Karin B. Busch

Nanoscale Organization of Mitochondrial Microcompartments Revealed by Combining Tracking and Localization Microscopy

While detailed information on the nanoscale-organization 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.

Triple‐Color Super‐Resolution Imaging of Live Cells: Resolving Submicroscopic Receptor Organization in the Plasma Membrane

In living color: efficient intracellular covalent labeling of proteins with a photoswitchable dye using the HaloTag for dSTORM super-resolution imaging in live cells is described. The dynamics of cellular nanostructures at the plasma membrane were monitored with a time resolution of a few seconds. In combination with dual-color FPALM imaging, submicroscopic receptor organization within the context of the membrane skeleton was resolved.

Respiratory chain complexes in dynamic mitochondria display a patchy distribution in life cells.

Background Mitochondria, the main suppliers of cellular energy, are dynamic organelles that fuse and divide frequently. Constraining these processes impairs mitochondrial is closely linked to certain neurodegenerative diseases. It is proposed that functional mitochondrial dynamics allows the exchange of compounds thereby providing a rescue mechanism. Methodology/Principal Findings The question discussed in this paper is whether fusion and fission of mitochondria in different cell lines result in re-localization of respiratory chain (RC) complexes and of the ATP synthase. This was addressed by fusing cells containing mitochondria with respiratory complexes labelled with different fluorescent proteins and resolving their time dependent re-localization in living cells. We found a complete reshuffling of RC complexes throughout the entire chondriome in single HeLa cells within 2–3 h by organelle fusion and fission. Polykaryons of fused cells completely re-mixed their RC complexes in 10–24 h in a progressive way. In contrast to the recently described homogeneous mixing of matrix-targeted proteins or outer membrane proteins, the distribution of RC complexes and ATP synthase in fused hybrid mitochondria, however, was not homogeneous but patterned. Thus, complete equilibration of respiratory chain complexes as integral inner mitochondrial membrane complexes is a slow process compared with matrix proteins probably limited by complete fusion. In co-expressing cells, complex II is more homogenously distributed than complex I and V, resp. Indeed, this result argues for higher mobility and less integration in supercomplexes. Conclusion/Significance Our results clearly demonstrate that mitochondrial fusion and fission dynamics favours the re-mixing of all RC complexes within the chondriome. This permanent mixing avoids a static situation with a fixed composition of RC complexes per mitochondrion.

Restricted diffusion of OXPHOS complexes in dynamic mitochondria delays their exchange between cristae and engenders a transitory mosaic distribution

Summary Mitochondria are involved in cellular energy supply, signaling and apoptosis. Their ability to fuse and divide provides functional and morphological flexibility and is a key feature in mitochondrial quality maintenance. To study the impact of mitochondrial fusion/fission on the reorganization of inner membrane proteins, oxidative phosphorylation (OXPHOS) complexes in mitochondria of different HeLa cells were tagged with fluorescent proteins (GFP and DsRed-HA), and cells were fused by polyethylene glycol treatment. Redistribution of the tagged OXPHOS complexes was then followed by means of immunoelectron microscopy, two color super-resolution fluorescence microscopy and single molecule tracking. In contrast to outer membrane and matrix proteins, which mix quickly and homogeneously upon mitochondrial fusion, the mixing of inner membrane proteins was decelerated. Our data suggest that the composition of cristae is preserved during fusion of mitochondria and that cristae with mixed OXPHOS complexes are only slowly and successively formed by restricted diffusion of inner membrane proteins into existing cristae. The resulting transitory mosaic composition of the inner mitochondrial membrane illuminates mitochondrial heterogeneity and potentially is linked to local differences in function and membrane potential.

Lateral pH gradient between OXPHOS complex IV and F 0 F 1 ATP-synthase in folded mitochondrial membranes

Ion-driven ATP synthesis by rotary F0F1 ATP-synthase powers aerobic life. Since Mitchells seminal hypothesis, this synthesis has been discussed in terms of the proton-motive force between two bulk phases, each in equilibrium. In active mitochondria, a steady proton flow cycles between pumps and the distant ATP synthase. Here we determine the lateral pH profile along the p-side of cristae in situ by attaching a ratiometric fluorescent pH-sensitive GFP variant to OXPHOS complex IV, a proton pump, and the dimeric F0F1 ATP-synthase, a proton consumer. In respiring HeLa cells, we observe that the local pH at F0F1 dimers is 0.3 units less acidic than that at complex IV. This finding is consistent with the calculated pH profile for steady proton diffusion from CIV to F0F1. The observed lateral variation in the proton-motive force necessitates a modification to Peter Mitchells chemiosmotic proposal. The experimental technique can be extended to other pH-dependent reactions in membrane microcompartments.

