Letter pubs.acs.org/NanoLett
Nanoscale Organization of Mitochondrial Microcompartments Revealed by Combining Tracking and Localization Microscopy Timo Appelhans,† Christian P. Richter,‡ Verena Wilkens,† Samuel T. Hess,§ Jacob Piehler,‡ and Karin B. Busch*,† †
Division of Mitochondrial Dynamics and ‡Division of Biophysics, Department of Biology, University of Osnabrück, Barbarastrasse 11, 49076 Osnabrück, Germany § Department of Physics and Astronomy, 120 Bennett Hall, University of Maine, Orono, Maine, 04469 United States S Supporting Information *
ABSTRACT: While detailed information on the nanoscaleorganization of proteins within intracellular membranes has emerged from electron microcopy, information on their spatiotemporal dynamics is scarce. By use of a photostable rhodamine attached specifically to Halo-tagged proteins in mitochondrial membranes, we were able to track and localize single protein complexes such as Tom20 and ATP synthase in suborganellar structures in live cells. Individual membrane proteins in the inner and outer membrane of mitochondria were imaged over long time periods with localization precisions below 15 nm. Projection of single molecule trajectories revealed diffusion-restricting microcompartments such as individual cristae in mitochondria. At the same time, protein-specific diffusion characteristics within different mitochondrial membranes could be extracted. KEYWORDS: Super-resolution imaging, single molecule tracking, mitochondria, ATP synthase, Tom20, membrane protein dynamics, TALM
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accessible zones, not only the dynamics but also the morphology and the connectivity of different microcompartments within the membranes were revealed, as well as the dwell time in these structures. We established this approach for probing the spatiotemporal organization of cellular microcompartments, that is, subcellular units that provide the platform for signaling and activity and, more importantly, are the base of functional heterogeneity. A key example for the intimate linkage between heterogeneity and functionality are mitochondria, the cells’ main energy suppliers. Mitochondria are heterogeneous in terms of structure and function and this heterogeneity is closely linked to the dynamics, mobility and exchange of mitochondrial proteins. Moreover, these dynamics maintain the functionality of mitochondria.16 Their impairment is closely associated with age-related neuro-degenerative and certain neuro-muscular diseases.17 For treating these maladies, an understanding of the spatiotemporal organization of its functional compounds, such as TOM (the translocase of the outer membrane) and the respiratory chain complexes (RCC) in the context of mitochondrial compartmentation, is crucial. Because of their evolutionary origin, mitochondria possess two distinct membranes, the outer mitochondrial membrane (OMM) that
uper-resolution fluorescence imaging techniques based on single molecule localization have opened tremendous insights into submicrometer organization of the cell.1−7 Among these techniques, fluorescence photoactivation localization microscopy (FPALM)3 can be applied in living cells and has been shown to provide information on local diffusion properties.8−12 However, prolonged tracking of individual fluorescent proteins is limited by their modest photostability. For high-density mapping of protein mobilities, trajectories from many molecules are required, which are irreversibly bleached after readout.11 Stochastic optical reconstruction microscopy (STORM) techniques based on reversible photoswitching1,7 have also been successfully applied for live cell super-resolution imaging,13,14 but these are not suitable for tracking individual probes due to their fast blinking kinetics. We here report an approach for single molecule localization-based super-resolution imaging, which employs bioorthogonal posttranslational labeling of proteins with a bright and photostable fluorescent probe. To this end, target proteins in mitochondrial membranes were covalently labeled with tetramethylrhodamine (TMR) at the HaloTag (Halo/TMR). The HaloTag reporter protein is a derivative of a 33 kDa, monomeric hydrolase not endogenous to mammalian cells that irreversibly reacts with chlorhexane derivative (HaloTag) in a rapid manner.15 Owing to the brightness of TMR, the so-labeled membrane protein could be localized with accuracy below 15 nm. The organic dye was also sufficiently photostable to permit acquisition of long trajectories. As these diffusive probes rapidly explored © 2011 American Chemical Society
