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Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells
Biochemical and Biophysical Research Communications 405 (2011) 638–643
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells Toshikazu Tsuji a,1, Shigeko Kawai-Noma a, Chan-Gi Pack b, Hideki Terajima a, Junichiro Yajima c, Takayuki Nishizaka c, Masataka Kinjo d, Hideki Taguchi a,⇑ a Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan b Cellular Informatics Laboratory, RIKEN Advanced Science Institute, Wako-shi, Saitama 351-0198, Japan c Department of Physics, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan d Laboratory of Molecular Cell Dynamics, Graduate School of Life Sciences, Hokkaido University, Sapporo 001-0021, Japan
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Article history: Received 8 January 2011 Available online 28 January 2011 Keywords: Quantum dots Single-particle tracking Yeast Yeast prion proteins
a b s t r a c t Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Understanding the dynamical behaviors of individual proteins of interest in living cells is one of the demands for cell biology in the post-genome era. Yeast cells are a quite attractive organism for monitoring individual protein motions, as budding and fission yeasts are model eukaryotes with a wide variety of biological resources, including vast amounts of global genetic and proteomic data, protein–protein interaction networks and so on [1]. Watching individual proteins inside cells usually requires the incorporation of proteins that are linked to a very bright fluorescent molecule, such as a quantum dot (QD) [2–4]. However, the incorporation of proteins from outside the yeast cells is considered to be difficult due to the very rigid yeast cell wall, although a unique ‘‘injection’’ method of proteins into fission yeast was recently reported, using a microfabrication system [5]. In the budding yeast Saccharomyces cerevisiae, the non-Mendelian genetic elements such as [PSI+] and [URE3] are typical prionlike protein-based genetic elements [6–10]. In [PSI+] cells, the altered conformations of Sup35, which is the [PSI+] determinant, Abbreviations: QD, quantum dot; FCS, fluorescence correlation spectroscopy; FRAP, fluorescence recovery after photobleaching; PEG, polyethylene glycol. ⇑ Corresponding author. Fax: +81 45 924 5785. E-mail address: [email protected] (H. Taguchi). 1 Present address: Central Laboratories for Frontier Technology, Kirin Holdings Co., 1-13-5 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan. 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.01.083
are self-propagating aggregates and are transmitted to daughter cells [7–12]. Earlier single-cell approaches using fluorescence correlation spectroscopy (FCS) or fluorescence recovery after photobleaching (FRAP) revealed that the aggregated forms of Sup35 inside living cells are highly dynamic, in which continuous remodeling of Sup35 oligomers are required to maintain and transmit the prion phenotype [13–19]. Since FCS and FRAP are ensemble method for calculating the diffusion properties of fluorescent molecules [20,21], it cannot be used to unmask the individual behaviors of the molecules, which would provide unique insights into prion biology. To overcome the limitation of those ensemble techniques, here we developed a simple and versatile method to incorporate QDconjugated proteins into living budding yeast using the yeast prion Sup35 as a model protein, and analyzed the individual motions of the Sup35-QD conjugates inside living yeast cells. 2. Material and methods 2.1. Protein expression and purification The DNA encoding SUP35NM was amplified by polymerase chain reaction (PCR) using pET-SUP35NMHC, which contains the N and M domains of the SUP35 gene [22], as a template. Sup35NHMC was purified in a similar manner as Sup35NMHC [22]. Further details are described in Supplementary Information.
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2.2. QDs labeling
2.3. Yeast spheroplast preparation
The amino group of Qdot 605 ITK Amino (PEG) Quantum Dots (Invitrogen) was converted to a maleimide group with Sulfosuccinimidyl-trans-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC, Pierce). For the conversion, 8 lM Qdot 605 ITK Amino (PEG) was incubated with 1 mM Sulfo-SMCC at room temperature. After 1 h incubation, 100 mM glycine was added to quench the succinimidyl group of Sulfo-SMCC. A NAP-5 column equilibrated with A-buffer was used to remove the unreacted SMCC and the excess glycine from the maleimidederivatized QDs. Sup35NHMC was dissolved in B-buffer (PBS with 8 M guanidine hydrochloride (GdnHCl)) containing Tris(2-carboxyethyl)-phosphine (TCEP) at a 10-fold molar excess relative to Sup35NHMC. After 1 h incubation at room temperature, the protein solution was applied to a NAP-5 (GE Healthcare) column equilibrated with B-buffer to remove the excess TCEP before the QD labeling. An excess of the maleimide-derivatized QDs was added to the Sup35NHMC and incubated for 1 h at room temperature. The reaction mixture was fractionated on a Ni–NTA agarose column to separate the free QDs from the QD-labeled Sup35NHMC. After washing the column with A-buffer, the Sup35NHMC labeled with QD (Sup35-QD) was eluted with A-buffer containing 500 mM imidazole. The Sup35-QD conjugates were concentrated by ultracentrifugation (TLA100.3 rotor, Beckman).
