GuHCl-treated [PSI+] cells was similar to that in the [psi–] cells (Fig. 1c,e). Although a transmission bias for Sup35 in Hsp104-impaired [PSI+] cells was previous reported (Satpute-Krishnan et al. 2007), the diffusion of Sup35 has not been analysed in detail. Therefore, we next tried to investigate the diffusion properties of Sup35 in individual mother–daughter pairs, to directly analyse the curing process in a single-cell manner. For efficient FCS measurements, we have to search the cells that might be affected by the GuHCl treatment. On this purpose, we used 74-D694 [PSI+] cells, in which Sup35NM–GFP was induced to form visible aggregates for an easy discrimination by our eyes. When the Sup35NM–GFP was expressed in the 74-D694 [PSI+] cells, the dot-like spherical aggregates were formed (Fig. 2aleft), and were found in 67% of the cells in the culture (Table 1). The GuHCl-treated [PSI+] cells also contained the spherical aggregates (24% of the cells), but they primarily contained the rod-like aggregates (43% of the cells) (Fig. 2aright, Table 1). The [PSI+] curing assay (Cox et al. 2003; Byrne et al. 2007) confirmed the gradual curing of [PSI+] even after the induction of Sup35NM–GFP in the GuHCl-treated 74-D694 [PSI+] cells (Fig. 2b).
|
|
We then used FCS to analyse the intracellular molecular dynamics of Sup35NM–GFP in mother–daughter pairs with the spherical or rod-shaped aggregates in the mother (Fig. 3a,b). The noninvasive character of FCS allows us to measure the diffusion profiles of Sup35NM–GFP in the mother and the adjacent daughter cell simultaneously, as shown in our previous FCS measurements, which demonstrated that the diffuse Sup35NM–GFP oligomers are transmissible to the daughter cells (Kawai-Noma et al. 2006).
|
Two-component fitting of FAFs in the cells with spherical aggregates showed that the diffusion properties of Sup35NM–GFP in the mother and the daughter were roughly the same (Fig. 3a), and corresponded to that in [PSI+] cells without GuHCl, as reported previously (Kawai-Noma et al. 2006). In contrast, the cells with the rod aggregates contained a larger percentage of faster moving species than those with the spherical aggregates. The mother [PSI+] cells with the rod aggregates contained 66% of the faster species (919 µs) (Fig. 3b), whereas the mother cells with the spherical aggregates contained 9% of the faster ones (2.9 ms) (Fig. 3a), suggesting an increase in the population of smaller Sup35 aggregates in the mother cell with the rod aggregates. In addition, we observed a further increase in the percentage of the faster species in the daughters of the rod-containing mother cells. The percentage of the faster diffusing species increased from 66% in the mother to 91% in the daughter cell (Fig. 3b).
In an extreme case, we were able to measure FCS through five generations simultaneously (Fig. 3c). After confirming the parent–child relation, by checking a series of z-sectional confocal images, we performed the FCS. In the analysis, the diffusion of Sup35NM–GFP gradually shifted to the faster type. In particular, a drastic change occurred between generations 3 and 4. In conclusion, the FCS analysis revealed that the GuHCl-treated cells with the rod-shaped aggregates contained smaller Sup35 molecules, and the ongoing cell divisions accelerated the size reduction. Taken together, these results suggest that the cells with the rod-shaped aggregates are under a curing process.
Mother–daughter FRAP to analyse Sup35 aggregate flux to the daughter
In addition to the previous observation of the segregation bias of the Sup35–GFP aggregates in the Hsp104-inactivated cells (Satpute-Krishnan et al. 2007), we showed that the daughter cells tended to contain smaller forms of the Sup35 aggregates. Although FCS (Kawai-Noma et al. 2006) as well as FRAP analyses (Wu et al. 2005, 2006; Satpute-Krishnan et al. 2007) revealed the dynamics of Sup35–GFP in individual cells, those analyses could not address the flux of Sup35 from mother to daughter cells. To directly measure the flux between mother and daughter cells, we modified the FRAP technique. In the conventional FRAP technique, fluorescent proteins in a small region of the cell are photobleached by a laser beam, and the recovery is measured in the bleached region to assess the diffusion properties of the fluorescent proteins (Lippincott-Schwartz et al. 2001; Wu et al. 2006). 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 (MD-FRAP, Fig. 4a, Movies S1 and S2 in Supporting Information).
