STED microscopy of cardiac membrane nanodomains

Heart muscle cells (cardiomyocytes) have to fulfill a demanding task. They have to ensure continuous and regular heartbeat and maintain their integrity despite undergoing constant mechanical stress during muscle contraction. To accomplish this, they feature a characteristic membrane architecture: the Transverse-Axial Tubular System (TATS), a network of membrane invaginations – the Transverse Tubules (TT) – which allows the fast translation of an electrical stimulus into a mechanical response. Further, specialized membrane protein complexes ensure membrane flexibility and stability during cycles of contraction and relaxation. Structural alterations of the TATS and the membrane protein complexes are linked to cardiac pathologies. This thesis presents the subdiffraction image based investigations of the cardiac membrane architecture and membrane nanodomains using STimulated Emission Depletion (STED) microscopy of mouse ventricular cardiomyocytes (VM). Experimental and analytical methods comprising single- and multicolor, one- and two-photon-excitation STED microscopy are developed and applied. Special focus is laid on the three-dimensional (3D) TATS topology, on the membrane lipids Cholesterol (Chol) and Ganglioside GM1 (GM1), and on the membrane associated proteins Caveolin-3 (Cav-3) and Dystrophin (Dyst). Novel fluorescent Chol analogs are characterized and established as a class of membrane labels with superior properties for STED microscopy of living VM. These dye compounds allow the visualization of the TATS with an unprecedented lateral resolution of below 35 nm and can be used for both membrane bulk staining and labeling of nanoscopic membrane compartments. Using a custom-built two-photon-excitation-STED (2P-Exc-STED) microscope, the new Chol dyes enable the acquisition of 3D subdiffraction images of the TATS of living VM. These 3D images reveal that TT bud from Chol rich membrane domains and that these Chol rich domains can also form shallow membrane invaginations which are hypothesized to be caveolae. The signal patterns of the caveolae-associated protein Cav-3 and of Chol are comparatively investigated and their similarities quantitatively evaluated. The dramatic effect of membrane Chol depletion on the nanoscopic Cav-3 signal distribution is assessed. The correlation between the Cav-3 and Chol membrane patterns is further supported by two-color STED microscopy of VM labeled for Cav-3 and GM1, and Chol and GM1. Finally, the spatial association between Cav-3 and the cytoskeletal protein Dyst is studied in detail. For this, two- and three-color STED imaging protocols and image analysis procedures are developed. To determine the molecular orientation of the Dyst protein with respect to Cav-3 and with respect to the cardiac membrane, a multicolor “intra-protein” labeling protocol is developed that is based on immunofluorescence staining using different primary antibodies that target specific epitopes along the Dyst protein. A cardiac membrane nanodomain model summarizing the presented observations and findings is derived, validated, and discussed in detail

Axial tubule junctions control rapid calcium signaling in atria.

The canonical atrial myocyte (AM) is characterized by sparse transverse tubule (TT) invaginations and slow intracellular Ca2+ propagation but exhibits rapid contractile activation that is susceptible to loss of function during hypertrophic remodeling. Here, we have identified a membrane structure and Ca2+-signaling complex that may enhance the speed of atrial contraction independently of phospholamban regulation. This axial couplon was observed in human and mouse atria and is composed of voluminous axial tubules (ATs) with extensive junctions to the sarcoplasmic reticulum (SR) that include ryanodine receptor 2 (RyR2) clusters. In mouse AM, AT structures triggered Ca2+ release from the SR approximately 2 times faster at the AM center than at the surface. Rapid Ca2+ release correlated with colocalization of highly phosphorylated RyR2 clusters at AT-SR junctions and earlier, more rapid shortening of central sarcomeres. In contrast, mice expressing phosphorylation-incompetent RyR2 displayed depressed AM sarcomere shortening and reduced in vivo atrial contractile function. Moreover, left atrial hypertrophy led to AT proliferation, with a marked increase in the highly phosphorylated RyR2-pS2808 cluster fraction, thereby maintaining cytosolic Ca2+ signaling despite decreases in RyR2 cluster density and RyR2 protein expression. AT couplon "super-hubs" thus underlie faster excitation-contraction coupling in health as well as hypertrophic compensatory adaptation and represent a structural and metabolic mechanism that may contribute to contractile dysfunction and arrhythmias