Yeast strains (74-D694: MATa ura3–52 leu2–3112. trp1–289 his3-D200 ade1–14 [PSI+] or [psi ]) were grown in 50 ml YPD (1% yeast extract, 2% glucose, 2% bacto peptone) at 30 °C to an OD600 = 0.5 (7 106 cells/ml), pelleted at 1500g for 5 min at room temperature, and successively washed with 20 ml of 0.1 M EDTA, 1.2 M sorbitol and SCEM buffer (1.2 M sorbitol, 10 mM EDTA, 100 mM citrate, pH 5.8, 30 mM 2-mercaptoethanol). The cells were resuspended in 15 ml SCEM buffer and spheroplasted with 2 ll lyticase (10 U/ll in 20% glycerol; Sigma, L-5263) for 30 min at 30 °C. Spheroplasts were pelleted at 300g for 5 min at room temperature, and were washed twice with 20 ml of 1.2 M sorbitol and STC buffer (1.2 M sorbitol, 10 mM CaCl2, 10 mM Tris–HCl, pH 7.5). The pelleted cells were resuspended in 1 ml STC buffer. 2.4. Incorporation of QDs or QD-proteins into the spheroplasts A 100 ll portion of the spheroplast suspension was mixed with either QDs or Sup35-QDs (<10 ll) and 5 ll of 20 mg/ml bovine serum albumin (Sigma, filtration), to minimize the nonspecific adsorption of QDs or Sup35-QDs to the yeast cell membrane. The mixture was incubated for 30 min at room temperature. The incorporation of QDs or Sup35-QDs was induced by the addition of 900 ll PEG buffer (20% (w/v) PEG 8000 (Fluka), 10 mM Tris–HCl,
Fig. 1. (A) Schematic representation of the method. Cultured yeast cells are spheroplasted with lyticase to remove the cell walls. Amino groups of Qdot605 ITK amino (PEG) quantum dots (QDs) are converted to cysteine-reactive maleimide groups with Sulfosuccinimidyl-trans-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC). The maleimide-derivatized QDs are used to modify the recombinant Sup35NM protein with a unique cysteine residue at the C-terminus. The incorporation of QDs or Sup35QD conjugates into the yeast spheroplast is promoted by the addition of CaCl2 and polyethylene glycol. (B) Snapshots of incorporated QDs inside the yeast [psi ] and [PSI+] spheroplast cells, observed by fluorescence microscopy. Dotted red lines show cell shapes. See also Movies S1 and S2. (C) Fluorescence in the cytoplasms of [psi ] and [PSI+] cells was measured. The autocorrelation curves correlation spectroscopy (FCS) of QDs inside the cells. Normalized autocorrelation curves of QDs in PBS buffer and in living yeast cells. Diffusion profiles of QDs in PBS and in the cells were fitted by a one-component model and a two-component model, respectively. (D) QDs in the daughter and further progeny cells after the recovery of the cell wall (see also Movie S3). Arrows show the QDs in the progeny cells.
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T. Tsuji et al. / Biochemical and Biophysical Research Communications 405 (2011) 638–643
10 mM CaCl2, pH 7.5, filtered). After 30 min incubation at room temperature, the spheroplasts were collected by centrifugation at 300g for 5 min at room temperature. For single-particle tracking in single yeast cells, the QD-incorporated cells were washed twice with 1.2 M sorbitol and collected by centrifugation at 200g for 4 min, to remove the QDs that were not incorporated into cells. An observation chamber was made from a glass slide (24 36 mm NEO micro cover glass, Matsunami), a coverslip with two slivers of parafilm acting as spacers, and a cover glass (18 18 mm NEO micro cover glass, Matsunami). To attach the yeast cells, the chamber was coated with 0.1% concanavalin A (Wako) before the infusion of yeast cells.