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Several previous reports have described the dynamics of Sup35 fused with GFP in GuHCl-treated [PSI+] cells (Wegrzyn et al. 2001; Song et al. 2005; Wu et al. 2005; Satpute-Krishnan et al. 2007). Satpute-Krishnan et al. observed that the Sup35–GFP complexes increase in size, as initially detected by brighter Sup35–GFP fluorescence, and are mostly immobile (Satpute-Krishnan et al. 2007). Based on this apparent immobility of the large complexes, as well as the segregation bias of the Sup35–GFP fluorescence in the microcolony observation, they suggested the diminished transmission of propagons to daughters in the Hsp104-inactivated cells, eventually resulting in the loss of [PSI+] (Wegrzyn et al. 2001; Satpute-Krishnan et al. 2007). Our FCS results, which revealed the asymmetrical distribution of the Sup35NM–GFP diffusion profiles in individual mother–daughter cells (Fig. 3), provided a direct demonstration of the biased transmission. Technically, the advantage of FCS is the simultaneous measurement of the diffusion properties of Sup35–GFP in both the mother and daughter cells (Kawai-Noma et al. 2006).
From the FCS analysis of mother–daughter pairs, we selected the cells that contained rod-shaped aggregates as candidates for the cells undergoing the curing process. Relating to this, we note that Zhou et al. observed that the rod-shaped aggregates in the GuHCl-treated [PSI+] cells, although they have not investigated the detail (Zhou et al. 2001). Several studies have reported the formation of the rod aggregates when Sup35-GFP was overexpressed in [psi–][PIN+] cells (Zhou et al. 2001; Ganusova et al. 2006). In this case, the rod aggregates are regarded as a sign of the [PSI+] induction. We suggest that the cells with the rod aggregates were in the process of being cured under our conditions, for the following reasons. First, the [PSI+] curing assay showed the gradual curing of [PSI+] even after the induction of Sup35NM–GFP in the GuHCl-treated [PSI+] cells (Fig. 2b). Second, we found that the diffuse oligomers in the GuHCl-treated cells, which had the rod-shaped visible aggregates, were smaller than those in the cells with the spherical aggregates (Fig. 3a,b). Finally, in the mother–daughter pairs, in which the mother contained the rod aggregates, ongoing cell divisions accelerated the size reduction of the diffuse oligomers, resulting in a strong bias between the mother and daughter cells (Fig. 3b,c). Taken together, the mother–daughter pairs with the rod-shaped aggregates seem to be in the process of being cured.
We note that the rod-shaped aggregates described in our study are similar to those observed in a Sup35
22-69 derivative (Borchsenius et al. 2001). The similarity provides insight into the mechanism of the rod aggregate formation, since Borchsenius et al. previously suggested that the defect in aggregate shearing, due to the low sensitivity to Hsp104, produces the rod aggregates. This explanation can be applied to the rod aggregates in the GuHCl-treated cells, as GuHCl inactivates Hsp104 (Eaglestone et al. 2000; Ferreira et al. 2001; Jung & Masison 2001; Wegrzyn et al. 2001; Jung et al. 2002; Ness et al. 2002; Grimminger et al. 2004). The defect in aggregate shearing by the Hsp104 inactivation seems to result in the formation of a long fibril, which might correspond to the rod aggregates.
Our FCS observations revealed two distinct distributions in GuHCl-treated [PSI+] cells (Fig. 1d). As the cells were undergoing the curing process, we consider the two distributions to reflect [PSI+] and cured [psi–] cells. In an extreme case, the FCS analysis of multiple generations (Fig. 3c) revealed a large decrease in the diffusion ensembles from three to four cell generations. This drastic decrease would correspond to the loss of [PSI+]. In addition, the lag before the drastic decrease in the diffusion ensembles might explain the well-known fact that there is a lag before [psi–] cells appear in the GuHCl treatment (Eaglestone et al. 2000; Ferreira et al. 2001).
