Refine
Has Fulltext
- yes (55)
Is part of the Bibliography
- yes (55)
Year of publication
Document Type
- Journal article (55)
Language
- English (55) (remove)
Keywords
- super-resolution microscopy (8)
- active zone (5)
- dSTORM (4)
- ceramides (3)
- imaging (3)
- immunotherapy (3)
- superresolution (3)
- BDNF (2)
- Bruchpilot (2)
- Expansion microscopy (2)
- SARS-CoV-2 (2)
- T cells (2)
- biology (2)
- fluorescence imaging (2)
- fluorescence microscopy (2)
- fluorescent probes (2)
- imaging the immune system (2)
- induced pluripotent stem cells (2)
- infectious diseases (2)
- localization microscopy (2)
- microscopy (2)
- neurotransmitter release (2)
- sphingolipids (2)
- tumour immunology (2)
- 3D tissue model (1)
- AI (1)
- Alzheimers disease (1)
- Amyotrophic-lateral-sclerosis (1)
- Aspergillus fumigatus (1)
- Automated analysis (1)
- Axon degeneration (1)
- Axonal transport (1)
- C.376A>G (p.S126G) (1)
- CA2+ channels (1)
- CA3 pyrimidal cells (1)
- CCL3 (1)
- CCL4 (1)
- CCL5 (1)
- CD28 (1)
- CENP-A (1)
- CSE4, CENP-A (1)
- Ca\(^{2+}\) channels (1)
- Cadherin-13 (CDH13) (1)
- DNA (1)
- DNA double-strand breaks (1)
- DNA nanotechnology (1)
- DNA-binding (1)
- DRD1 (1)
- FLIMbee (1)
- Fabry disease (1)
- G-protein-coupled receptors (1)
- GABA-A receptor (1)
- GPCR (1)
- HDBSCAN (1)
- ImageJ plugin (1)
- Intermediate filaments (1)
- Ionizing radiation (1)
- Lacking neurofilaments (1)
- Latrophilin (1)
- MiMIC (1)
- Microtubules (1)
- Missense mutation (1)
- Motoneuron disease (1)
- Mouse model (1)
- NF-KAPPA-B (1)
- Neisseria (1)
- Neisseria meningitidis (1)
- Neurofilament (1)
- Open-source tool (1)
- PALM (1)
- PKCζ, (1)
- Progressive motor neuronopathy (1)
- RIM-binding protein (1)
- RNA metabolism (1)
- RNA-binding protein (1)
- Radiation biology (1)
- S-type anion channel (1)
- SLAH3 (1)
- SMLM (1)
- STORM (1)
- Si-rhodamine (1)
- Stat3 (1)
- Stathmin (1)
- Superagonistic antibody (1)
- T-cadherin (1)
- Th1 cells (1)
- Transgenic mice (1)
- Translation (1)
- TrkB (1)
- Unc-13 (1)
- acid sphingomyelinase (1)
- actin (1)
- acute myeloid leukaemia (1)
- adhesion GPCR (1)
- alpha (1)
- antimicrobials (1)
- assay systems (1)
- azido-ceramides (1)
- bacterial infection (1)
- bacterial meningitis (1)
- basal ganglia (1)
- biological fluorescence (1)
- biological techniques (1)
- brain endothelial cells (1)
- cadherin-13 (CDH13) (1)
- caenorhabditis elegans (1)
- cancer imaging (1)
- cancer immunotherapy (1)
- cancer microenvironment (1)
- cell membranes (1)
- cell migration (1)
- centomeres (1)
- centromere (1)
- ceramidase (1)
- ceramide (1)
- ceramide analogs (1)
- chemotherapy (1)
- chlamydia (1)
- chromatin (1)
- click-chemistry (1)
- compaction (1)
- complexes (1)
- compressed sensing (1)
- conformational restriction (1)
- correlative light and electron microscopy (1)
- cortico-striatal synapse (1)
- corticosteroids (1)
- cristae (1)
- cyanine dyes (1)
- cytokine release (1)
- cytosolic acidification (1)
- dCIRL (1)
- dSPN (1)
- deep learning–artificial neural network (DL-ANN) (1)
- dendritic cell (1)
- developmental forms (1)
- diffraction limit (1)
- direct pathway (1)
- direct stochasticoptical reconstruction microscopy (1)
- disease model (1)
- diseases (1)
- dorsal raphe (1)
- dynamics (1)
- electrolytes (1)
- excitation-secretion coupling (1)
- expansion microscopy (1)
- fission yeast (1)
- flooding (1)
- fluorescent dyes (1)
- fluorescent protein (1)
- fluorescent-probes (1)
- fluorophore (1)
- fluoxetine (1)
- fungal infection (1)
- fungi (1)
- gamma H2AX-foci (1)
