Quantitative and spatio-temporal features of protein aggregation in Escherichia coli and consequences on protein quality control and cellular ageing
2010; Springer Nature; Volume: 29; Issue: 5 Linguagem: Inglês
10.1038/emboj.2009.412
ISSN1460-2075
AutoresJuliane Winkler, Anja Seybert, Lars M. König, Sabine Pruggnaller, Uta Haselmann, Victor Sourjik, Matthias Weiß, Achilleas S. Frangakis, Axel Mogk, Bernd Bukau,
Tópico(s)Microbial Metabolic Engineering and Bioproduction
ResumoArticle21 January 2010free access Quantitative and spatio-temporal features of protein aggregation in Escherichia coli and consequences on protein quality control and cellular ageing Juliane Winkler Juliane Winkler Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany Search for more papers by this author Anja Seybert Anja Seybert EMBL Meyerhofstrasse 1, Heidelberg, Germany Search for more papers by this author Lars König Lars König Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, GermanyPresent address: Institute of Cellular and Molecular Immunology, Georg-August-University, Humboldtallee 34, Göttingen 37073, Germany Search for more papers by this author Sabine Pruggnaller Sabine Pruggnaller EMBL Meyerhofstrasse 1, Heidelberg, Germany Search for more papers by this author Uta Haselmann Uta Haselmann EMBL Meyerhofstrasse 1, Heidelberg, Germany Search for more papers by this author Victor Sourjik Victor Sourjik Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany Search for more papers by this author Matthias Weiss Matthias Weiss Cellular Biophysics Group, German Cancer Research Center, c/o BIOQUANT, Heidelberg, Germany Search for more papers by this author Achilleas S Frangakis Achilleas S Frangakis EMBL Meyerhofstrasse 1, Heidelberg, Germany Institute of Biophysics, Goethe University Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Axel Mogk Corresponding Author Axel Mogk Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany Search for more papers by this author Bernd Bukau Corresponding Author Bernd Bukau Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany Search for more papers by this author Juliane Winkler Juliane Winkler Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany Search for more papers by this author Anja Seybert Anja Seybert EMBL Meyerhofstrasse 1, Heidelberg, Germany Search for more papers by this author Lars König Lars König Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, GermanyPresent address: Institute of Cellular and Molecular Immunology, Georg-August-University, Humboldtallee 34, Göttingen 37073, Germany Search for more papers by this author Sabine Pruggnaller Sabine Pruggnaller EMBL Meyerhofstrasse 1, Heidelberg, Germany Search for more papers by this author Uta Haselmann Uta Haselmann EMBL Meyerhofstrasse 1, Heidelberg, Germany Search for more papers by this author Victor Sourjik Victor Sourjik Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany Search for more papers by this author Matthias Weiss Matthias Weiss Cellular Biophysics Group, German Cancer Research Center, c/o BIOQUANT, Heidelberg, Germany Search for more papers by this author Achilleas S Frangakis Achilleas S Frangakis EMBL Meyerhofstrasse 1, Heidelberg, Germany Institute of Biophysics, Goethe University Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Axel Mogk Corresponding Author Axel Mogk Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany Search for more papers by this author Bernd Bukau Corresponding Author Bernd Bukau Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany Search for more papers by this author Author Information Juliane Winkler1, Anja Seybert2, Lars König1, Sabine Pruggnaller2, Uta Haselmann2, Victor Sourjik1, Matthias Weiss3, Achilleas S Frangakis2,4, Axel Mogk 1 and Bernd Bukau 1 1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany 2EMBL Meyerhofstrasse 1, Heidelberg, Germany 3Cellular Biophysics Group, German Cancer Research Center, c/o BIOQUANT, Heidelberg, Germany 4Institute of Biophysics, Goethe University Frankfurt, Frankfurt am Main, Germany *Corresponding authors. Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Ruprecht-Karls-Universitaet, Universitat Heidelberg, Im Neuenheimer Feld 282, Heidelberg 69120, Germany. Tel.: +49 6221 546863; Fax: +49 6221 545894; E-mail: [email protected] or Tel.: +49 6221 546795; Fax: +49 6221 545894; E-mail: [email protected] The EMBO Journal (2010)29:910-923https://doi.org/10.1038/emboj.2009.412 There is a Have you seen ...? (March 2010) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The aggregation of proteins as a result of intrinsic or environmental stress may be cytoprotective, but is also linked to pathophysiological states and cellular ageing. We analysed the principles of aggregate formation and the cellular strategies to cope with aggregates in Escherichia coli using fluorescence microscopy of thermolabile reporters, EM