Received: September 26, 2011 Revised: December 23, 2011 Published: December 28, 2011 610
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Figure 1. Nanometric localization of Tom20 and respiratory chain complex I in the OMM and IMM, respectively, of living HeLa cells revealed by single molecule localization microscopy. (A) Schematic view of a mitochondrion with OMM and IMM adapted from Mannella et al.18 The inner membrane is further divided into the CM and the IBM microcompartments that are adjacent to the OMM. Respiratory chain complexes are preferentially located in the CM. (B) Comparison of diffraction-limited widefield and superresolution imaging based on single molecule localization of a mitochondrion with Tom20-Halo (TMR-labeled, red) and CI-paGFP (green). The super-resolved part is a rendered image from a series of images with single signals of Tom20-Halo/TMR and CI-paGFP molecules each fitted by a modified 2D Gaussian. (C) Localization precision for CIpaGFP and Tom20-Halo/TMR calculated according to Thompson et al.26 (D) Fluorescence intensity plot of cross sections through the averaged widefield image (thin lines) and the TALM image (thick lines) of the mitochondrion from (B). Scale bar: 500 nm (B).
the spatiotemporal dynamics of these proteins. To address the localization as well as dynamics of mitochondrial membrane proteins we employed tracking and localization microscopy (TALM) of individual mitochondrial proteins and protein complexes. To this end, OMM proteins Tom20 and hFis1 as well as subunits of the respiratory chain complexes II (succinate dehydrogenase, CII) and V (ATP synthase, CV) in the IMM were fused to the HaloTag (Tom20-Halo, Halo-hFis1, CIIHalo, and CV-Halo, respectively). HeLa cells stably transfected with one of these constructs could be specifically and efficiently labeled with 1−10 nM HTL-TMR (Supporting Information Figures 1 and 2). In order to confirm the correct localization of tagged and labeled Tom20 in the OMM, double transfection with Tom20-Halo and respiratory complex I tagged with paGFP (CI-paGFP) located in the IMM was performed. Upon substoichiometric labeling of Tom20-Halo with HTL-TMR (0.5 nM), individual TOM complexes could be discerned. By rendering of individual localizations from 200 to 500 frames (6−16 s), a super-resolution image of the OMM was obtained (Figure 1B). Since the mitochondrial diameter in average is 500 nm and the diffraction limited resolution is about 500−800 nm in z, all signals assigned to a mitochondrion likely derived from this specific mitochondrion. To further confine the focal position without 3D equipment, exclusively single emitters with a signal of 8 times above the mean background were accepted. The position of each molecule was then plotted as a Gaussian blurred spot with the amplitude proportional to the observed
encloses the organelle in tubular structures (∼500 nm in diameter) and the inner mitochondrial membrane (IMM) that intrudes with numerous invaginations (∼1 crista/60 nm) into the interior of mitochondrial space, thereby enlarging the surface manifold (Figure 1A). These cristae membranes (CM) of the IMM are likely separated from the inner boundary membrane (IBM) part of the IMM by so-called cristae junctions that probably hinder diffusion of solutes between intercristae space and intermembrane space.18,19 It is not known whether or to what extent these junctions represent diffusion barriers to inner membrane proteins, too, but experimental data have shown that mitochondria display a patterned distribution of respiratory complexes in dynamic mitochondria indicating an at least partially restricted diffusibility.20,21 TOM as a major compound of the import machinery of mitochondria is located in the OMM. In contrast, the complexes of the respiratory chain (RCC) are situated in the CM of the IMM. Owing to the nanoscopic dimensions of these structures, the dynamics of protein diffusion in CM cannot be explored by diffraction-limited fluorescence techniques such as fluorescence recovery after photobleaching (FRAP). The submicroscopic organization of Tom20 and ATP synthase (CV) in mitochondria has already been explored by super-resolution fluorescence microscopy such as stimulated emission depletion (STED) microscopy, PALM, and STORM.22−25 However, these methods were applied to fixed, detergent-treated cells and thus did not provide information on 611