We also observed two distinct distributions in the MD-FRAP experiment (Fig. 4). The mother–daughter pairs that have fast Sup35–GFP flux rates would be cured [psi–] cells, as the flux rates are almost identical to those in [psi–] cells. In contrast, the mother–daughter pairs that have extremely slower flux rates seem to be [PSI+] cells that are not cured, although the flux rates are slower than those in [PSI+] cells without GuHCl treatment. The delayed transmission in the GuHCl-treated cells can induce the asymmetric distribution, observed by FCS analyses (Fig. 3). The asymmetric distribution caused by the GuHCl treatment probably eventually cures [PSI+].
Finally, the conclusion presented here provides new insight into the propagon counting method, as the method mainly relies on the assumption of an equal distribution of transmissible entities (Cox et al. 2003; Byrne et al. 2007). Our conclusion strongly suggests that the mother cells tend to retain more aggregates than the daughter cells. If this is the case, then the conventional propagon counting method would result in an underestimation of the propagon number (see alsoTanaka et al. (2006)).
|
Experimental procedures |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
The strains used in this study were 74-D694 and G74-D694 ([PSI+] and [psi–]), derivatives of the 74-D694 (MATaade1-14 leu2-3112 his3-
200 trp1-289 ura3-52 [PSI+] and [psi–]) strains respectively. In G74-D694, GFP was inserted into the chromosomal SUP35 gene in between the N and M domains. G74-D694 was constructed by transforming 74-D694 with the integration plasmid YIp211-NGMC (see below), which is a URA3-based plasmid carrying SUP35NGMC. Transformants were selected on 5'-FOA, and then the integration of GFP in the targeted position was verified by PCR and subsequent sequencing. Standard rich medium (YPD) and synthetic complete medium (using Difco yeast nitrogen base) lacking leucine (SC-Leu) were used (Sherman 2002). SRaf-Leu medium contained 2% raffinose instead of glucose. To induce the expression of the GAL1 promoter, galactose was added to a final concentration of 2% (w/v). For the GuHCl treatment, GuHCl was added to the medium at 5 mM. Yeast strains were grown at 30 °C.
Plasmids
The yeast plasmid YCp-GAL1p-SUP35 (NM)-GFP [LEU2], expressing the Sup35NM domain conjugated to GFP by the galactose-inducible GAL1 promoter, was previously described (Ayano et al. 2004; Kawai-Noma et al. 2006). YIp211-NGMC, for the integration of GFP into the SUP35 locus, was constructed from YIp211. SUP35 tagged GFP from the plasmid pJ510, which was a kind gift from Daniel C. Masison (Song et al. 2005), was inserted into YIp211. The plasmids thus obtained were verified by DNA sequencing.
Single-cell imaging
The on-chip microculture system is the same as that previously described (Ayano et al. 2004; Kawai-Noma et al. 2006), except for the microchamber design, which was modified for the long-term cultivation of individual, isolated yeast cells and for the efficient exchange of fresh medium. The cultivation system was used with a bright-field optical microscopy system (IX-71 inverted microscopy with a 
100 Å objective lens; Olympus, Tokyo, Japan) with an EM CCD camera (iXon; Andor Technology, Belfast, UK) to obtain differential interference contrast and fluorescent images. During on-chip cultivation, fresh medium was continuously supplied to the chamber system at a constant rate of 1 mL/min, by a peristaltic pump. Yeast cells were grown in SRaf-Leu medium. To induce the expression of Sup35NM–GFP, 2% galactose was added to the SRaf-Leu medium. The temperature of the system was maintained at 30 °C throughout the observations by a heated chamber surrounding the microscope.
