Astrocytes are no longer considered subservient to neurons, and are, instead, now understood to play an active role in brain signaling. The intercellular communication of astrocytes with neurons and other non-neuronal cells involves the exchange of molecules by exocytotic and endocytotic processes. This type of communication is being the subject of intensive research. In this presentation I will first elucidate our study of the anatomy of single vesicles in astrocytes using super-resolution stimulated emission depletion (STED) microscopy and structured illumination microscopy (SIM). Then, I will focus on the interaction of these vesicles with the plasma membrane, measured by a high-resolution membrane capacitance approach. Last, I will show results, which indicate that certain disorders, like X-linked non-syndromic intellectual disability (XLID), that have traditionally been linked to neurons, exhibit strong vesicle related phenotypes also in astrocytes. These findings will contribute to the debate on gliotransmission.
B.04 Guest lecture
COBISS.SI-ID: 32774617Exocytic transmitter release is most likely regulated by the SNARE complex, which contains a vesicular protein, synaptobrevin2 (Sb2). We first used super-resolution stimulated emission depletion (STED) microscopy and structured illumination microscopy (SIM) to study the anatomy of single vesicles in astrocytes. Smaller vesicles with diameters of ~65 nm contained amino acid and peptidergic transmitters and larger vesicles with diameters of ~230 nm contained ATP. To estimate the number of Sb2 proteins per vesicle and the quantity necessary for vesicular fusion, we transfected astrocytes with yellow synaptopHluorin (YSpH). Then we stimulated astrocytes with ATP to promote Ca2+-dependent exocytosis and performed photo bleaching of YSpH. Fluorescence intensity analysis revealed that the average number of YSpH molecules is 3–5 per vesicle, while the average number of endogenous Sb2 molecules is 15–25 per vesicle. The density of Sb2 molecules appeared to be lower in larger vesicles. Moreover, the distribution of Sb2 molecules in vesicles is non-uniform and one YSpH molecule appeared necessary for fusion pore formation. To determine unitary exocytic properties of single vesicles, discrete increases in membrane capacitance, indicating single-vesicle fusion, revealed that astrocyte stimulation increases the frequency of predominantly transient fusion events in smaller vesicles, whereas larger vesicles transitioned to full fusion. To determine whether this reflects a lower density of SNARE proteins in larger vesicles, we treated astrocytes with botulinum neurotoxins D and E, which reduced exocytotic events of both vesicle types. dnSNARE peptide stabilized the fusion-pore diameter to narrow, release-unproductive diameters in both vesicle types, regardless of vesicle diameter.
B.03 Paper at an international scientific conference
COBISS.SI-ID: 32775385Fluorescent microscopy has become an essential tool in the modern research of cell structures and physiological processes. In fluorescent microscopy, the resolution that can be obtained is limited by the diffraction of light to ~200 nm. Stimulated emission depletion microscopy (STED) was the first physical concept to break the diffraction barrier in the far-field fluorescence microscopy. In STED microscopy, the diffraction-limited focal spot can be made infinitely small. Nonetheless, in biological samples lateral resolution of 20–40 nm is typically achieved. For his contribution to the invention of super-resolved fluorescence microscopy, Stefan W. Hell received the Nobel Prize in Chemistry in 2014, together with Eric Betzig and William Moerner. In this presentation I will first explain the basic concept of STED microscopy, with special focus on the specific technical implementation of a two-colour STED microscope, which we developed in the collaboration with dr. Stefan W. Hell, dr. Alexander Egner and dr. Claudia Geisler. Then, I will present our study of the anatomy of single vesicles in astrocytes using confocal microscopy, structured illumination microscopy (SIM; lateral resolution of ~100 nm) and STED microscopy. These findings will contribute to the debate on gliotransmission.
B.04 Guest lecture
COBISS.SI-ID: 32961497