tomography and mathematical modelling. Misfolded proteins deposited at the cell poles lead to selective re-localization of the DnaK/DnaJ/ClpB disaggregating chaperones, but not of GroEL and Lon to these sites. Polar aggregation of cytosolic proteins is mainly driven by nucleoid occlusion and not by an active targeting mechanism. Accordingly, cytosolic aggregation can be efficiently re-targeted to alternative sites such as the inner membrane in the presence of site-specific aggregation seeds. Polar positioning of aggregates allows for asymmetric inheritance of damaged proteins, resulting in higher growth rates of damage-free daughter cells. In contrast, symmetric damage inheritance of randomly distributed aggregates at the inner membrane abrogates this rejuvenation process, indicating that asymmetric deposition of protein aggregates is important for increasing the fitness of bacterial cell populations. Introduction Protein misfolding is an inevitable process in all cells that is enhanced by internal and environmental stress such as heat shock. In response to such problems, cells have evolved powerful protein quality control systems, composed of molecular chaperones and proteolytic machineries, to eliminate misfolded proteins and thereby maintain protein homeostasis. In the cytosol of Escherichia coli, the DnaK chaperone with its DnaJ and GrpE co-chaperones and the GroEL–GroES chaperonin have central functions in the refolding of misfolded proteins (Hartl and Hayer-Hartl, 2002). Alternatively, misfolded proteins are degraded by overlapping activities of different ATP-dependent proteases that harbour AAA+ chaperone and peptidase components or domains (ClpAP, ClpXP, HslUV, Lon and FtsH). Despite its adaptability, this proteostasis network represents a delicate equilibrium, which can be perturbed by severe stress, leading to the accumulation of misfolded conformers, which form aggregates. Protein aggregates in E. coli were first identified as inclusion bodies resulting from the overproduction of heterologous proteins (Carrio et al, 1998) and are even detectable in wild-type cells cultivated at physiological temperatures in the absence of protein overproduction (Lindner et al, 2008). Protein aggregation can be reverted in the cytosol of bacteria, yeast and plants by a powerful disaggregation machinery, composed of an Hsp70 chaperone system (DnaK in bacteria) and the AAA+ chaperone Hsp104 (ClpB in bacteria) (Glover and Lindquist, 1998). This bi-chaperone system solubilizes and refolds aggregated proteins, an activity that is essential for thermotolerance development (Sanchez and Lindquist, 1990; Weibezahn et al, 2004). However, when the refolding and degradation capacity of the cell is persistently limited, an initial strategy seems to involve the sequestration of aggregated proteins at specific sites, thereby protecting the cellular environment from potentially deleterious protein species (Wigley et al, 1999; Kaganovich et al, 2008; Kirstein et al, 2008). Such sequestration may also allow for an asymmetric inheritance of damaged proteins if the aggregated proteins cannot be eliminated before cell division (Rujano et al, 2006; Kaganovich et al, 2008). In E. coli, protein aggregates are deposited at the polar sites and this spatial sequestration was suggested to allow for asymmetric damage inheritance (Lindner et al, 2008; Rokney et al, 2009). The underlying mechanisms for the sequestration of aggregated proteins and the resulting consequences for the protein quality control system, however, remain elusive. Furthermore, it is not evident whether asymmetric damage segregation is beneficial compared with symmetric inheritance, as it was so far not possible to manipulate the site of cellular protein aggregation. Here, we present a rigorous analysis of the principles of protein aggregation caused by physiological heat stress in E. coli and the relationship between aggregate deposition and bacterial ageing. Results Quantitative analysis of protein aggregation using thermolabile reporters Heat treatment of E. coli cells within their growth temperature range can lead to protein aggregation, providing a means to study the aggregation process at physiological stress conditions. To monitor protein aggregation in vivo, we constructed N- or C-terminal fusions between thermostable yellow or cyan fluorescent proteins (YFP, CFP) and thermolabile model proteins (Photinus pyralis Luciferase, E. coli MetA), which aggregate in E. coli at temperatures above 44°C (Gur et al, 2002; Weibezahn et al, 2004). Immunoblot analysis of cell extracts prior and after heat treatment revealed only single protein bands corresponding to the sizes of the fusion proteins, assuring that fluorescence microscopy images of the fusion proteins reflect the cellular localization of the full-length proteins. To exclude the possibility that protein aggregation