Engineered Upconversion Nanoparticles for Resolving Protein Interactions inside Living Cells

Upconversion nanoparticles (UCNPs) convert near-infrared into visible light at much lower excitation densities than those used in classic two-photon absorption microscopy. Here, we engineered <50 nm UCNPs for application as efficient lanthanide resonance energy transfer (LRET) donors inside living cells. By optimizing the dopant concentrations and the core-shell structure for higher excitation densities, we observed enhanced UCNP emission as well as strongly increased sensitized acceptor fluorescence. For the application of these UCNPs in complex biological environments, we developed a biocompatible surface coating functionalized with a nanobody recognizing green fluorescent protein (GFP). Thus, rapid and specific targeting to GFP-tagged fusion proteins in the mitochondrial outer membrane and detection of protein interactions by LRET in living cells was achieved.

Selective Targeting of Fluorescent Nanoparticles to Proteins Inside Live Cells

Single-molecule localization and tracking techniques have contributed towards observing the spatiotemporal organization of proteins in the plasma membrane. Individual proteins labeled with fluorescent nanoparticles (FNPs) can be imaged over long time with ultrahigh spatial and temporal resolution. A key challenge for the biophysical application of FNPs, however, is to site-specifically target the nanoparticles to proteins in living cells. For selective labeling of cell surface proteins with FNPs, biomolecules such as antibodies and streptavidin have been employed as well as chemical recognition based on immobilized transition-metal ions. These recognition units, however, are not compatible with FNP targeting to proteins inside living cells, because the structural integrity of antibodies is often affected by the reducing conditions in the cytoplasm, streptavidin is blocked with endogenous biotin, and transition-metal ions are coordinated by cysteine-rich proteins. Intracellular FNP targeting is furthermore challenging, because blocking of nonspecific binding and washing out of nonbound FNPs is not possible in intact cells. Here, we aimed to establish a highly specific and efficient approach for FNP targeting inside live cells, which overcomes these particular challenges. As a biochemical recognition system compatible with the cytoplasm, we employed an enzymatic covalent labeling approach based on the HaloTag. This engineered dehalogenase irreversibly reacts with a chlorohexane moiety (HaloTag ligand, HTL) attached to fluorescent dyes and other probes. This highly specific reaction has been exploited for protein labeling in live cells. We attempted functionalization of FNPs with HTL through maleimide/thiol-chemistry as well as by using different biotin derivatives. However, neither significant specific binding to the immobilized HaloTag was observed in vitro, nor efficient targeting upon microinjection into live cells expressing HaloTag fusion proteins using a microcapillary. For this reason, we characterized the association kinetics of different derivatives of the HTL in more detail using realtime surface-sensitive detection by simultaneous reflectance interference (RIF) and total internal reflection fluorescence spectroscopy (TIRFS) detection. For this purpose, purified HaloTag with a His-tag (His= histidine, HaloTag-H12) was site-specifically immobilized on a polyethylene glycol (PEG) polymer brush functionalized with tris(nitrilotriacetic acid), tris-NTA, and binding of fluorescent substrates was monitored in real time (see Figure S1 in the Supporting Information). Rapid binding of HTL conjugated with the fluorescent dye AlexaFluor 488 (HTL) was detected by TIRFS (see Figure S1 in the Supporting Information), yielding a reaction rate constant of 1 10m 1 s . To directly compare the reaction rate constants of fluorescent and nonfluorescent HTL conjugates (see Scheme S1 in the Supporting Information), a competition assay was established with HTL as a fluorescent tracer (see Figure S2 in the Supporting Information). These rate constants are summarized in Table S1 in the Supporting Information. Strikingly, a substantially faster reaction rate constant of 1 10m 1 s 1 was observed for HTL conjugated with tetramethylrhodamine (HTL), which is similar to the published rate constant of this reaction measured in solution (2.7 10m 1 s ). Surprisingly, an elongated ethylene glycol linker substantially reduced the reaction rate constant. Even slower rate constants were obtained for biotinylated and for unmodified HTL (around 10m 1 s ). These results suggested that the conjugated fluorescence dyes play a critical role for the association kinetics, probably by stabilizing the noncovalent enzyme–substrate complex, by hydrophobic interactions, prior to the ester formation by reaction with D106 in the binding pocket of the HaloTag. Strikingly, engineering of the HaloTag from the original dehalogenase involved incorporation of hydrophobic residues in the proximity of the reactive site. Based on the observation that hydrophobic as well as positively charged residues increase the reaction rate constant of HTL derivatives, we implemented a novel approach for surface functionalization with the HTL based on click chemistry using commercially available dibenzocyclooctynelike (DBCO) derivatives and azide-functionalized HTL (Figure 1a and Scheme S2 in the Supporting Information). Thus a hydrophobic moiety was integrated to HTL similar to the HTL-dye conjugates. The reaction kinetics of the HTL derivative obtained by this reaction (clickHTL) was compared with other HTL-derivatives of the competitive binding [*] D. Lise, Dr. C. You, Prof. Dr. J. Piehler Division of Biophysics, Department of Biology Universit t Osnabr ck, Barbarastrasse 11 49076 Osnabr ck (Germany) E-mail: piehler@uos.de Homepage: http://www.biologie.uni-osnabrueck.de/Biophysik/ Piehler/

Determination of protein mobility in mitochondrial membranes of living cells.