dichroic mirror and D605/40M as the emitter. Alexa488-labeled Sup35NM fibrils were observed as described previously [22,24]. 3. 3D tracking microscopy Tracking of QDs in three dimensions was accomplished by a custom-made 3D tracking system [25]. Briefly, a wedge prism was placed at the back-focal plane of the objective in a standard epi-fluorescence microscope, leading to the conversion of z-directed movements into x-directed movements, which was used for the calculation of the z-directed movements. 3.1. Data analysis
2.5. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) All of the FCS measurements were performed at 25 °C on an LSM510 confocal microscope equipped with a ConfoCor 2 (Zeiss), as essentially described in our previous study[13,17,23]. Further details on FCS and FCCS are described in Supplementary information. 2.5.1. Fluorescence microscopy To visualize the QDs or Sup35-QD conjugates in vitro and in vivo, a bright-field optical microscopy system (IX-70 inverted microscope with a 100 objective lens, Olympus) with a cooled CCD camera iXon (Andor Technology) was used. The fluorescence cubes for QDs detection were E460SPUV as the exciter, 475DCXRU as the
The recorded QD dynamics in living yeast cells were analyzed by the ImageJ (http://rsb.info.nih.gov/ij/) and Igor (Wave Metrics) software. Details are described in Supplementary Information. 4. Results and discussion 4.1. Incorporation of quantum dots (QDs) into yeast cells To incorporate QDs or QD-conjugated proteins into living yeast cells, we employed a transfection method using polyethylene glycol (PEG), which was originally developed for genetic transformation [26] and later used for the introduction of yeast prion Sup35 protein [27,28]. We first incorporated QDs that were not conjugated with a protein, and then evaluated the efficiency and
Fig. 2. (A) Representative sequential time-lapse fluorescent images of Sup35-QD in [psi ] cells. The snap shots were taken from Movie S4. The intervals between images are 6 ms. The first 10 frames were merged with bright-field images to show the cell shape. Bar indicates 2 lm. (B) Representative trajectories of individual Sup35-QD motions in [psi ] (left) and [PSI+] (right) cells. The intervals between points are 6 ms, and 100 points are shown in individual trajectories.
T. Tsuji et al. / Biochemical and Biophysical Research Communications 405 (2011) 638–643
cytotoxicity of the QD incorporation (Fig. 1A). After optimization of the method, we reproducibly incorporated the QDs into the yeast spheroplast cells (strain 74-D694 [psi ] or [PSI+], which have nonprion and prion phenotypes, respectively) (Fig. 1B, supporting information (SI) Movies S1 and S2). The incorporation efficiency was dose-dependent, but we limited the incorporation to one-third of the cells to avoid increased background fluorescence that interferes the individual tracking of QDs. To determine whether the observed QDs diffused in the cells or were immobilized by attachment to a cell surface or an organelle, the ensemble behaviors of QDs were measured by FCS, which can analyze the diffusional properties of fluorescent molecules inside a living cell in a non-invasive manner [13,17]. Fitting analysis of the autocorrelation functions (Fig. 1C) revealed that the diffusion constants of QDs inside the cells (0.4–0.5 lm2/s) were 10-times slower than that in a solution (5 lm2/s) in both [psi ] and [PSI+] cells. The slower diffusion inside the spheroplast cells can be ascribed to the viscous environment of the cytoplasm, which was previously estimated [13]. Taken together, we concluded that the QD diffused in the cytosol. We note that the diffusion constants of the QDs were larger than the expected value calculated from a core of QDs described in the manufacturer’s catalogue, probably due to the bulky polymer coating and the long linkers for the chemical modification. The viability of the QD-incorporated spheroplasts was 80% of that of the spheroplast cells without the incorporation (Fig. S1), suggesting that the incorporation of QDs affects cell growth, but not seriously. In fact, we could observe the QDs that were transmitted to the daughter and further progeny cells after the cell-wall recovery of the spheroplasts (Fig. 1D, Movie S3).
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observed the motion for several seconds with a time resolution of 6 ms (Fig. 2A, Movies S4 and S5). Trajectories of individual QD motions were obtained by fitting an intensity profile of the QD fluorescence with a two-dimensional Gaussian curve (Fig. 2B). We then analyzed the motion by plotting the mean-squared displacement (MSD) against time, to evaluate the stochastic behavior of the molecules (Fig. S4). Typically, the motions were initially linear, but eventually reached a plateau, suggesting that the motion was basically Brownian, but confined within the cell volume. In addition, the [PSI+] cells tended to have slower moving molecules than the [psi ] cells (Fig. S5), reflecting the larger particles in [PSI+] cells.
