In JoVE (1)
Other Publications (10)
- Nano Letters
- Angewandte Chemie (International Ed. in English)
- Biological Chemistry
- Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences
- ACS Chemical Biology
- Biochimica Et Biophysica Acta
- Methods in Molecular Biology (Clifton, N.J.)
- Biophysical Reviews
- Current Biology : CB
Articles by Karin B. Busch in JoVE
Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells Timo Appelhans1, Felix R.M. Beinlich1, Christian P. Richter1, Rainer Kurre2, Karin B. Busch1,3 1School of Biology, University of Osnabrück, 2Center of Cellular Nanoanalytics, Integrated Bioimaging Facility, University of Osnabrück, 3Department of Biology, WWU Münster Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18 nm.
Other articles by Karin B. Busch on PubMed
Nanoscale Organization of Mitochondrial Microcompartments Revealed by Combining Tracking and Localization Microscopy Nano Letters. Feb, 2012 | Pubmed ID: 22201267 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 Angewandte Chemie (International Ed. in English). May, 2012 | Pubmed ID: 22488831 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.
Dynamics of Bioenergetic Microcompartments Biological Chemistry. Feb, 2013 | Pubmed ID: 23104839 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.
Quality Matters: How Does Mitochondrial Network Dynamics and Quality Control Impact on MtDNA Integrity? Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. Jul, 2014 | Pubmed ID: 24864312 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.
Clustering and Dynamics of Phototransducer Signaling Domains Revealed by Site-directed Spin Labeling Electron Paramagnetic Resonance on SRII/HtrII in Membranes and Nanodiscs Biochemistry. Jan, 2015 | Pubmed ID: 25489970 In halophilic archaea the photophobic response is mediated by the membrane-embedded 2:2 photoreceptor/-transducer complex SRII/HtrII, the latter being homologous to the bacterial chemoreceptors. Both systems bias the rotation direction of the flagellar motor via a two-component system coupled to an extended cytoplasmic signaling domain formed by a four helical antiparallel coiled-coil structure. For signal propagation by the HAMP domains connecting the transmembrane and cytoplasmic domains, it was suggested that a two-state thermodynamic equilibrium found for the first HAMP domain in NpSRII/NpHtrII is shifted upon activation, yet signal propagation along the coiled-coil transducer remains largely elusive, including the activation mechanism of the coupled kinase CheA. We investigated the dynamic and structural properties of the cytoplasmic tip domain of NpHtrII in terms of signal transduction and putative oligomerization using site-directed spin labeling electron paramagnetic resonance spectroscopy. We show that the cytoplasmic tip domain of NpHtrII is engaged in a two-state equilibrium between a dynamic and a compact conformation like what was found for the first HAMP domain, thus strengthening the assumption that dynamics are the language of signal transfer. Interspin distance measurements in membranes and on isolated 2:2 photoreceptor/transducer complexes in nanolipoprotein particles provide evidence that archaeal photoreceptor/-transducer complexes analogous to chemoreceptors form trimers-of-dimers or higher-order assemblies even in the absence of the cytoplasmic components CheA and CheW, underlining conservation of the overall mechanistic principles underlying archaeal phototaxis and bacterial chemotaxis systems. Furthermore, our results revealed a significant influence of the NpHtrII signaling domain on the NpSRII photocycle kinetics, providing evidence for a conformational coupling of SRII and HtrII in these complexes.
Shuttling of PINK1 Between Mitochondrial Microcompartments Resolved by Triple-Color Superresolution Microscopy ACS Chemical Biology. Sep, 2015 | Pubmed ID: 26046594 The cytosolic phosphatase and tensin homologue Pten-kinase PINK1 involved in mitochondrial quality control undergoes a proteolytic process inside mitochondria. It has been suggested that the protein is not fully imported into mitochondria during this maturation. Here, we have established live cell triple-color super-resolution microscopy by combining FPALM and tracking and localization microscopy (TALM) in order to unravel the spatiotemporal organization of the C-terminal kinase domain of PINK1 during this process. We find that the kinase domain is imported into active mitochondria and colocalizes with respiratory complex I at the inner mitochondrial membrane. When the processing step inside mitochondria is inhibited or mitochondria are de-energized, full length PINK1 distributes between the outer and the inner mitochondrial membranes, indicating a holdup of import. These findings give the molecular base for a dual role of PINK1-inside energized mitochondria and outside of de-energized mitochondria.