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Figure 2. Tracking and localization of single Tom20 in the OMM of living HeLa cells revealing that the majority of Tom20 is mobile. (A) Superresolution image showing the localization of Tom20-Halo/TMR in the OMM of a bifurcated mitochondrion. The bifurcated mitochondrion is part of a mitochondrial reticulum as demonstrated by the averaged fluorescence wide-field image (inset). The bifurcated mitochondrion (framed in red) was further analyzed by single molecule tracking and localization microscopy (TALM). (B) Magnified detail from the previous image (A, red framed) to show the rather homogeneous distribution of single Tom20-Halo/TMR along site the mitochondrion. The image was obtained by localizing and rendering hundreds of single molecules in subsequent frames by a 2D-Gaussian-fit. (C) Trajectories of individual Tom20-Halo/TMR in the same part of the mitochondrion. (D) Trajectory map of Tom20 in the entire bifurcated mitochondrion with accented Tom20 trajectories characterized by different diffusion behavior (red, confined; blue, freely diffusive). (E−F) Single trajectories typical for restricted and freely diffusive Tom20 molecules with their specific diffusivities calculated from their respective mean square displacement plots. In both cases, anomalous diffusion behavior (α ≠ 1) describes the mobility best. Distribution of step lengths resulting in different diffusion constants by appropriate preseparation into confined and freely diffusing molecules. Scale bars: 1 μm (A,D), 500 nm (B,C).
confined (red) and mobile (blue) species. By analysis of the mean square displacement (MSD) as a function of the lag time,9,11,27,28 we found that long-range diffusion data were not adequately described by a simple linear function. In contrast, minimum systematic deviation was observed when a power fit was used (Supporting Information Figure 8B) implying an anomalous diffusion behavior.27 This was confirmed by the analysis of single finite trajectories29 (Supporting Information Figure 8C,D). Albeit, to better identify and display extreme diffusion behavior of distinct single molecules (Figure 2D,E) we chose the preconfinement analysis (pre- and postprocessed jump size distributions). Following, diffusion constants were determined from decomposing the obtained jump size histograms to a mixture model of increasing complexity. The final complexity was validated by applying a χ 2 -test. Independent of the analysis method (fraction fit, single finite trajectory MSD analysis, and postprocessed jump size distributions, respectively), the majority of Tom20 was found to be mobile (52 ± 5% with Dapp 0.14 ± 0.01 μm2/s; 34 ± 5% less mobile) and only 14 ± 1% were immobile with an apparent diffusion constant Dapp < 0.005 μm2/s (e.g. trajectory a in Figure 2F, and Supporting Information Figure 3A). Restriction of mobility could be partially associated with a confinement at the tips of mitochondria (Figure 2Dc). We cannot exclude that in some cases confinement might be apparent, though, because
intensity and width equal to the localization precision estimated according to Thompson et al.26 Simultaneous FPALM imaging of CI-paGFP revealed clearly its distinct localization in the IMM of the same mitochondrion. Tom20 was localized with a mean accuracy of 12 nm, while the mean accuracy of CI-paGFP localized with FPALM was 24 nm (Figure 1C). The good localization precision of TMR-tagged proteins was possible because of the brightness of TMR and because the cells' autofluorescence emitted in this wavelength regime was low and thus a better S/N ratio could be achieved.26 A transverse fluorescence line plot through the widefield image in comparison to the superresolved image visibly demonstrates the improvement in resolution (Figure 1D); in the superresolved image, molecules located in the IMM can be clearly distinguished from molecules in the OMM. In addition to the localization with high precision (Figure 2A,B), trajectories of individual Tom20-Halo/TMR could be reconstructed over extended time periods due to the high photostability of TMR (Figure 2C,D and Supporting Information Videos 1 (si_003) and 1B online). Distinct types of trajectories were observed: molecules with a rather confined movement (Figure 2D−F red) and molecules with higher mobility (Figure 2D−F blue). To further analyze this, jump size distributions of Tom20molecules were generated. Clearly, at least two fractions of Tom20 characterized by distinct mobilities were identified: 612