- gangliosides and lipid rafts (1)
- gap junction (1)
- gene expression (1)
- genetic code expansion (1)
- glioblastoma multiforme (1)
- globotriaosylceramide (1)
- hippocampal (1)
- hippocampal neurons (1)
- homeostasis (1)
- homodimers (1)
- human induced pluripotent stem cell (hiPSC) (1)
- hyphae (1)
- hypotonic (1)
- hypotonic solutions (1)
- hypoxia (1)
- identification (1)
- illumination microscopy (1)
- image data (1)
- imaging and sensing (1)
- in vitro (1)
- in vivo (1)
- inflammation (1)
- ingle-molecule microscopy (1)
- innexins (1)
- interferon γ (1)
- isotonic (1)
- junction proteins (1)
- laboratory techniques and procedures (1)
- leptomeningeal cells (1)
- localization micoscopy (1)
- measles virus (1)
- mechanotransduction (1)
- median and dorsal raphe (1)
- medical and biological imaging (1)
- membrane proteins (1)
- meningeal blood-csf barrier (1)
- metabotropic signalling (1)
- microbiology (1)
- microbiology techniques (1)
- microtubule cytoskeleton (1)
- migration (1)
- mitochondria (1)
- molecular mobility (1)
- mossy fiber synapses (1)
- motor learning (1)
- nanoarchitecture (1)
- nanoscale imaging (1)
- natural killer cell (1)
- natural killer cells (1)
- neisseria meningitidis (1)
- nervous-system (1)
- neural circuits (1)
- neurodevelopment (1)
- neuromuscular junction (1)
- neuropsychiatric disorders (1)
- neutral sphingomyelinase 2 (1)
- nuclear proe (1)
- nucleosome (1)
- optical reconstruction microscopy (1)
- pH (1)
- pattern recognition receptors (1)
- permeability (1)
- phlorizin (1)
- photoactivation (1)
- photodynamic therapy (1)
- plasticity (1)
- platform (1)
- prefrontal cortex (1)
- presynapse (1)
- presynaptic calcium (1)
- presynaptic differentiation (1)
- presynaptic homeostasis (1)
- presynaptic plasticity (1)
- propagation (1)
- proteins (1)
- psychiatric disorders (1)
- radial glia (1)
- reconstruction (1)
- release (1)
- resolution limit (1)
- reveals (1)
- schizosaccaromyces-pombe (1)
- secreted effectors (1)
- sensory neurons (1)
- sensory physiology (1)
- serotonin (1)
- serotonin-specific neurons (1)
- single molecule localization microscopy (1)
- single-molecule fluorescence spectroscopy (1)
- single-molecule tracking (1)
- sphingolipid expansion microscopy (1)
- sphingosine-1-phosphate (1)
- sporidia (1)
- stem cell transplantation (1)
- stoichiometry (1)
- structure-function relationships (1)
- structured illumination (1)
- structured illumination microscope (1)
- structured illumination microscopy (1)
- super-resolution (1)
- super-resolution imaging (1)
- super-resolution microscopy (SRM) (1)
- synapse formation (1)
- synapse structure (1)
- synaptic delay (1)
- synaptic localization (1)
- synaptic plasticity (1)
- synaptic transmission (1)
- synaptotagmin (1)
- tag fusion proteins (1)
- temporal range (1)
- the microtubule-organizing center (1)
- three-dimensional microscopy (1)
- tonicity (1)
- translational research (1)
- transportome (1)
- treatment (1)
- ultrastructure (1)
- variants of unknown significance (1)
- volume regulation (1)
- α-Galactosidase A (1)
Institute
- Theodor-Boveri-Institut für Biowissenschaften (55) (remove)
Using Expansion Microscopy to Visualize and Characterize the Morphology of Mitochondrial Cristae
(2020)