4.2. Incorporation of QD-protein conjugates into yeast cells Next, a target protein was specifically conjugated with the QDs. Although non-covalent binding methods, such as biotin-streptavidin or antigen–antibody interactions, have been conventionally used to label the QDs [2], we covalently modified the QDs with proteins to minimize their possible detachment from the QD-protein conjugates (Fig. 1A). As a target protein, we used the recombinant histidine-tagged NM domain of the Sup35 protein, with a unique cysteine introduced at the C-terminus [22]. The guanidine hydrochloride-denatured Sup35 protein was modified with the maleimide-derivatized QDs (Fig. 1A). The Sup35-QD conjugates were purified by Ni-chelating affinity chromatography under the denaturing conditions. The Sup35-QD conjugates formed a typical amyloid fibril in vitro (Fig. S2), indicating that the behavior of the Sup35-QD conjugates was similar to that of the unlabeled recombinant Sup35. In addition to the fibril-forming assay, interactions between Sup35-QD conjugates and Sup35 in yeast lysates were confirmed by fluorescence cross-correlation spectroscopy (FCCS). FCCS is an extension of FCS, which can measure interactions between two different fluorescences [29–31]. We prepared yeast lysates, in which Sup35NM-GFP was expressed, and added the Sup35-QDs for FCCS measurement. FCCS of the lysates revealed that Sup35-QDs and Sup35-GFP were positively cross-correlated in [PSI+] but not in [psi ] lysates (Fig. S3). Taken together, these results suggest that Sup35-QDs interact with Sup35 in [PSI+] cells. 4.3. Single-particle tracking of Sup35-QDs in living yeast cells The Sup35-QD conjugates were incorporated into [PSI+] and [psi ] cells by the PEG-assisted transfection protocol. The efficiency of the Sup35-QD incorporation was independent of the prion phenotypes and was comparable to that of the unlabeled QDs. We selected the cells in which the incorporation of the QD conjugates was sparse, to facilitate the tracking of the individual QDs, and
Fig. 3. (A) Representative ‘‘Stop and Go’’ behavior of Sup35-QD motion in a [PSI+] cell (see also Movie S6). (B) Time-lapse fluorescent images of Sup35-QDs in ‘‘Division’’ behavior of Sup35-QD particles in a [PSI+] cell (see also Movie S7). Arrows show the timing of the division of the particle. (C) Representative threedimensional trajectory of Sup35-QD in a [PSI+] cell, with 100 points. The intervals between points are 2 ms.
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movements between [PSI+] and [psi ] cells further supported the retention of the Sup35 moiety in the Sup35-QDs in the cells. Acknowledgments We thank K. Mizutani for technical assistance with the 3D tracking analysis. This work was supported by Grants-in-Aid for Scientific Research (B) and on Priority Areas (17370034, 18031007, 19058002 to H.T.) from JSPS and MEXT, Japan, and was also supported in part by a grant to T. N. from the New Energy and Industrial Technology Development Organization (NEDO). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2011.01.083.
Fig. 4. Distribution of diffusion coefficients (D) derived from Sup35-QD motions in [psi ] and [PSI+] cells. Both distributions are shown with the same horizontal scale. The diffusion coefficients were determined from the trajectories of individual molecules in the cells.
4.4. Irregular motions of Sup35-QDs in [PSI+] cells The unique advantage of single-particle tracking is that it allowed us to watch irregular motions that are usually obscured by the conventional ensemble-averaging method. During the observation we noticed that some of the QDs moved irregularly and did not display typical diffusion patterns. Intriguingly, such irregular motions were only observed in [PSI+] cells, suggesting that the irregular movements are characteristic of the prion. The irregular motions included a repeated ‘‘Go-Stop’’ motion (Fig. 3A, Movie S6) and the multiplication of smaller QDs from a clustered QD particle (Fig. 3B, Movie S7). 4.5. Three-dimensional movements of Sup35-QDs in [PSI+] cells To gain higher spatial dimensions, we also applied an epi-illumination based three-dimensional (3D) tracking technique, which analyzes the xyz-movement without reducing the time resolution [25]. The 3D trajectory of the Sup35-QD movement in vivo showed that the movement was in all directions (Fig. 3C, Movies S8 and S9, Fig. S6), confirming that the movement was not on the surface of the membrane but inside the cell. In some cases, we observed the confined movement of Sup35-QDs in a certain space (Fig. S7), suggesting that the Sup35-QD movements were in the cytoplasm, but not in other organelles, such as the nucleus or vacuole. 4.6. Histograms of the movements in [psi ] and [PSI+] cells Calculation of the diffusion constants (D) from the MSD (Fig. S4) provided the statistics of the individual motions (N 100), as summarized in a histogram (Fig. 4). The histograms of both the [PSI+] and [psi ] cells had a similar distribution at around –0.6 of log D, which corresponds to the diffusion constant of QD itself in the cell (Fig. 1C). We note that the diffusion constant of QDs is comparable to that for Sup35-GFP in [PSI+] cells, which were obtained from FCS analysis [13], since QDs in the cells tended to form clusters. However, the histogram of [PSI+] cells had a second distribution with slower motion (log D 2.2). The slower distribution in [PSI+] cells, as well as the probability density distribution based on the histograms (Fig. S8), indicates the overall slower movement of Sup35-QDs in [PSI+] cells, which is consistent with the FCCS results (Fig. S3). More importantly, the overall difference in the