Fluorescence correlation spectroscopy
All of the FCS measurements were performed at 25 °C on an LSM510 confocal microscope combined with a ConfoCor 2 (Carl Zeiss, Jena, Germany), as described in our previous study (Kawai-Noma et al. 2006; Pack et al. 2006). Briefly, the FAF [G()], from which the average residence time (
) and the absolute number of fluorescent proteins in the detection volume are calculated, are obtained as follows:
|
| (1) |
) is the fluorescence intensity obtained by the single photon counting method in a detection volume at a delay time (brackets denote ensemble averages). The curve fitting for the multi-component model is given by:
|
| (2) |
i are the fraction and the diffusion time of the component i, respectively, N is the total number of fluorescent molecules in the detection volume defined by the beam waist w0 and the axial radius z0, and s is the structure parameter representing the ratio of w0 and z0. The structure parameter was determined with the standard Rh6G solution. The GFP fluorescence in living cells was excited with the minimal total power needed for sufficient signal to noise by adjusting the acousto-optical tunable filter. Three or five sequential measurements of 10 or 15 sec were performed in a single cell. The effect of photobleaching on FCS analysis was minimized by lowering the excitation intensity and by selecting cells with low fluorescence. The FAFs were normalized by the number of particles, N, for the comparison of apparent differences in the diffusion of GFP-fused proteins from cell to cell. Fluorescence recovery after photobleaching
All of the FRAP experiments were performed at 25 °C with a Zeiss LSM510 confocal microscope, as described in a previous study (Song et al. 2005). A defined region (0.5 µm in the diameter) of the photobleaching within the cytoplasm of mother cells was selected, excluding the fluorescent foci and the vacuoles. The photobleached region for the whole daughter cell was fixed at 2 µm in diameter, and the daughter cells smaller than 2 µm were selected. The laser power was set to 0.5% for the image collection for the fluorescence recovery and 100% for the bleaching. For each condition, 8–15 cells were photobleached.
[PSI+] curing assay
The curing assay of [PSI+] by GuHCl was performed according to the procedure described by Cox et al. (2003) and Byrne et al. (2007). Logarithmic cultures of [PSI+]/GAL1pSUP35NM-GFP were grown in SRaf-Leu+ 5 mM GuHCl for 15 h, and then 2% galactose was added. Samples were taken at 4-h intervals over 40 h after galactose induction. Percentage of [PSI+] cells were determined by plating 100 cells on SC-Ade plates. The yeast generations were calculated using a haemocytometer and by counting the colonies on SC-Ade plates at each sample time.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
These authors contributed equally.
* taguchi{at}k.u-tokyo.ac.jp |
References |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Bagriantsev, S. & Liebman, S.W. (2004) Specificity of prion assembly in vivo. [PSI+] and [PIN+] form separate structures in yeast. J. Biol. Chem. 279, 51042–51048.
Bagriantsev, S.N., Gracheva, E.O., Richmond, J.E. & Liebman, S.W. (2008) Variant-specific [PSI+] infection is transmitted by Sup35 polymers within [PSI+] aggregates with heterogeneous protein composition. Mol. Biol. Cell 19, 2433–2443.
Borchsenius, A.S., Wegrzyn, R.D., Newnam, G.P., Inge-Vechtomov, S.G. & Chernoff, Y.O. (2001) Yeast prion protein derivative defective in aggregate shearing and production of new ‘seeds’. EMBO J. 20, 6683–6691.[CrossRef][Medline]
Bosl, B., Grimminger, V. & Walter, S. (2006) The molecular chaperone Hsp104–a molecular machine for protein disaggregation. J. Struct. Biol. 156, 139–148.[Medline]
Byrne, L.J., Cox, B.S., Cole, D.J., Ridout, M.S., Morgan, B.J. & Tuite, M.F. (2007) Cell division is essential for elimination of the yeast [PSI+] prion by guanidine hydrochloride. Proc. Natl Acad. Sci. USA 104, 11688–11693.