is the consequence of protein overproduction, we produced YFP–Luciferase and MetA–YFP from IPTG-controlled expression plasmids only to low/intermediate levels (2800 and 1500 molecules/cell, respectively) that were even below the endogenous levels in case of MetA (Supplementary Figure 1; data not shown). Spectinomycin was added to the cells before heat shock to block de novo protein synthesis, ensuring that any differences in cellular localization reflect the re-distribution of pre-existing fusion protein. YFP–Luciferase and MetA–YFP showed a uniform cytosolic staining at 30°C, whereas after temperature upshift to 45°C, both fusion proteins quantitatively re-localized to form foci at the cell poles (>75% of total YFP fluorescence; Figure 1A and B). For YFP–Luciferase, we determined that foci formation was accompanied by the complete loss of Luciferase activity (data not shown). In contrast, heat-induced polar localization was not observed when YFP alone was produced in control experiments (data not shown), together showing that the observed re-distribution is driven by the thermolabile fusion partner. During a recovery period at 30°C, polar YFP–Luciferase and MetA–YFP foci were reverted to a uniform cytosolic staining and polar foci were no longer observed after 45–60 min. This process was directly coupled to the regain of Luciferase activity, indicating that the disappearance of fluorescent foci reflects ClpB-mediated protein disaggregation. Accordingly, the disintegration of heat-induced YFP–Luciferase foci was not detectable in ΔclpB mutant cells (data not shown), thereby qualifying YFP–Luciferase and MetA–YFP as valuable reporter to study protein aggregation and disaggregation in vivo. Figure 1.Heat-induced protein aggregation in E. coli. (A) MC4100 cells expressing either YFP–Luciferase or MetA–YFP were grown at 30°C and shifted to 45°C for 20 min. The images show an overlay of YFP signal (false coloured in green) and membrane stain FM4-64 (red). Scale bar: 1 μm. (B) Localization of YFP–Luciferase and MetA–YFP aggregates after heat shock (n=200). Positioning of protein aggregates is indicated with blue dots in the inset. (C) 3D electron tomographic reconstructions of heat-treated, serial-sectioned plastic embedded wild-type and ΔclpB mutant cells harbouring protein aggregates. ΔclpB mutant cells were expressing YFP–Luciferase. Cell surfaces are visualized in grey colour and aggregates in red. Scale bar: 100 nm. Quantifications of aggregate size and calculations of the copy numbers of aggregated protein species are given. (D) Obliquely sectioned 7.2 nm thick cryo-electron tomographic slices of vitreous sections of heat-shocked wild-type cells. Various electron dense complexes are visualized in the cytosol (presumably ribosomes), whereas the protein aggregates (indicated through the red dotted line) seem more electron lucent excluding these complexes. Scale bar: 100 nm. Download figure Download PowerPoint Using a heating device for the microscope, we directly followed the kinetics of YFP–Luciferase and MetA–YFP aggregation in individual cells. The first fluorescent foci of YFP–Luciferase appeared after 2–5 min and aggregation was completed after 10 min; MetA–YFP aggregated slower, exhibiting the first visible aggregates after 4–8 min (Supplementary Figure 2). Statistical evaluation revealed that 62% of all heat-treated cells (n=200) exhibited two fluorescent foci at both poles, 22% contained only one focus at one pole, 1% showed one focus in mid-cell position, whereas ⩽15% contained three fluorescent foci (Figure 1B). In the latter case, two foci were localized to poles and the third one located at mid-cell, probably representing a future septation site. Aggregation was also analysed without addition of antibiotics and resulted in the same number and localization of fluorescent foci after heat treatment, showing that de novo protein synthesis is not required for the polar deposition of aggregates. EM tomography of protein aggregates To elucidate the molecular features of the protein aggregates, we used electron tomography (ET) of plastic embedded wild-type and ΔclpB mutant cells, with the latter expressing YFP–Luciferase, which allows to compare the extent of protein aggregation in cells with and without disaggregating chaperone activity. Aggregates were detected as electron dense deposits close to the poles of heat-treated cells (20 min at 45°C), but not in cells kept at 30°C. Using serial sectioning (Supplementary Movies 1–3), we generated 3D reconstructions of complete cells (Figure 1C). Number and localization of the aggregates detected by EM tomography in the reconstructed cells agreed well with the characteristics of those detected by fluorescence microscopy in the living cells. The 3D reconstructions revealed that protein aggregates have amorphous structures and strongly varying sizes. They also