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells Toshikazu Tsuji a,1, Shigeko Kawai-Noma a, Chan-Gi Pack b, Hideki Terajima a, Junichiro Yajima c, Takayuki Nishizaka c, Masataka Kinjo d, Hideki Taguchi a,⇑ a Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan b Cellular Informatics Laboratory, RIKEN Advanced Science Institute, Wako-shi, Saitama 351-0198, Japan c Department of Physics, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan d Laboratory of Molecular Cell Dynamics, Graduate School of Life Sciences, Hokkaido University, Sapporo 001-0021, Japan
a r t i c l e
i n f o
Article history: Received 8 January 2011 Available online 28 January 2011 Keywords: Quantum dots Single-particle tracking Yeast Yeast prion proteins
a b s t r a c t Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Understanding the dynamical behaviors of individual proteins of interest in living cells is one of the demands for cell biology in the post-genome era. Yeast cells are a quite attractive organism for monitoring individual protein motions, as budding and fission yeasts are model eukaryotes with a wide variety of biological resources, including vast amounts of global genetic and proteomic data, protein–protein interaction networks and so on [1]. Watching individual proteins inside cells usually requires the incorporation of proteins that are linked to a very bright fluorescent molecule, such as a quantum dot (QD) [2–4]. However, the incorporation of proteins from outside the yeast cells is considered to be difficult due to the very rigid yeast cell wall, although a unique ‘‘injection’’ method of proteins into fission yeast was recently reported, using a microfabrication system [5]. In the budding yeast Saccharomyces cerevisiae, the non-Mendelian genetic elements such as [PSI+] and [URE3] are typical prionlike protein-based genetic elements [6–10]. In [PSI+] cells, the altered conformations of Sup35, which is the [PSI+] determinant, Abbreviations: QD, quantum dot; FCS, fluorescence correlation spectroscopy; FRAP, fluorescence recovery after photobleaching; PEG, polyethylene glycol. ⇑ Corresponding author. Fax: +81 45 924 5785. E-mail address: [email protected] (H. Taguchi). 1 Present address: Central Laboratories for Frontier Technology, Kirin Holdings Co., 1-13-5 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan. 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.01.083
are self-propagating aggregates and are transmitted to daughter cells [7–12]. Earlier single-cell approaches using fluorescence correlation spectroscopy (FCS) or fluorescence recovery after photobleaching (FRAP) revealed that the aggregated forms of Sup35 inside living cells are highly dynamic, in which continuous remodeling of Sup35 oligomers are required to maintain and transmit the prion phenotype [13–19]. Since FCS and FRAP are ensemble method for calculating the diffusion properties of fluorescent molecules [20,21], it cannot be used to unmask the individual behaviors of the molecules, which would provide unique insights into prion biology. To overcome the limitation of those ensemble techniques, here we developed a simple and versatile method to incorporate QDconjugated proteins into living budding yeast using the yeast prion Sup35 as a model protein, and analyzed the individual motions of the Sup35-QD conjugates inside living yeast cells. 2. Material and methods 2.1. Protein expression and purification The DNA encoding SUP35NM was amplified by polymerase chain reaction (PCR) using pET-SUP35NMHC, which contains the N and M domains of the SUP35 gene [22], as a template. Sup35NHMC was purified in a similar manner as Sup35NMHC [22]. Further details are described in Supplementary Information.
T. Tsuji et al. / Biochemical and Biophysical Research Communications 405 (2011) 638–643
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2.2. QDs labeling
2.3. Yeast spheroplast preparation
The amino group of Qdot 605 ITK Amino (PEG) Quantum Dots (Invitrogen) was converted to a maleimide group with Sulfosuccinimidyl-trans-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC, Pierce). For the conversion, 8 lM Qdot 605 ITK Amino (PEG) was incubated with 1 mM Sulfo-SMCC at room temperature. After 1 h incubation, 100 mM glycine was added to quench the succinimidyl group of Sulfo-SMCC. A NAP-5 column equilibrated with A-buffer was used to remove the unreacted SMCC and the excess glycine from the maleimidederivatized QDs. Sup35NHMC was dissolved in B-buffer (PBS with 8 M guanidine hydrochloride (GdnHCl)) containing Tris(2-carboxyethyl)-phosphine (TCEP) at a 10-fold molar excess relative to Sup35NHMC. After 1 h incubation at room temperature, the protein solution was applied to a NAP-5 (GE Healthcare) column equilibrated with B-buffer to remove the excess TCEP before the QD labeling. An excess of the maleimide-derivatized QDs was added to the Sup35NHMC and incubated for 1 h at room temperature. The reaction mixture was fractionated on a Ni–NTA agarose column to separate the free QDs from the QD-labeled Sup35NHMC. After washing the column with A-buffer, the Sup35NHMC labeled with QD (Sup35-QD) was eluted with A-buffer containing 500 mM imidazole. The Sup35-QD conjugates were concentrated by ultracentrifugation (TLA100.3 rotor, Beckman).