STED nanoscopy of Interfaces and Interactions between Nanostructure Arrays and Living Cells

STED Nanoscopy of Interfaces and Interactions between Nanostructure Arrays and Living Cells The specific arrangement of membrane lipids and proteins in a living cell at the interface to high-aspect ratio nanostructures (nanowires and nanostraws) is still unknown – as are the dynamic structural adaptations and molecular rearrangements of living cells in the vicinity of such nanostructures. Whether the nanostructures actually pierce through the cell membrane or how introduced changes in membrane curvature change the biophysical properties of the cell membrane is of particular interest for investigations of the efficacy and safety of nano-sized tissue implants and for studying the delivery of substances into living cells via hollow nanostraws. To elucidate these questions, STimulated Emission Depletion (STED) nanoscopy is the ideal technique because it is live-cell compatible, target-specific, and offers a lateral resolution on the protein level (<30 nm). Here we present STED based investigations of the live-cell membrane and the cytoskeletal Actin signal in the presence of hollow Alumina nanostraws with diameter of 100 nm. As cellular model system we chose the lung-cancer derived A549 culture cell line. The cells were incubated on the nanostraws and subsequently fluorescence-tagged with live-cell compatible labels targeting the cell membrane and filamentous Actin, respectively. We find that the cellular membrane forms ring structures of about 100 nm in diameter, wrapping tightly around the nanostraws. On the other hand, the Actin cytoskeleton forms intricate, coil-like nanometric structures around the nanostraws; these structures strongly vary in diameters between 250-600 nm and appear to widen with increasing distance from the nanostraw substrate. In addition, STED images of living cells stained for both membrane and Actin signal reveal a significant degree of co-localization at the apical cell membrane, i.e. further away from the nanostraws. This co-localization is almost entirely lost at the basal membrane close to the nanostraws which is due to a strongly reduced Actin signal on that side of the cell. In conclusion, our sub-diffraction STED imaging based investigations of the behavior of single living cells cultured on nanostraws reveals a strong response of the cellular membrane and the Actin cytoskeleton – two of the main structure-giving features of the cell. In a next step, we will extend our studies to additional scaffolding proteins to arrive at a more detailed map of the topology of living cells at the interface to nanostructures of different geometries

STED Nanoscopy of Interfaces and Interactions between Nanostructure Arrays and Living Cells

STED Nanoscopy of Interfaces and Interactions between Nanostructure Arrays and Living Cells The specific arrangement of membrane lipids and proteins in a living cell at the interface to high-aspect ratio nanostructures (nanowires and nanostraws) is still unknown – as are the dynamic structural adaptations and molecular rearrangements of living cells in the vicinity of such nanostructures. Whether the nanostructures actually pierce through the cell membrane or how introduced changes in membrane curvature change the biophysical properties of the cell membrane is of particular interest for investigations of the efficacy and safety of nano-sized tissue implants and for studying the delivery of substances into living cells via hollow nanostraws. To elucidate these questions, STimulated Emission Depletion (STED) nanoscopy is the ideal technique because it is live-cell compatible, target-specific, and offers a lateral resolution on the protein level (<30 nm). Here we present STED based investigations of the live-cell membrane and the cytoskeletal Actin signal in the presence of hollow Alumina nanostraws with diameter of 100 nm. As cellular model system we chose the lung-cancer derived A549 culture cell line. The cells were incubated on the nanostraws and subsequently fluorescence-tagged with live-cell compatible labels targeting the cell membrane and filamentous Actin, respectively. We find that the cellular membrane forms ring structures of about 100 nm in diameter, wrapping tightly around the nanostraws. On the other hand, the Actin cytoskeleton forms intricate, coil-like nanometric structures around the nanostraws; these structures strongly vary in diameters between 250-600 nm and appear to widen with increasing distance from the nanostraw substrate. In addition, STED images of living cells stained for both membrane and Actin signal reveal a significant degree of co-localization at the apical cell membrane, i.e. further away from the nanostraws. This co-localization is almost entirely lost at the basal membrane close to the nanostraws which is due to a strongly reduced Actin signal on that side of the cell. In conclusion, our sub-diffraction STED imaging based investigations of the behavior of single living cells cultured on nanostraws reveals a strong response of the cellular membrane and the Actin cytoskeleton – two of the main structure-giving features of the cell. In a next step, we will extend our studies to additional scaffolding proteins to arrive at a more detailed map of the topology of living cells at the interface to nanostructures of different geometries