allowed to determine the volumes of the aggregates, which in turn allowed to estimate the number of molecules trapped in each aggregate. The average molecular weight of E. coli proteins is approximately 35 kDa (Netzer and Hartl, 1998). A typical globular protein of that size occupies a volume of 4.3 × 10−23 l (Harpaz et al, 1994). Under the assumptions that the aggregates contain proteins with similar size average and that the volume of these proteins is not dramatically altered on heat denaturation, we estimate that individual aggregates are composed of approximately 2400–16500 protein molecules. The total number of heat-aggregated proteins within a cell (wild-type or clpB-expressing YFP–Luciferase) was less diverse between cells, ranging from 17500 to 33000 aggregated proteins/cell, which corresponds to 1.5–3% of total cytosolic proteins. We note that the presence of YFP–Luciferase did not significantly alter the degree of aggregation of endogenous thermolabile proteins, consistent with its relatively low production level. To ensure that the preparation of cells for electron microscopy did not affect the existing protein aggregates, we performed in addition cryo-ET of vitreous sections of cells in a near-native state. Using this technique, we obtained similar data with respect to number, size and localization of heat-induced protein aggregates, showing that the reconstruction of E. coli cells provides a valuable model for analysing protein aggregation (Figure 1D). Selective re-distribution of quality control components to polar aggregates We analysed the localization of the protein quality control machinery prior and after heat treatment using C-terminal fusions of DnaK, DnaJ, ClpB, ClpX, HslU, Lon and ClpP to YFP or CFP. If not stated differently, all fusion proteins were produced from IPTG-controlled expression plasmids in respective knockout cells to approximately wild-type levels (at 30°C) and their functional active state was verified (Supplementary Figure 3). Immunoblot analysis revealed only single protein bands corresponding to the sizes of the fusion proteins. As GroEL cannot be fused to YFP/CFP in its functional active state, we monitored its cellular localization (and that of DnaK for comparison) by immunofluorescence. We first monitored the localization of YFP/CFP fusions to DnaK, DnaJ and ClpB, which constitute the central bacterial disaggregation machinery (Mogk et al, 1999). Although at 30°C the fusion proteins exhibited diffuse cytosolic staining, on heat shock to 45°C, they extensively re-localize to the poles (Figure 2A). Confirming these results, DnaK also exhibited polar localization after heat treatment when analysed by immunofluorescence. GroEL instead did not change its localization on heat stress treatment and remained distributed throughout the cytosol (Figure 2B). Figure 2.Re-localization of chaperones and proteases on heat treatment. (A) Indicated chaperone fusion proteins were produced in respective knockout cells, except DnaK–YFP that was produced in wild-type cells to approximately 1000 molecules. Cells were grown at 30°C to mid-logarithmic growth phase and heat shocked to 45°C for 20 min. The images show an overlay of the YFP signal (false coloured in green) and the membrane stain FM4-64 (red). Scale bar: 1 μm. (B) Wild-type cells were incubated at 30°C or heat shocked to 45°C for 20 min. GroEL and DnaK localizations were determined by immunofluorescence using specific antibodies. Scale bar: 1 μm. (C) Indicated AAA+ protein and peptidase fusions were produced in respective knockout cells that were grown at 30°C to mid-logarithmic growth phase and shifted to 45°C for 20 min. The images show an overlay of YFP signal (false coloured in green) and membrane stain FM4-64 (red). Scale bar: 1 μm. (D) Calculated ratio of polar and cytosolic fluorescence intensity for the indicated fusion proteins after heat shock. The fluorescence intensities of 20 individual cells harbouring two polar aggregates of the respective fusion proteins were determined. Download figure Download PowerPoint We next analysed the localization of the proteolytic machineries. At 30°C, the fusion proteins to ClpX, HslU and ClpP all exhibited uniform cytosolic staining. The only exception was Lon–YFP, which showed a more condensed fluorescence coinciding with the DAPI staining of the chromosome that is reminiscent of nucleoid-associated proteins (data not shown). After temperature upshift to 45°C, polar foci were observed for all fusion proteins except Lon–YFP, which did not change its localization (Figure 2C). Numbers and positions of the fluorescent foci generated by the different chaperone/protease fusion proteins on temperature upshift were comparable with those formed by YFP–Luciferase and MetA–YFP, suggesting that the polar localization of quality control components is directed by the occurrence of protein