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Figure 3. TALM as a tool to reveal hidden mitochondrial morphology. (A) Halo/TMR-hFis1 distribution in the OMM of tubular mitochondria (inset) on the single molecule level. (B) Distinct trajectories of hFis1 molecules (colored) projected on the localized molecules of hFis revealing the existence of a single circular mitochondrion (green arrowhead) buried between the two branches of another bifurcated mitochondrion (white arrowhead). The course of the hFis trajectories in the circular mitochondrion (orange, green, blue) is unique and demonstrates that a single hFis molecule can orbit a circular mitochondrion randomly. (C) Two distinct finite trajectories, one of a hFis1 molecule moving along the circular mitochondrion (orange), one of a single hFis1 molecule traveling along the tubular mitochondrion (magenta). The mobility in both cases is nearly unhindered (α ∼ 1), albeit the analysis of the finite trajectories by msd analysis states a higher mobility for the hFis1 molecule on the tubular mitochondrion than for the molecule on the circular mitochondrion. All images were recorded with a frame rate of 30 Hz using HiLo excitation in a TIRF setup. Scale bars: 1000 nm (A), and 500 nm (B).
Figure 4. Tracking and localization microscopy as a versatile tool to dissect the spatiotemporal microcompartmentation of single respiratory complexes in living mitochondria. Trajectory analysis of respiratory complexes V and II in the IMM. (A) Rendered image of localized CV-Halo/ TMR clearly indicating a compartmented localization of CV but without revealing clear details. (B) Trajectory map of CV-Halo/TMR in mitochondria with a significant proportion of track courses orthogonal to the longitudinal axis of mitochondria (superimposed on an averaged intensity projection from raw data). (C) Step length analysis showing two subpopulations of diffusive CV-TMR molecules representing free (blue) and confined diffusion (red). (D) Orthogonal tracks of CV-Halo/TMR in single cristae. The typical shape of a crista is depicted in a gray dotted line. (E) Trajectories of four CII-Halo/TMR molecules as representatives for localization and diffusion in the IBM and CM, respectively. Scale bars: 500 nm (A,B), 300 nm (E).
rather uniformly distributed across single mitochondria in live cells (Figure 3A) probably because hFis1 moved along the OMM without a significant immobile fraction indicating pure Brownian motion (Supporting Information Figure 3B and Supporting Information Video 2 (si_004)). Consequently, the
molecules moving in the depth of the mitochondrion would be projected on a spot. In contrast to Tom20, hFis1, a protein anchored in the OMM by a single trans-membrane domain, is not part of a larger complex. Super-resolution images confirmed that hFis1 is 613
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Figure 5. Localization of respiratory complexes in the IMM by single molecule live-cell microscopy enables the study of nanoscale organization of mitochondrial microcompartments such as cristae. (A) Localization of complex V (CV-Halo/TMR) in a live mitochondrion, depicted in different subsequent time windows (4.8 s). The repetitive orthogonal patterns indicate a predominant localization in the cristae membrane (localized SM are superimposed on an averaged image from raw data). (B) Overlay of subsequent time windows showing CV localization (same mitochondrion as in A). CV is preferentially arranged in perpendicular rows compared to the longitudinal axis of the mitochondrion. The arrowheads indicate the rather stable arrangement of some of these cristae rows during 24 s. (C) TEM micrograph showing the regular arrangement of cristae over large areas in a typical HeLa mitochondrion. The insert depicts the distances between outward and inward membranes from two adjacent cristae, respectively. (D) Plot of measured distances between adjacent cristae in electron and TALM micrographs. Scale bars: 300 nm (B,C).