Mitochondria are double membrane bound organelles indispensable for biological processes such as apoptosis, cell signaling, and the production of many important metabolites, which includes ATP that is generated during the process known as oxidative phosphorylation (OXPHOS). The inner membrane contains folds called cristae, which increase the membrane surface and thus the amount of membrane-bound proteins necessary for the OXPHOS. These folds have been of great interest not only because of their importance for energy conversion, but also because changes in morphology have been linked to a broad range of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. With a distance between opposing cristae membranes often below 100 nm, conventional fluorescence imaging cannot provide a resolution sufficient for resolving these structures. For this reason, various highly specialized super-resolution methods including dSTORM, PALM, STED, and SIM have been applied for cristae visualization. Expansion Microscopy (ExM) offers the possibility to perform super-resolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded by a factor of 4–4.5, improving the resolution to 60–70 nm on conventional confocal microscopes, which can be further increased to ∼ 30 nm laterally using SIM. Here, we demonstrate that the expression of the mitochondrial creatine kinase MtCK linked to marker protein GFP (MtCK-GFP), which localizes to the space between the outer and the inner mitochondrial membrane, can be used as a cristae marker. Applying ExM on mitochondria labeled with this construct enables visualization of morphological changes of cristae and localization studies of mitochondrial proteins relative to cristae without the need for specialized setups. For the first time we present the combination of specific mitochondrial intermembrane space labeling and ExM as a tool for studying internal structure of mitochondria.
G-protein-coupled receptors (GPCRs) are hypothesized to possess molecular mobility over a wide temporal range. Until now the temporal range has not been fully accessible due to the crucially limited temporal range of available methods. This in turn, may lead relevant dynamic constants to remain masked. Here, we expand this dynamic range by combining fluorescent techniques using a spot confocal setup. We decipher mobility constants of β\(_{2}\)-adrenergic receptor over a wide time range (nanosecond to second). Particularly, a translational mobility (10 µm\(^{2}\)/s), one order of magnitude faster than membrane associated lateral mobility that explains membrane protein turnover and suggests a wider picture of the GPCR availability on the plasma membrane. And a so far elusive rotational mobility (1-200 µs) which depicts a previously overlooked dynamic component that, despite all complexity, behaves largely as predicted by the Saffman-Delbrück model.
Immune checkpoint blockade therapy is beneficial and even curative for some cancer patients. However, the majority don’t respond to immune therapy. Across different tumor types, pre-existing T cell infiltrates predict response to checkpoint-based immunotherapy. Based on in vitro pharmacological studies, mouse models and analyses of human melanoma patients, we show that the cytokine GDF-15 impairs LFA-1/β2-integrin-mediated adhesion of T cells to activated endothelial cells, which is a pre-requisite of T cell extravasation. In melanoma patients, GDF-15 serum levels strongly correlate with failure of PD-1-based immune checkpoint blockade therapy. Neutralization of GDF-15 improves both T cell trafficking and therapy efficiency in murine tumor models. Thus GDF-15, beside its known role in cancer-related anorexia and cachexia, emerges as a regulator of T cell extravasation into the tumor microenvironment, which provides an even stronger rationale for therapeutic anti-GDF-15 antibody development.