aggregates (Supplementary Figure 4). Indeed, co-expression of YFP–Luciferase and ClpB–CFP showed complete co-localization of both fusion proteins at polar foci after heat shock (Supplementary Figure 5). DnaJ, DnaK and ClpB dynamically associate with protein aggregates The extent of polar localization differed significantly between the individual fusion proteins and was most pronounced in case of DnaK–YFP and ClpB–YFP (⩾75% of total fluorescence) (Figure 2D). These differences in the degree of heat-induced polar fluorescence between refolding (DnaK/ClpB) and degrading machineries (ClpX/ClpP/HslU) may imply differences in their binding kinetics to aggregates. To investigate this possibility, we determined the dynamics of the relevant components at the cell poles using fluorescence recovery after photobleaching (FRAP) experiments (Figure 3). The fluorescence of individual polar foci was bleached followed by a monitoring of the time course of fluorescence recovery. Such experiments were performed using heat-treated cells harbouring fluorescent foci at both poles, thereby allowing for fluorescence recovery through the non-bleached focus and the cytosolic fluorescent fraction. Before heat shock, spectinomycin was added to ensure that the fluorescence recovery is solely resulting from the re-localization of pre-existing fusion protein. The kinetics of fluorescence recovery showed at least two phases. The first phase represented an initial fast recovery because of the re-equilibration of cytosolic fusion proteins within this compartment. This phase represented a fraction of free diffusing cytosolic proteins (Df) and was observed to a similar degree (approximately 40% of regained fluorescence) for all analysed fusion proteins. Second was a slower recovery phase that correlated with a loss of fluorescence at the non-bleached focus. This regain in fluorescence includes two steps: the dissociation of the respective fusion protein from the non-bleached focus and the re-association with the bleached focus, thereby representing the mobile aggregate-associated fraction (Maggregatef). We defined the Df and the Maggregatef as the mobile fraction (Mf). The immobile fraction (If) resulted from non-exchangeable aggregate-associated proteins. We first monitored the recovery of polar fluorescence of YFP–Luciferase aggregates in heat-treated wild-type cells on temperature downshift to 30°C. YFP–Luciferase fluorescence was only regained during the initial fast recovery phase showing that the aggregated molecules are immobile (40% Mf) (Figure 3). A similar result was obtained in case of ClpX–YFP, indicating that the AAA+ component of the ClpX/ClpP proteolytic machinery is irreversibly sequestered within the aggregates (Supplementary Figure 6). In case of HslU–YFP, we observed a minor Mf (59% Mf), whereas the majority of HslU–YFP stayed immobile (Supplementary Figure 6). In contrast, a pronounced polar fluorescence was regained for DnaK–YFP, DnaJ–YFP and ClpB–YFP (100% Mf), concomitant with a loss of fluorescence at the unbleached pole, which declined with similar kinetics (Figure 3). This finding shows that the disaggregating chaperones are rapidly recruited from the opposite cell pole and exchange between sites of protein damage within the entire cell. The kinetics of the recovery (Mf) was fast for all three chaperone components, with ClpB being slower than DnaK and DnaJ. Together, these data illustrate that DnaK, DnaJ and ClpB associate dynamically with polar-localized aggregated proteins, whereas the occurrence of ClpX and HslU at the poles rather indicates co-aggregation by binding to misfolded protein species. Figure 3.Disaggregating chaperones dynamically interact with aggregated proteins. FRAP measurements of Luciferase and chaperone (ClpB, DnaK, DnaJ) YFP fusion proteins were carried out in corresponding knockout strains that were heat shocked to 45°C for 10 min. Analysis of DnaK–YFP was performed in wild-type cells. De novo synthesis of fusion proteins was inhibited by addition of 100 μg/ml spectinomycin before heat shock. One out of the two polar fluorescent foci was bleached when cells were shifted back to 30°C for recovery. Representative pre- and post-bleach images of the indicated fusion proteins were recorded at the indicated time periods and recovery curves were calculated based on 10–20 cells. Download figure Download PowerPoint Nucleoid occlusion determines polar localization of protein aggregates As protein aggregates are specifically deposited at cell poles, but are not randomly distributed throughout the cytosol, it seemed an intriguing possibility that a cellular machinery is actively engaged in aggregate depositioning, by either transporting aggregates to the poles and/or providing a polar retention system. To initially differentiate between these possibilities, we microscopically followed protein aggregation