mitochondrion, implies a higher mobility of hFis1 in the tubular mitochondrion than in the circular one (Figure 3C). The connectivity between mitochondria as well as mitochondrial subcompartments is important for inter- and intramitochondrial equilibration and remixing to avoid the local accumulation of damaged compounds. The importance of this property is stressed by the fact that single isolated mitochondria, which lost the ability to fuse and exchange compounds, often in combination with a functional decline, are prone to be eliminated from the mitochondrial population.16 In this context, we next investigated the spatiotemporal dynamics of respiratory chain complexes (RCC) within inner-mitochondrial microcompartments. Individual ATP synthase complexes were localized with high precision upon labeling of CV-Halo with HTL-TMR (Supporting Information Figure 4A). The distribution of CV in rendered images was clearly distinct from the distribution of Tom20 and already indicated a compartmentalization (Figure 4A and Supporting Information Video 3 (si_010)). The trajectory map of individual CV complexes confirmed the following impression: The majority of CV displayed a restricted diffusion and a major fraction of CV trajectories displayed courses orthogonal to the longitudinal axis of the mitochondrion (Figure 4B, Supporting Information Figure 5, and Supporting Information Video 4 (si_005)). Step length analysis of CV resulted in at least two fractions with different mobilities with a significantly lower step length distribution for CV (Figure 4C) than observed for Tom20 (Figure 2F). The time course of five orthogonal CV-traces
apparent diffusion constant (Dapp) of hFis1 was higher compared to Tom20. As mentioned before, projection of a 3D movement on a tubular organelle in 2D probably underestimates the step-length for movements into the depth of the mitochondrion. Monte Carlo simulations suggested that the error in calculation of D when the curvature is not taken into account depends on the mobility of the particle, the diameter of the tubular surface, and the image acquisition rate.30 In the case of mitochondria with an average diameter of 500 nm of the OMM and a Dapp between 0.1 and 0.25 μm2/s of OMM proteins, a corrected D of 0.215 μm2/s for Tom20 and 0.410 μm2/s for hFis1, were estimated. This is still below the value, which was obtained from FRAP measurements (Tom7 D = 0.7 μm2/s; hFis1 0.6 μm2/s),20 but (i) FRAP is an ensemble method and (ii) only measures mobile molecules,6,31,32 neglecting the immobile or less mobile ones. Single molecule tracking in contrast includes all probed molecules likewise. Tracking individual proteins over extended time periods not only provided information on diffusion kinetics, but also proved to be suitable to reveal the connectivity of organelle structures (Figure 2D, blue track). Furthermore, by TALM it was possible to reveal hidden mitochondrial morphology, as shown in Figure 3B: the spatiotemporal mapping of three single hFis1 molecules exploring the shape of a circular mitochondrion (Figure 3B, green arrowhead) buried between the branches of a bifurcated mitochondrion (Figure 3B, white arrowhead). MSD analysis (short-range fit) of two different hFis1 molecules, one traveling along the tubular mitochondrion and one in the circular 614
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term tracking of few probes in order to use single molecule trajectories for exploring submicroscopic structures and their connectivities. While tracking of single molecules tagged with fluorescent proteins was previously shown in bacterial membranes 12,33 (that roughly have the size of short mitochondria), we have here succeeded to track membrane proteins inside organelles in living mammalian cells in a noninvasive manner. Localization for tracking single molecules in vitro has been achieved with localization accuracies below 10 nm.34 Inside living mammalian cells, however, localization and tracking of individual proteins are strongly limited by background fluorescence as well as the limited brightness and photostability of