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Fluorescence microscopy is an excellent tool to gain knowledge on cellular structures and biochemical processes. Stimulated emission depletion (STED) microscopy provides a resolution in the range of a few ten nanometers at relatively fast data acquisition. As cellular structures can be oriented in any direction, it is of great benefit if the microscope exhibits an isotropic resolution. Here, we present an isoSTED microscope that utilizes water-immersion objective lenses and enables imaging of cellular structures with an isotropic resolution of better than 60 nm in living samples at room temperature and without CO2 supply or another pH control. This corresponds to a reduction of the focal volume by far more than two orders of magnitude as compared to confocal microscopy. The imaging speed is in the range of 0.8 s/μm3. Since fluorescence signal can only be detected from a diffraction-limited volume, a background signal is inevitably observed at resolutions well beyond the diffraction limit. Therefore, we additionally present a method which allows to identify this unspecific background signal and to remove it from the image.

AlkB homolog 5 (Alkbh5) is one of nine members of the AlkB family, which are nonheme Fe(2+)/α-ketoglutarate-dependent dioxygenases that catalyze the oxidative demethylation of modified nucleotides and amino acids. Alkbh5 is highly selective for the N(6)-methyladenosine modification, an epigenetic mark that has spawned significant biological and pharmacological interest because of its involvement in important physiological processes, such as carcinogenesis and stem cell differentiation. Herein, we investigate the structure and dynamics of human Alkbh5 in solution. By using (15)N and (13)C(methyl) relaxation dispersion and (15)N-R(1) and R(1ρ) NMR experiments, we show that the active site of apo Alkbh5 experiences conformational dynamics on multiple timescales. Consistent with this observation, backbone amide residual dipolar couplings measured for Alkbh5 in phage pf1 are inconsistent with the static crystal structure of the enzyme. We developed a simple approach that combines residual dipolar coupling data and accelerated molecular dynamics simulations to calculate a conformational ensemble of Alkbh5 that is fully consistent with the experimental NMR data. Our structural model reveals that Alkbh5 is more disordered in solution than what is observed in the crystal state and undergoes breathing motions that expand the active site and allow access to α-ketoglutarate. Disordered-to-ordered conformational changes induced by sequential substrate/cofactor binding events have been often invoked to interpret biochemical data on the activity and specificity of AlkB proteins. The structural ensemble reported in this work provides the first atomic-resolution model of an AlkB protein in its disordered conformational state to our knowledge.

Eukaryotic cells respond to signaling molecules with picomolar to nanomolar sensitivities. However, molar concentrations give no suggestion of the sufficient number of molecules per cell and are confusing when referring to physiological situations in which signaling molecules act in an immobilized state. Here, we studied platelet adhesion by thrombin, a key step in normal hemostasis and pathological arterial thrombosis. We generated a biofunctional nanosheet surface to mimic the in vivo solid-state interaction between platelets and thrombin at sites of injured tissues. We observed that 1000-fold stoichiometric excess of thrombin. We conclude that immobilizing thrombin apposed to the membrane receptor allows platelets to respond with very high sensitivity. Moreover, we propose that irreversible cell activation may require several ligands to avoid activation by single, mislocalized signaling molecules.

Tetratricopeptide repeat (TPR) domains are protein-protein interaction domains present in all kingdoms of life and mutations of human TPR domains have been linked to disease. The TPR protein fission 1 (Fis1) is conserved in all species that contain mitochondria. In S. cerevisiae, Fis1 is essential for mitochondrial fission, acting as a scaffold for other fission proteins through its TPR domain. It remains unclear if the function of Fis1 is conserved in mammals. Fis1 oligomers exist in high molecular weight complexes on isolated mitochondria. However, the structural basis of these Fis1 oligomers remains largely unknown. The TPR and transmembrane domains of Fis1 have been shown to be essential for Fis1 oligomer formation on the mitochondrial membrane. We hypothesize that the ability of Fis1 to self-associate on the membrane is mediated by dimerization of the cytoplasmic domain and dimerization of the transmembrane domain. Here, we use biophysical and structural techniques to test this hypothesis. We demonstrate that Fis1 is able to self-associate via its cytoplasmic TPR domain and have developed reagents in order to reconstitute Fis1 in a membrane environment. This project will address the structure of the Fis1 oligomer on the membrane, which is an outstanding question in the field.