Yeast strains (74-D694: MATa ura3–52 leu2–3112. trp1–289 his3-D200 ade1–14 [PSI+] or [psi ]) were grown in 50 ml YPD (1% yeast extract, 2% glucose, 2% bacto peptone) at 30 °C to an OD600 = 0.5 (7 106 cells/ml), pelleted at 1500g for 5 min at room temperature, and successively washed with 20 ml of 0.1 M EDTA, 1.2 M sorbitol and SCEM buffer (1.2 M sorbitol, 10 mM EDTA, 100 mM citrate, pH 5.8, 30 mM 2-mercaptoethanol). The cells were resuspended in 15 ml SCEM buffer and spheroplasted with 2 ll lyticase (10 U/ll in 20% glycerol; Sigma, L-5263) for 30 min at 30 °C. Spheroplasts were pelleted at 300g for 5 min at room temperature, and were washed twice with 20 ml of 1.2 M sorbitol and STC buffer (1.2 M sorbitol, 10 mM CaCl2, 10 mM Tris–HCl, pH 7.5). The pelleted cells were resuspended in 1 ml STC buffer. 2.4. Incorporation of QDs or QD-proteins into the spheroplasts A 100 ll portion of the spheroplast suspension was mixed with either QDs or Sup35-QDs (<10 ll) and 5 ll of 20 mg/ml bovine serum albumin (Sigma, filtration), to minimize the nonspecific adsorption of QDs or Sup35-QDs to the yeast cell membrane. The mixture was incubated for 30 min at room temperature. The incorporation of QDs or Sup35-QDs was induced by the addition of 900 ll PEG buffer (20% (w/v) PEG 8000 (Fluka), 10 mM Tris–HCl,
Fig. 1. (A) Schematic representation of the method. Cultured yeast cells are spheroplasted with lyticase to remove the cell walls. Amino groups of Qdot605 ITK amino (PEG) quantum dots (QDs) are converted to cysteine-reactive maleimide groups with Sulfosuccinimidyl-trans-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC). The maleimide-derivatized QDs are used to modify the recombinant Sup35NM protein with a unique cysteine residue at the C-terminus. The incorporation of QDs or Sup35QD conjugates into the yeast spheroplast is promoted by the addition of CaCl2 and polyethylene glycol. (B) Snapshots of incorporated QDs inside the yeast [psi ] and [PSI+] spheroplast cells, observed by fluorescence microscopy. Dotted red lines show cell shapes. See also Movies S1 and S2. (C) Fluorescence in the cytoplasms of [psi ] and [PSI+] cells was measured. The autocorrelation curves correlation spectroscopy (FCS) of QDs inside the cells. Normalized autocorrelation curves of QDs in PBS buffer and in living yeast cells. Diffusion profiles of QDs in PBS and in the cells were fitted by a one-component model and a two-component model, respectively. (D) QDs in the daughter and further progeny cells after the recovery of the cell wall (see also Movie S3). Arrows show the QDs in the progeny cells.
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10 mM CaCl2, pH 7.5, filtered). After 30 min incubation at room temperature, the spheroplasts were collected by centrifugation at 300g for 5 min at room temperature. For single-particle tracking in single yeast cells, the QD-incorporated cells were washed twice with 1.2 M sorbitol and collected by centrifugation at 200g for 4 min, to remove the QDs that were not incorporated into cells. An observation chamber was made from a glass slide (24 36 mm NEO micro cover glass, Matsunami), a coverslip with two slivers of parafilm acting as spacers, and a cover glass (18 18 mm NEO micro cover glass, Matsunami). To attach the yeast cells, the chamber was coated with 0.1% concanavalin A (Wako) before the infusion of yeast cells.