fluorescent proteins.26,35 Single molecule tracking and localization at the plasma membrane of live cells has been achieved by using photoactivatible fluorescent proteins (sptPALM).5,7 Within organelles, much longer trajectories were obtained by TALM based on Halo/TMR labeling compared to sptPALM (Supporting Information Figure 6). Synthetic organic fluorescence probes for transiently staining membranes6,36 or membrane proteins37 have been used for super-resolution imaging, but these techniques are so far not applicable for probing organellar and suborganellar protein complexes. Thus, TALM complements and extends sptPALM to intracellular applications. Strikingly, rhodamines have been used for super-resolution imaging based on reversible photoswitching,38 too, and were reported to be efficiently photoswitched in the cytoplasm of living cells.39 However, under the conditions used in this study, TMR-labeled proteins were highly photostable and yielded long trajectories. Also, rhodamines have been used for tracking in artificial lipid bilayers before.40 Obviously, different photoswitching properties of TMR exist depending on the local environment given by the tagging systems or the subcellular localization (own observations and personal communication M. Sauer). By TALM, we for the first time identified characteristic differences in the diffusion and the localization of OMM proteins and respiratory chain complexes within cristae of mitochondria in live cells. hFis1 showed the highest mobility, while CV/FOF1 ATP synthase obviously was most restricted in diffusion. We further predict that use of real 3D tracking techniques based on astigmatic distortions, use of polarizers, or biplane detection6,31,32 will pronounce this difference between OMM and IMM protein diffusion even more. Tracking and localization of rapidly diffusing proteins in an organelle was possible with ultrahigh spatial resolution (12 nm). Since a small population of labeled proteins can be used for successively probing microcompartments by diffusion, overexpression artifacts can be minimized. This is particularly critical in the case of multisubunit membrane protein complexes, which require correct assembly of the fluorescence-tagged subunit. By combining HaloTag-specific labeling with other orthogonal posttranslational labeling techniques, multiplexed TALM detection inside organelles of live cells can be envisaged.
depicted in detail revealed dimensions that clearly fit the shape of individual cristae (Figure 3D), supporting the assumption that orthogonal trajectories reflect a movement within individual cristae. A similar observation was made when respiratory complex II was tracked; numerous trajectories displayed a course orthogonal to the longitudinal axis of a mitochondrion. However, movement alongside the longitudinal axis of the mitochondrion was also recorded (Figure 4E) (Supporting Information Video 5 (si_006)). We assigned this different type of motion to CII diffusion in the IBM microcompartment of the IMM, which is essentially located alongside the OMM. The calculation of diffusion constants from mean square displacements from single trajectories assigned to one of these compartments yielded a significantly faster diffusion for CII in the IBM (0.161 μm2/s) than for CII in the CM (0.003 μm2/s, Figure 4E) in typical cases. Diffusion within cristae, dependent on their real shape (discs or tubules),18 might not be fully comprehended by MSD analysis, though. Rendering a super-resolution image of CV distribution from the total ensemble of localized molecules could not properly reveal individual cristae (Figure 4A). However, this was possible by generating images taking into account that individual cristae were sequentially probed by single protein complexes. To this end, multiple images were rendered from consecutive localizations within a time window of several seconds, which clearly revealed mitochondrial cristae orthogonal to the longitudinal axis of the mitochondria (Figure 5A). In addition, by superimposition of subsequent time windows of localized CV-Halo/TMR, information about