Nanostraw-Assisted Cellular Injection of Fluorescent Nanodiamonds via Direct Membrane Opening

Due to their stable fluorescence, biocompatibility, and amenability to functionalization, fluorescent nanodiamonds (FND) are promising materials for long term cell labeling and tracking. However, transporting them to the cytosol remains a major challenge, due to low internalization efficiencies and endosomal entrapment. Here, nanostraws in combination with low voltage electroporation pulses are used to achieve direct delivery of FND to the cytosol. The nanostraw delivery leads to efficient and rapid FND transport into cells compared to when incubating cells in a FND-containing medium. Moreover, whereas all internalized FND delivered by incubation end up in lysosomes, a significantly larger proportion of nanostraw-injected FND are in the cytosol, which opens up for using FND as cellular probes. Furthermore, in order to answer the long-standing question in the field of nano-biology regarding the state of the cell membrane on hollow nanostructures, live cell stimulated emission depletion (STED) microscopy is performed to image directly the state of the membrane on nanostraws. The time-lapse STED images reveal that the cell membrane opens entirely on top of nanostraws upon application of gentle electrical pulses, which supports the hypothesis that many FND are delivered directly to the cytosol, avoiding endocytosis and lysosomal entrapment

Uptake of nanowires by human lung adenocarcinoma cells

Semiconductor nanowires are increasingly used in optoelectronic devices. However, their effects on human health have not been assessed fully. Here, we investigate the effects of gallium phosphide nanowires on human lung adenocarcinoma cells. Four different geometries of nanowires were suspended in the cell culture for 48 hours. We show that cells internalize the nanowires and that the nanowires have no effect on cell proliferation rate, motility, viability and intracellular ROS levels. By blocking specific internalization pathways, we demonstrate that the nanowire uptake is the result of a combination of processes, requiring dynamin and actin polymerization, which suggests an internalization through macropinocytosis and phagocytosis

Self-healing dyes for super-resolution fluorescence microscopy

In recent years, optical microscopy techniques have emerged that allow optical imaging at unprecedented resolution beyond the diffraction limit. These techniques exploit photostabilizing buffers to enable photoswitching and/or the enhancement of fluorophore brightness and stability. A major drawback with the use of photostabilizing buffers, however, is that they cannot be used in live cell imaging. In this paper, we tested the performance of self-healing organic fluorophores, which undergo intramolecular photostabilization, in super-resolution microscopy examining both targeted (stimulated emission depletion (STED) microscopy) and stochastic readout (stochastic optical reconstruction microscopy (STORM)). The overall goal of the study was to identify dyes and conditions that lead to improved spatial and temporal resolution of both techniques without the need for mixtures of photostabilizing agents in the imaging buffer. As a result of previously shown superior performance, we identified an ATTO647N-photostabilizer conjugate as a potential candidate for STED microscopy. We have here characterized the photostability and resulting performance of this nitrophenylalanine (NPA) conjugate of ATTO647N on oligonucleotides in STED microscopy. We found that the superior photophysical performance resulted in optimal STED imaging and demonstrated that single-molecule fluorescent transients of individual fluorophores can be obtained with both the excitation- and STED-laser. In similar experiments, we also tested a nitrophenylacetic acid conjugate of STAR635P, another frequently used dye in STED microscopy, and present a characterization of its photophysical properties. Finally, we performed an analysis of the photoswitching kinetics of self-healing Cy5 dyes (containing trolox, cyclooctatetraene and NPA-based stabilizers) in the presence of Tris(2-carboxyethyl)phosphine and cysteamine, which are typically used in STORM microscopy. In line with previous work, we found that intramolecular photostabilization strongly influences photoswitching kinetics and requires careful attention when designing STORM-experiments. In summary, this contribution explores the possibilities and limitations of self-healing dyes in super-resolution microscopy of differing modalities