dichroic mirror and D605/40M as the emitter. Alexa488-labeled Sup35NM fibrils were observed as described previously [22,24]. 3. 3D tracking microscopy Tracking of QDs in three dimensions was accomplished by a custom-made 3D tracking system [25]. Briefly, a wedge prism was placed at the back-focal plane of the objective in a standard epi-fluorescence microscope, leading to the conversion of z-directed movements into x-directed movements, which was used for the calculation of the z-directed movements. 3.1. Data analysis
2.5. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) All of the FCS measurements were performed at 25 °C on an LSM510 confocal microscope equipped with a ConfoCor 2 (Zeiss), as essentially described in our previous study[13,17,23]. Further details on FCS and FCCS are described in Supplementary information. 2.5.1. Fluorescence microscopy To visualize the QDs or Sup35-QD conjugates in vitro and in vivo, a bright-field optical microscopy system (IX-70 inverted microscope with a 100 objective lens, Olympus) with a cooled CCD camera iXon (Andor Technology) was used. The fluorescence cubes for QDs detection were E460SPUV as the exciter, 475DCXRU as the
The recorded QD dynamics in living yeast cells were analyzed by the ImageJ (http://rsb.info.nih.gov/ij/) and Igor (Wave Metrics) software. Details are described in Supplementary Information. 4. Results and discussion 4.1. Incorporation of quantum dots (QDs) into yeast cells To incorporate QDs or QD-conjugated proteins into living yeast cells, we employed a transfection method using polyethylene glycol (PEG), which was originally developed for genetic transformation [26] and later used for the introduction of yeast prion Sup35 protein [27,28]. We first incorporated QDs that were not conjugated with a protein, and then evaluated the efficiency and
Fig. 2. (A) Representative sequential time-lapse fluorescent images of Sup35-QD in [psi ] cells. The snap shots were taken from Movie S4. The intervals between images are 6 ms. The first 10 frames were merged with bright-field images to show the cell shape. Bar indicates 2 lm. (B) Representative trajectories of individual Sup35-QD motions in [psi ] (left) and [PSI+] (right) cells. The intervals between points are 6 ms, and 100 points are shown in individual trajectories.
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cytotoxicity of the QD incorporation (Fig. 1A). After optimization of the method, we reproducibly incorporated the QDs into the yeast spheroplast cells (strain 74-D694 [psi ] or [PSI+], which have nonprion and prion phenotypes, respectively) (Fig. 1B, supporting information (SI) Movies S1 and S2). The incorporation efficiency was dose-dependent, but we limited the incorporation to one-third of the cells to avoid increased background fluorescence that interferes the individual tracking of QDs. To determine whether the observed QDs diffused in the cells or were immobilized by attachment to a cell surface or an organelle, the ensemble behaviors of QDs were measured by FCS, which can analyze the diffusional properties of fluorescent molecules inside a living cell in a non-invasive manner [13,17]. Fitting analysis of the autocorrelation functions (Fig. 1C) revealed that the diffusion constants of QDs inside the cells (0.4–0.5 lm2/s) were 10-times slower than that in a solution (5 lm2/s) in both [psi ] and [PSI+] cells. The slower diffusion inside the spheroplast cells can be ascribed to the viscous environment of the cytoplasm, which was previously estimated [13]. Taken together, we concluded that the QD diffused in the cytosol. We note that the diffusion constants of the QDs were larger than the expected value calculated from a core of QDs described in the manufacturer’s catalogue, probably due to the bulky polymer coating and the long linkers for the chemical modification. The viability of the QD-incorporated spheroplasts was 80% of that of the spheroplast cells without the incorporation (Fig. S1), suggesting that the incorporation of QDs affects cell growth, but not seriously. In fact, we could observe the QDs that were transmitted to the daughter and further progeny cells after the cell-wall recovery of the spheroplasts (Fig. 1D, Movie S3).
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observed the motion for several seconds with a time resolution of 6 ms (Fig. 2A, Movies S4 and S5). Trajectories of individual QD motions were obtained by fitting an intensity profile of the QD fluorescence with a two-dimensional Gaussian curve (Fig. 2B). We then analyzed the motion by plotting the mean-squared displacement (MSD) against time, to evaluate the stochastic behavior of the molecules (Fig. S4). Typically, the motions were initially linear, but eventually reached a plateau, suggesting that the motion was basically Brownian, but confined within the cell volume. In addition, the [PSI+] cells tended to have slower moving molecules than the [psi ] cells (Fig. S5), reflecting the larger particles in [PSI+] cells.