Probing of Protein Localization and Shuttling in Mitochondrial Microcompartments by FLIM with Sub-diffraction Resolution Biochimica Et Biophysica Acta. Aug, 2016 | Pubmed ID: 27016377 The cell is metabolically highly compartmentalized. Especially, mitochondria host many vital reactions in their different microcompartments. However, due to their small size, these microcompartments are not accessible by conventional microscopy. Here, we demonstrate that time-correlated single-photon counting (TCSPC) fluorescence lifetime-imaging microscopy (FLIM) classifies not only mitochondria, but different microcompartments inside mitochondria. Sensor proteins in the matrix had a different lifetime than probes at membrane proteins. Localization in the outer and inner mitochondrial membrane could be distinguished by significant differences in the lifetime. The method was sensitive enough to monitor shifts in protein location within mitochondrial microcompartments. Macromolecular crowding induced by changes in the protein content significantly affected the lifetime, while oxidizing conditions or physiological pH changes had only marginal effects. We suggest that FLIM is a versatile and completive method to monitor spatiotemporal events in mitochondria. The sensitivity in the time domain allows for gaining substantial information about sub-mitochondrial localization overcoming diffraction limitation. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
Single Molecule Tracking and Localization of Mitochondrial Protein Complexes in Live Cells Methods in Molecular Biology (Clifton, N.J.). 2017 | Pubmed ID: 28276025 Mitochondria are the power plant of most non-green eukaryotic cells. An understanding of their function and regulation is only possible with the knowledge of the spatiotemporal dynamics of their proteins. Mitochondrial membrane proteins involved in diverse functions like protein import, cell respiration, metabolite transport, and mitochondrial morphology are mobile within membranes. Here, we provide a protocol for a superresolution fluorescence microscopy technique named tracking and localization microscopy (TALM) that allows for localization and diffusion analysis of single mitochondrial membrane proteins in situ in cell cultures. This noninvasive imaging technique is a useful tool to reveal the spatiotemporal organization of proteins in diverse mitochondrial membrane compartments in living cells. Proteins of interest are tagged with the HaloTag and specifically labeled with functionalized rhodamine dyes. The method profits from low abundance of proteins and therefore works better with substoichiometric labeling of HaloTag®-tagged proteins. In particular, the use of photostable bright rhodamine dyes enables the specific tagging and localization of single molecules with a calculated precision below 20 nm and the recording of single trajectories.
Dynamic Imaging of Mitochondrial Membrane Proteins in Specific Sub-organelle Membrane Locations Biophysical Reviews. Aug, 2017 | Pubmed ID: 28819924 Mitochondria are cellular organelles with multifaceted tasks and thus composed of different sub-compartments. The inner mitochondrial membrane especially has a complex nano-architecture with cristae protruding into the matrix. Related to their function, the localization of mitochondrial membrane proteins is more or less restricted to specific sub-compartments. In contrast, it can be assumed that membrane proteins per se diffuse unimpeded through continuous membranes. Fluorescence recovery after photobleaching is a versatile technology used in mobility analyses to determine the mobile fraction of proteins, but it cannot provide data on subpopulations or on confined diffusion behavior. Fluorescence correlation spectroscopy is used to analyze single molecule diffusion, but no trajectory maps are obtained. Single particle tracking (SPT) technologies in live cells, such as tracking and localization microscopy (TALM), do provide nanotopic localization and mobility maps of mitochondrial proteins in situ. Molecules can be localized with a precision of between 10 and 20 nm, and single trajectories can be recorded and analyzed; this is sufficient to reveal significant differences in the spatio-temporal behavior of diverse mitochondrial proteins. Here, we compare diffusion coefficients obtained by these different technologies and discuss trajectory maps of diverse mitochondrial membrane proteins obtained by SPT/TALM. We show that membrane proteins in the outer membrane generally display unhindered diffusion, while the mobility of inner membrane proteins is restricted by the inner membrane architecture, resulting in significantly lower diffusion coefficients. Moreover, tracking analysis could discern proteins in the inner boundary membrane from proteins preferentially diffusing in cristae membranes, two sub-compartments of the inner mitochondrial membrane. Thus, by evaluating trajectory maps it is possible to assign proteins to different sub-compartments of the same membrane.
Respiration: Life Without Complex I Current Biology : CB. May, 2018 | Pubmed ID: 29787729 Eukaryotic life has developed a fascinating and highly optimized system for energy transduction: the mitochondrial respiratory chain. Typically composed of five core protein complexes, we now learn from two studies that plant hemi-parasites of the type Viscum cope without Complex I, the entry point of the classical respiratory system.