Molecular mobility in membranes of intracellular organelles is poorly understood, due to the lack of experimental tools applicable for a great diversity of shapes and sizes such organelles can acquire. Determinations of diffusion within the plasma membrane or cytosol are based mostly on the assumption of an infinite flat space, not valid for curved membranes of smaller organelles. Here we extend the application of FRAP to mitochondria of living cells by application of numerical analysis to data collected from a small region inside a single organelle. The spatiotemporal pattern of light pulses generated by the laser scanning microscope during the measurement is reconstructed in silico and consequently the values of diffusion parameters best suited to the particular organelle are found. The mobility of the outer membrane proteins hFis and Tom7, as well as oxidative phosphorylation complexes COX and F(1)F(0) ATPase located in the inner membrane is analyzed in detail. Several alternative models of diffusivity applied to these proteins provide insight into the mechanisms determining the rate of motion in each of the membranes. Tom7 and hFis move along the mitochondrial axis in the outer membrane with similar diffusion coefficients (D=0.7μm(2)/s and 0.6μm(2)/s respectively) and equal immobile fraction (7%). The notably slower motion of the inner membrane proteins is best represented by a dual-component model with approximately equal partitioning of the fractions (F(1)F(0) ATPase: 0.4μm(2)/s and 0.0005μm(2)/s; COX: 0.3μm(2)/s and 0.007μm(2)/s). The mobility patterns specific for the membranes of this organelle are unambiguously distinguishable from those of the plasma membrane or artificial lipid environments: The parameters of mitochondrial proteins indicate a distinct set of factors responsible for their diffusion characteristics.

Quality matters: how does mitochondrial network dynamics and quality control impact on mtDNA integrity?

Mammalian mtDNA encodes for 13 core proteins of oxidative phosphorylation. Mitochondrial DNA mutations and deletions cause severe myopathies and neuromuscular diseases. Thus, the integrity of mtDNA is pivotal for cell survival and health of the organism. We here discuss the possible impact of mitochondrial fusion and fission on mtDNA maintenance as well as positive and negative selection processes. Our focus is centred on the important question of how the quality of mtDNA nucleoids can be assured when selection and mitochondrial quality control works on functional and physiological phenotypes constituted by oxidative phosphorylation proteins. The organelle control theory suggests a link between phenotype and nucleoid genotype. This is discussed in the light of new results presented here showing that mitochondrial transcription factor A/nucleoids are restricted in their intramitochondrial mobility and probably have a limited sphere of influence. Together with recent published work on mitochondrial and mtDNA heteroplasmy dynamics, these data suggest first, that single mitochondria might well be internally heterogeneous and second, that nucleoid genotypes might be linked to local phenotypes (although the link might often be leaky). We discuss how random or site-specific mitochondrial fission can isolate dysfunctional parts and enable their elimination by mitophagy, stressing the importance of fission in the process of mtDNA quality control. The role of fusion is more multifaceted and less understood in this context, but the mixing and equilibration of matrix content might be one of its important functions.

Dynamics of bioenergetic microcompartments

Abstract The vast majority of life on earth is dependent on harvesting electrochemical potentials over membranes for the synthesis of ATP. Generation of membrane potential often relies on electron transport through membrane protein complexes, which vary among the bioenergetic membranes found in living organisms. In order to maximize the efficient harvesting of the electrochemical potential, energy loss must be minimized, and this is achieved partly by restricting certain events to specific microcompartments, on bioenergetic membranes. In this review we will describe the characteristics of the energy-converting supramolecular structures involved in oxidative phosphorylation in mitochondria and bacteria, and photophosphorylation. Efficient function of electron transfer pathways requires regulation of electron flow, and we will also discuss how this is partly achieved through dynamic re-compartmentation of the membrane complexes into different supercomplexes. In addition to supercomplexes, the supramolecular structure of the membrane, and in particular the role of water layers on the surface of the membrane in the prevention of wasteful proton escape (and therefore energy loss), is discussed in detail. In summary, the restriction of energetic processes to specific microcompartments on bioenergetic membranes minimizes energy loss, and dynamic rearrangement of these structures allows for regulation.