in real time using YFP–Luciferase and MetA–YFP as fluorescent monitors. In most cells subjected to a temperature upshift to 45°C (76%, n=100), the fluorescent foci formed directly at the poles without showing any movement over longer distance. Still, in a minority of cells (24%), the sites of initial and final positioning of foci differed, and occasionally (10%) we observed the fusion of smaller foci to bigger aggregates. Thus, a minority of aggregates does not form directly at their final polar destination, but instead appears to move within the cell (Supplementary Movie 4/5). To detect a possible transport machinery that moves aggregates within the cell, we used several approaches. We first tested whether the cytoskeleton is involved in polar foci formation by inactivating the bacterial actin homologue MreB with the inhibitor A22 (Iwai et al, 2002). Treatment of cells with A22 abrogated the helical structure of a YFP–MreB fusion protein without affecting cell shape within the time frame of the experiment. Addition of A22 to the cells before a heat treatment neither changed the number nor the positioning of protein aggregates, largely excluding a function of the cytoskeleton in the aggregation process (Supplementary Figure 7A). We then investigated whether the polar positioning of aggregates is an active, energy-driven process by treating cells with the uncouplers 2.4-dinitrophenol (DNP) and CCCP or sodium azide, leading to strongly reduced ATP levels (to 20–34% as compared with non-treated cells) before heat shock. However, when monitoring YFP–Luciferase aggregation in such cells, we did not observe differences in the aggregation process (Supplementary Figure 7B–E). In contrast, the solubilization of aggregated proteins by DnaK/ClpB was abolished on ATP depletion by DNP, showing that the disaggregation, but not the aggregation, process requires normal ATP levels. We also speculated that molecular chaperones are directly involved in aggregate deposition by targeting misfolded protein species to polar sites. Here, we tested for an essential function of the DnaK chaperone machinery in aggregate deposition, as this chaperone system represents the central holder chaperone in E. coli cells at elevated temperatures (Mogk et al, 1999). Polar aggregate formation of MetA–YFP was largely unaffected in ΔdnaJ or ΔdnaK mutant cells, as dominant polar fluorescent foci were still detectable (Supplementary Figure 8). The existence of additional fluorescent foci in some of these cells is likely caused by the strong increase in protein aggregation and cell size of the filamentous mutant cells (data not shown). Next, we analysed whether septum formation, which generates cell poles on division, has an impact on the positioning of MetA–YFP aggregates. This was carried out by treating cells with cephalexin, which inhibits FtsI, thereby causing the formation of filamentous, multi-nucleated cells (Pogliano et al, 1997). In cephalexin-treated cells, multiple MetA–YFP foci were detectable at regular positions within the entire cell body in regions free of bacterial nucleoid (Figure 4A). This finding indicates that the positioning of cytosolic protein aggregates is not restricted to poles and thus not determined by cell division. Instead, it shows that protein aggregates are only formed in the nucleoid-free space. Figure 4.Nucleoid occlusion determines the positioning of protein aggregates. (A) Bacterial nucleoids and heat-induced MetA–YFP aggregates do not co-localize in filamentous cells. Filamentation of cells producing MetA–YFP was induced by addition of cephalexin. Cells were either kept at 30°C or shifted to 45°C for 20 min. Images were recorded and show membrane staining using FM4-64 (red), DNA staining using DAPI (blue) and the MetA–YFP signal. A merge of the individual images is given. Scale bar: 1 μm. (B) Nucleoid-free ΔmukB cells predominantly contain only one stress-induced MetA–YFP protein aggregate at either mid-cell or polar position. ΔmukB cells and isogenic YK1100 control cells producing MetA–YFP were grown at 37°C and shifted to 45°C for 20 min. Anucleoid cells were spontaneously formed in the DNA segregation-deficient ΔmukB strain. Images were recorded as described in (A). Scale bar: 1 μm. Numbers of fluorescent MetA–YFP foci per cell and their respective localizations were determined (n=100, lower panel). The positioning of protein aggregates is indicated (blue dots). (C) Re-localization of the bacterial nucleoid to the inner membrane by the nucleoid disruption protein (Ndd) leads to the formation of one stress-induced MetA–YFP aggregate at the mid-cell or the polar region of cells. BL21 Rosetta cells producing MetA–YFP and harbouring a plasmid encoding for Ndd were grown at 30°C to mid-logarithmic growth phase. The bacterial culture was splitted and Ndd
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