Chernoff, Y.O. (2007) Stress and prions: lessons from the yeast model. FEBS Lett. 581, 3695–3701.[CrossRef][Medline]
Chernoff, Y.O., Lindquist, S.L., Ono, B., Inge-Vechtomov, S.G. & Liebman, S.W. (1995) Role of the chaperone protein Hsp104 in propagation of the yeast prion-like factor [psi+]. Science 268, 880–884.
Chien, P., Weissman, J.S. & DePace, A.H. (2004) Emerging principles of conformation-based prion inheritance. Annu. Rev. Biochem. 73, 617–656.[CrossRef][Medline]
Cox, B.S. (1965) [PSI], a cytoplasmic suppressor of supersuppression in yeast. Heredity 20, 505–521.[CrossRef]
Cox, B., Ness, F. & Tuite, M. (2003) Analysis of the generation and segregation of propagons: entities that propagate the [PSI+] prion in yeast. Genetics 165, 23–33.
Eaglestone, S.S., Ruddock, L.W., Cox, B.S. & Tuite, M.F. (2000) Guanidine hydrochloride blocks a critical step in the propagation of the prion-like determinant [PSI+] of Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA 97, 240–244.
Ferreira, P.C., Ness, F., Edwards, S.R., Cox, B.S. & Tuite, M.F. (2001) The elimination of the yeast [PSI+] prion by guanidine hydrochloride is the result of Hsp104 inactivation. Mol. Microbiol. 40, 1357–1369.[CrossRef][Medline]
Ganusova, E.E., Ozolins, L.N., Bhagat, S., Newnam, G.P., Wegrzyn, R.D., Sherman, M.Y. & Chernoff, Y.O. (2006) Modulation of prion formation, aggregation, and toxicity by the actin cytoskeleton in yeast. Mol. Cell. Biol. 26, 617–629.
Grimminger, V., Richter, K., Imhof, A., Buchner, J. & Walter, S. (2004) The prion curing agent guanidinium chloride specifically inhibits ATP hydrolysis by Hsp104. J. Biol. Chem. 279, 7378–7383.
Horwich, A.L. & Weissman, J.S. (1997) Deadly conformations – protein misfolding in prion disease. Cell 89, 499–510.[CrossRef][Medline]
Jung, G. & Masison, D.C. (2001) Guanidine hydrochloride inhibits Hsp104 activity in vivo: a possible explanation for its effect in curing yeast prions. Curr. Microbiol. 43, 7–10.[CrossRef][Medline]
Jung, G., Jones, G. & Masison, D.C. (2002) Amino acid residue 184 of yeast Hsp104 chaperone is critical for prion-curing by guanidine, prion propagation, and thermotolerance. Proc. Natl Acad. Sci. USA 99, 9936–9941.
Kawai-Noma, S., Ayano, S., Pack, C.G., Kinjo, M., Yoshida, M., Yasuda, K. & Taguchi, H. (2006) Dynamics of yeast prion aggregates in single living cells. Genes Cells 11, 1085–1096.
Kryndushkin, D.S., Alexandrov, I.M., Ter-Avanesyan, M.D. & Kushnirov, V.V. (2003) Yeast [PSI+] prion aggregates are formed by small Sup35 polymers fragmented by Hsp104. J. Biol. Chem. 278, 49636–49643.
Kushnirov, V.V. & Ter-Avanesyan, M.D. (1998) Structure and replication of yeast prions. Cell 94, 13–16.[CrossRef][Medline]
Lippincott-Schwartz, J., Snapp, E. & Kenworthy, A. (2001) Studying protein dynamics in living cells. Nat. Rev. Mol. Cell Biol. 2, 444–456.[CrossRef][Medline]
Ness, F., Ferreira, P., Cox, B.S. & Tuite, M.F. (2002) Guanidine hydrochloride inhibits the generation of prion "seeds" but not prion protein aggregation in yeast. Mol. Cell. Biol. 22, 5593–5605.