By promoting ceramide release at the cytosolic membrane leaflet, the neutral sphingomyelinase 2 (NSM) is capable of organizing receptor and signalosome segregation. Its role in T cell receptor (TCR) signaling remained so far unknown. We now show that TCR-driven NSM activation is dispensable for TCR clustering and initial phosphorylation, but of crucial importance for further signal amplification. In particular, at low doses of TCR stimulatory antibodies, NSM is required for Ca\(^{2+}\) mobilization and T cell proliferation. NSM-deficient T cells lack sustained CD3ζ and ZAP-70 phosphorylation and are unable to polarize and stabilize their microtubular system. We identified PKCζ as the key NSM downstream effector in this second wave of TCR signaling supporting dynamics of microtubule-organizing center (MTOC). Ceramide supplementation rescued PKCζ membrane recruitment and MTOC translocation in NSM-deficient cells. These findings identify the NSM as essential in TCR signaling when dynamic cytoskeletal reorganization promotes continued lateral and vertical supply of TCR signaling components: CD3ζ, Zap70, and PKCζ, and functional immune synapses are organized and stabilized via MTOC polarization.
SARS-CoV-2 variants such as the delta or omicron variants, with higher transmission rates, accelerated the global COVID-19 pandemic. Thus, novel therapeutic strategies need to be deployed. The inhibition of acid sphingomyelinase (ASM), interfering with viral entry by fluoxetine was reported. Here, we described the acid ceramidase as an additional target of fluoxetine. To discover these effects, we synthesized an ASM-independent fluoxetine derivative, AKS466. High-resolution SARS-CoV-2–RNA FISH and RTqPCR analyses demonstrate that AKS466 down-regulates viral gene expression. It is shown that SARS-CoV-2 deacidifies the lysosomal pH using the ORF3 protein. However, treatment with AKS488 or fluoxetine lowers the lysosomal pH. Our biochemical results show that AKS466 localizes to the endo-lysosomal replication compartments of infected cells, and demonstrate the enrichment of the viral genomic, minus-stranded RNA and mRNAs there. Both fluoxetine and AKS466 inhibit the acid ceramidase activity, cause endo-lysosomal ceramide elevation, and interfere with viral replication. Furthermore, Ceranib-2, a specific acid ceramidase inhibitor, reduces SARS-CoV-2 replication and, most importantly, the exogenous supplementation of C6-ceramide interferes with viral replication. These results support the hypotheses that the acid ceramidase is a SARS-CoV-2 host factor.
Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 mu m, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 mu m thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons. Pauli et al. develop targeted volumetric dSTORM in order to image large hippocampal mossy fiber boutons (MFBs) in brain slices. They can identify synaptic targets of individual MFBs and measured size and density of Bassoon clusters within individual untruncated MFBs at nanoscopic resolution.
Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications, but their photon output can be limited by trans-to-cis photoisomerization. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to the parent cyanine derivatives in a range of contexts. These include their use in the plasmonic hotspot of a DNA-nanoantenna, in single-molecule Förster-resonance energy transfer (FRET) applications, far-red fluorescence-lifetime imaging microscopy (FLIM), and single-molecule localization microscopy (SMLM). These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance.
Synthesis of a far-red photoactivatable silicon-containing rhodamine for super-resolution microscopy
(2016)
The rhodamine system is a flexible framework for building small‐molecule fluorescent probes. Changing N‐substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si‐containing analogue of Q‐rhodamine. This probe is the first example of a “caged” Si‐rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red‐shifted to allow multicolor imaging. The dye is a useful label for super‐resolution imaging and constitutes a new scaffold for far‐red fluorogenic molecules.
Like human Th1 cells, mouse Th1 cells also secrete IFN‐γ upon stimulation with a superagonistic anti‐CD28 monoclonal antibody (CD28‐SA). Crosslinking of the CD28‐SA via FcR and CD40‐CD40L interactions greatly increased IFN‐γ release. Our data stress the utility of the mouse as a model organism for immune responses in humans.