Astrocytes actively participate in brain signalling by releasing a plethora of gliotransmitters which can modulate synaptic transmission. Vesicle-based mechanisms mediate the release of gliotransmitters. However, the anatomy and nature of exocytotic vesicle interaction with the plasma membrane is unclear. Using STED and SIM super-resolution microscopies, we studied the morphology of distinct gliotransmitter vesicles, whereas the interaction between a single vesicle with the plasma membrane was monitored by measurements of membrane capacitance (Cm), a parameter linearly related to the surface area of the plasma membrane. Immunolabelling of vesicles containing D-serine, glutamate, atrial natriuretic peptide (ANP) and brain derived neurotrophic factor (BDNF) yielded their diameter to be ∼70 nm, whereas ATP was found in larger vesicles (∼200 nm diameter). Cell-attached measurements have shown the predominant reversible unitary exocytotic events exhibiting transient fusion were found in two different populations, the first corresponding to smaller vesicles with diameters around 70 nm and the second corresponding to bigger vesicles with the median at 200 nm, consistent with the STED and SIM measurements. Upon stimulation with ATP, which increases intracellular Ca2+ concentration, the smaller vesicles persisted in transient fusion, whereas the bigger vesicles proceeded to full fusion. This was interpreted previously to be due to different SNARE protein densities in different sized vesicles and was tested here by astrocyte treatment with botulinum neurotoxin D (BotD), which significantly reduced the occurrence of unitary exocytotic events for both vesicle types. Additionally, when we expressed dominant-negative SNARE in astrocytes, the fusion-pore diameter was narrower in both vesicle types. Taken together, this work shows that vesicle content discharge is modulated by vesicle size, however independently of the functional integrity of the SNARE proteins, indicating that SNARE-dependent mechanisms determining vesicle merger with the plasmalemma are not strictly part of those modulating fusion-pore geometry.

Astrocytes, the most abundant type of glial cells, play a number of important functions in information processing in the brain. They ensheath synapses and modulate synaptic transmission by many mechanisms which include the exocytotic release of gliotransmitters into the extracellular space. However, the anatomy and nature of exocytotic vesicle interaction with the plasma membrane is unclear. Using stimulated emission depletion (STED) and structured illumination (SIM) super-resolution microscopies, we studied the morphology of distinct gliotransmitter vesicles, whereas the interaction between a single vesicle with the plasma membrane was monitored by the high-resolution cell-attached patch-clamp measurements of membrane capacitance (Cm), a parameter linearly related to the surface area of the plasma membrane. Immunolabelling of vesicles containing D-serine, glutamate, atrial natriuretic peptide (ANP) and brain derived neurotrophic factor (BDNF) yielded their diameter to be < 100 nm, whereas ATP was found in larger vesicles (∼200 nm diameter). Direct electrophysiologic measurements have shown that the predominant mode of vesicle interaction with the plasma membrane consisted of reversible capacitance steps, reflecting transient exocytosis, whereas irreversible capacitance steps, reflecting full-fusion exocytosis, were less abundant. The amplitude of discrete step increases in Cm reflects the size of interacting vesicles. By assuming the specific capacitance of 10 fF/μm2 and spherical morphology, the diameters of vesicles fusing with the plasma membrane ranged from 40 - 800 nm, consistent with the STED and SIM measurements. Reduction of extracellular Ca2+ attenuated the occurrence of unitary exocytotic events and treatment of astrocytes with Botulinum neurotoxins, which specifically inhibit the SNARE complex formation, attenuated the frequency of transient and full fusion events. These results show that in astrocytes the occurrence of unitary exocytotic events of small and large vesicles is Ca2+- and SNARE- dependent.