Pack, C., Saito, K., Tamura, M. & Kinjo, M. (2006) Microenvironment and effect of energy depletion in the nucleus analyzed by mobility of multiple oligomeric EGFPs. Biophys. J. 91, 3921–3936.[CrossRef][Medline]
Parsell, D.A., Kowal, A.S. & Lindquist, S. (1994a) Saccharomyces cerevisiae Hsp104 protein. Purification and characterization of ATP-induced structural changes. J. Biol. Chem. 269, 4480–4487.
Parsell, D.A., Kowal, A.S., Singer, M.A. & Lindquist, S. (1994b) Protein disaggregation mediated by heat-shock protein Hsp104. Nature 372, 475–478.[CrossRef][Medline]
Prusiner, S.B. (1998) Prions. Proc. Natl Acad. Sci. USA 95, 13363–13383.
Salnikova, A.B., Kryndushkin, D.S., Smirnov, V.N., Kushnirov, V.V. & Ter-Avanesyan, M.D. (2005) Nonsense suppression in yeast cells overproducing Sup35 (eRF3) is caused by its non-heritable amyloids. J. Biol. Chem. 280, 8808–8812.
Sanchez, Y. & Lindquist, S.L. (1990) HSP104 required for induced thermotolerance. Science 248, 1112–1115.
Satpute-Krishnan, P., Langseth, S.X. & Serio, T.R. (2007) Hsp104-dependent remodeling of prion complexes mediates protein-only inheritance. PLoS Biol. 5, e24.[CrossRef][Medline]
Serio, T.R. & Lindquist, S.L. (1999) [PSI+]: an epigenetic modulator of translation termination efficiency. Annu. Rev. Cell Dev. Biol. 15, 661–703.[CrossRef][Medline]
Sherman, F. (2002) Getting started with yeast. Methods Enzymol. 350, 3–41.[Medline]
Song, Y., Wu, Y.X., Jung, G., Tutar, Y., Eisenberg, E., Greene, L.E. & Masison, D.C. (2005) Role for Hsp70 chaperone in Saccharomyces cerevisiae prion seed replication. Eukaryot. Cell 4, 289–297.
Tanaka, M., Collins, S.R., Toyama, B.H. & Weissman, J.S. (2006) The physical basis of how prion conformations determine strain phenotypes. Nature 442, 585–589.[CrossRef][Medline]
Tuite, M.F. & Cox, B.S. (2003) Propagation of yeast prions. Nat. Rev. Mol. Cell Biol. 4, 878–890.[CrossRef][Medline]
Wegrzyn, R.D., Bapat, K., Newnam, G.P., Zink, A.D. & Chernoff, Y.O. (2001) Mechanism of prion loss after Hsp104 inactivation in yeast. Mol. Cell. Biol. 21, 4656–4669.
Wickner, R.B. (1994) [URE3] as an altered URE2 protein: evidence for a prion analog in Saccharomyces cerevisiae. Science 264, 566–569.
Wickner, R.B., Edskes, H.K., Shewmaker, F. & Nakayashiki, T. (2007) Prions of fungi: inherited structures and biological roles. Nat. Rev. Microbiol. 5, 611–618.[CrossRef][Medline]
Wu, Y.X., Greene, L.E., Masison, D.C. & Eisenberg, E. (2005) Curing of yeast [PSI+] prion by guanidine inactivation of Hsp104 does not require cell division. Proc. Natl Acad. Sci. USA 36, 12789–12794.
Wu, Y.X., Masison, D.C., Eisenberg, E. & Greene, L.E. (2006) Application of photobleaching for measuring diffusion of prion proteins in cytosol of yeast cells. Methods 39, 43–49.[CrossRef][Medline]
Zhou, P., Derkatch, I.L. & Liebman, S.W. (2001) The relationship between visible intracellular aggregates that appear after overexpression of Sup35 and the yeast prion-like elements [PSI+] and [PIN+]. Mol. Microbiol. 39, 37–46.[CrossRef][Medline]
Received: 6 February 2009
Accepted: 1 June 2009
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | ADVANCED SEARCH | TABLE OF CONTENTS |