4.2. Incorporation of QD-protein conjugates into yeast cells Next, a target protein was specifically conjugated with the QDs. Although non-covalent binding methods, such as biotin-streptavidin or antigen–antibody interactions, have been conventionally used to label the QDs [2], we covalently modified the QDs with proteins to minimize their possible detachment from the QD-protein conjugates (Fig. 1A). As a target protein, we used the recombinant histidine-tagged NM domain of the Sup35 protein, with a unique cysteine introduced at the C-terminus [22]. The guanidine hydrochloride-denatured Sup35 protein was modified with the maleimide-derivatized QDs (Fig. 1A). The Sup35-QD conjugates were purified by Ni-chelating affinity chromatography under the denaturing conditions. The Sup35-QD conjugates formed a typical amyloid fibril in vitro (Fig. S2), indicating that the behavior of the Sup35-QD conjugates was similar to that of the unlabeled recombinant Sup35. In addition to the fibril-forming assay, interactions between Sup35-QD conjugates and Sup35 in yeast lysates were confirmed by fluorescence cross-correlation spectroscopy (FCCS). FCCS is an extension of FCS, which can measure interactions between two different fluorescences [29–31]. We prepared yeast lysates, in which Sup35NM-GFP was expressed, and added the Sup35-QDs for FCCS measurement. FCCS of the lysates revealed that Sup35-QDs and Sup35-GFP were positively cross-correlated in [PSI+] but not in [psi ] lysates (Fig. S3). Taken together, these results suggest that Sup35-QDs interact with Sup35 in [PSI+] cells. 4.3. Single-particle tracking of Sup35-QDs in living yeast cells The Sup35-QD conjugates were incorporated into [PSI+] and [psi ] cells by the PEG-assisted transfection protocol. The efficiency of the Sup35-QD incorporation was independent of the prion phenotypes and was comparable to that of the unlabeled QDs. We selected the cells in which the incorporation of the QD conjugates was sparse, to facilitate the tracking of the individual QDs, and
Fig. 3. (A) Representative ‘‘Stop and Go’’ behavior of Sup35-QD motion in a [PSI+] cell (see also Movie S6). (B) Time-lapse fluorescent images of Sup35-QDs in ‘‘Division’’ behavior of Sup35-QD particles in a [PSI+] cell (see also Movie S7). Arrows show the timing of the division of the particle. (C) Representative threedimensional trajectory of Sup35-QD in a [PSI+] cell, with 100 points. The intervals between points are 2 ms.
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movements between [PSI+] and [psi ] cells further supported the retention of the Sup35 moiety in the Sup35-QDs in the cells. Acknowledgments We thank K. Mizutani for technical assistance with the 3D tracking analysis. This work was supported by Grants-in-Aid for Scientific Research (B) and on Priority Areas (17370034, 18031007, 19058002 to H.T.) from JSPS and MEXT, Japan, and was also supported in part by a grant to T. N. from the New Energy and Industrial Technology Development Organization (NEDO). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2011.01.083.
Fig. 4. Distribution of diffusion coefficients (D) derived from Sup35-QD motions in [psi ] and [PSI+] cells. Both distributions are shown with the same horizontal scale. The diffusion coefficients were determined from the trajectories of individual molecules in the cells.
4.4. Irregular motions of Sup35-QDs in [PSI+] cells The unique advantage of single-particle tracking is that it allowed us to watch irregular motions that are usually obscured by the conventional ensemble-averaging method. During the observation we noticed that some of the QDs moved irregularly and did not display typical diffusion patterns. Intriguingly, such irregular motions were only observed in [PSI+] cells, suggesting that the irregular movements are characteristic of the prion. The irregular motions included a repeated ‘‘Go-Stop’’ motion (Fig. 3A, Movie S6) and the multiplication of smaller QDs from a clustered QD particle (Fig. 3B, Movie S7). 4.5. Three-dimensional movements of Sup35-QDs in [PSI+] cells To gain higher spatial dimensions, we also applied an epi-illumination based three-dimensional (3D) tracking technique, which analyzes the xyz-movement without reducing the time resolution [25]. The 3D trajectory of the Sup35-QD movement in vivo showed that the movement was in all directions (Fig. 3C, Movies S8 and S9, Fig. S6), confirming that the movement was not on the surface of the membrane but inside the cell. In some cases, we observed the confined movement of Sup35-QDs in a certain space (Fig. S7), suggesting that the Sup35-QD movements were in the cytoplasm, but not in other organelles, such as the nucleus or vacuole. 4.6. Histograms of the movements in [psi ] and [PSI+] cells Calculation of the diffusion constants (D) from the MSD (Fig. S4) provided the statistics of the individual motions (N 100), as summarized in a histogram (Fig. 4). The histograms of both the [PSI+] and [psi ] cells had a similar distribution at around –0.6 of log D, which corresponds to the diffusion constant of QD itself in the cell (Fig. 1C). We note that the diffusion constant of QDs is comparable to that for Sup35-GFP in [PSI+] cells, which were obtained from FCS analysis [13], since QDs in the cells tended to form clusters. However, the histogram of [PSI+] cells had a second distribution with slower motion (log D 2.2). The slower distribution in [PSI+] cells, as well as the probability density distribution based on the histograms (Fig. S8), indicates the overall slower movement of Sup35-QDs in [PSI+] cells, which is consistent with the FCCS results (Fig. S3). More importantly, the overall difference in the
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