cristae stability was obtained; the overlay of consecutive time windows (Figure 3A) reproduced a rather stable arrangement of cristae over at least 24 s (Figure 5B arrowheads). However, with increasing length of recorded time series, the motility of mitochondria as entire organelles progressively played a role (Supporting Information Video 6b (si_007)). To underline the power of the method presented here, we compared ultrastructural images of mitochondria obtained by TALM with the typical morphology of mitochondria observed by transmission electron microscopy. Mitochondria in HeLa cells show, over large areas, a regular pattern of cristae as illustrated by a typical TEM micrograph shown in Figure 5C. The localization and trajectory map of CV obtained by TALM reflects this regular arrangement well (Figures 4B,D and 5A,B, Supporting Information Figure 5, and Supporting Information Video 3 (si_010). In addition, the overlay of the plotted distances between adjacent orthogonal signals from our TALM images in comparison to the values derived from the TEM micrographs showed an almost identical distribution with two peaks at 53 and 71 nm, respectively (Figure 5D). These correspond to the distances between two adjacent cristae: from the outward membranes to each other (71 nm) and from the inward membranes (53 nm) (Figure 5C, inset). Even with a localization precision of 12 nm it is not possible to distinguish the two membrane sites of a single crista, which are 17 ± 2 nm apart. The third peak at 100 nm in Figure 5D probably reflects the distance between second order neighborhood cristae. Taken together, we have shown that the combined evaluation of tracking and localization data derived from TALM with Halo/TMR-labeled proteins gives versatile information about the morphology and transient dynamics of cellular microcompartments as well as the spatiotemporal dynamics of proteins within those. This method requires long-
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(26) Thompson, R. E.; Larson, D. R.; Webb, W. W. Biophys. J. 2002, 82, 2775−83. (27) Schutz, G. J.; Schindler, H.; Schmidt, T. Biophys. J. 1997, 73, 1073−80. (28) You, C.; Wilmes, S.; Beutel, O.; Lochte, S.; Podoplelowa, Y.; Roder, F.; Richter, C.; Seine, T.; Schaible, D.; Uze, G.; Clarke, S.; Pinaud, F.; Dahan, M.; Piehler, J. Angew. Chem., Int. Ed. 2010, 49, 4108−12. (29) Qian, H.; Sheetz, M. P.; Elson, E. L. Biophys. J. 1991, 60, 910− 21. (30) Renner, M.; Domanov, Y.; Sandrin, F.; Izeddin, I.; Bassereau, P.; Triller, A. PLoS One 2011, 6, e25731. (31) Holtzer, L.; Meckel, T.; Schmidt, T. Appl. Phys. Lett. 2007, 90. (32) Mlodzianoski, M. J.; Juette, M. F.; Beane, G. L.; Bewersdorf, J. Opt. Express 2009, 17, 8264−77. (33) Deich, J.; Judd, E. M.; McAdams, H. H.; Moerner, W. E. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 15921−6. (34) Yildiz, A.; Selvin, P. R. Acc. Chem. Res. 2005, 38, 574−82. (35) Lord, S. J.; Lee, H. L.; Moerner, W. E. Anal. Chem. 2010, 82, 2192−203. (36) Sharonov, A.; Hochstrasser, R. M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 18911−6. (37) Giannone, G.; Hosy, E.; Levet, F.; Constals, A.; Schulze, K.; Sobolevsky, A. I.; Rosconi, M. P.; Gouaux, E.; Tampe, R.; Choquet, D.; Cognet, L. Biophys. J. 2010, 99, 1303−10. (38) Heilemann, M.; van de Linde, S.; Schuttpelz, M.; Kasper, R.; Seefeldt, B.; Mukherjee, A.; Tinnefeld, P.; Sauer, M. Angew. Chem., Int. Ed. 2008, 47, 6172−6. (39) Klein, T.; Loschberger, A.; Proppert, S.; Wolter, S.; van de Linde, S.; Sauer, M. Nat. Methods 2011, 8, 7−9. (40) Schmidt, T.; Schutz, G. J.; Baumgartner, W.; Gruber, H. J.; Schindler, H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 2926−9.
ACKNOWLEDGMENTS We thank Wladislaw Kohl and Markus Staufenbiel for excellent technical assistance. It is thankfully acknowledged that complex I subunit 30kD template was obtained from Ulrich Brandt, paGFP from Jennifer Lippincott-Schwarz, and hFis1 from Black Hill. This project was supported by the DFG (BU2288/1-1 and SFB 944).
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dx.doi.org/10.1021/nl203343a | Nano Lett. 2012, 12, 610−616
