TY  - JOUR
A1  - Paul, Mila M.
A1  - Pauli, Martin
A1  - Ehmann, Nadine
A1  - Hallermann, Stefan
A1  - Sauer, Markus
A1  - Kittel, Robert J.
A1  - Heckmann, Manfred
T1  - Bruchpilot and Synaptotagmin collaborate to drive rapid glutamate release and active zone differentiation
JF  - Frontiers in Cellular Neuroscience
N2  - The active zone (AZ) protein Bruchpilot (Brp) is essential for rapid glutamate release at Drosophila melanogaster neuromuscular junctions (NMJs). Quantal time course and measurements of action potential-waveform suggest that presynaptic fusion mechanisms are altered in brp null mutants (brp\(^{69}\)). This could account for their increased evoked excitatory postsynaptic current (EPSC) delay and rise time (by about 1 ms). To test the mechanism of release protraction at brp\(^{69}\) AZs, we performed knock-down of Synaptotagmin-1 (Syt) via RNAi (syt\(^{KD}\)) in wildtype (wt), brp\(^{69}\) and rab3 null mutants (rab3\(^{rup}\)), where Brp is concentrated at a small number of AZs. At wt and rab3\(^{rup}\) synapses, syt\(^{KD}\) lowered EPSC amplitude while increasing rise time and delay, consistent with the role of Syt as a release sensor. In contrast, syt\(^{KD}\) did not alter EPSC amplitude at brp\(^{69}\) synapses, but shortened delay and rise time. In fact, following syt\(^{KD}\), these kinetic properties were strikingly similar in wt and brp\(^{69}\), which supports the notion that Syt protracts release at brp\(^{69}\) synapses. To gain insight into this surprising role of Syt at brp\(^{69}\) AZs, we analyzed the structural and functional differentiation of synaptic boutons at the NMJ. At tonic type Ib motor neurons, distal boutons contain more AZs, more Brp proteins per AZ and show elevated and accelerated glutamate release compared to proximal boutons. The functional differentiation between proximal and distal boutons is Brp-dependent and reduced after syt\(^{KD}\). Notably, syt\(^{KD}\) boutons are smaller, contain fewer Brp positive AZs and these are of similar number in proximal and distal boutons. In addition, super-resolution imaging via dSTORM revealed that syt\(^{KD}\) increases the number and alters the spatial distribution of Brp molecules at AZs, while the gradient of Brp proteins per AZ is diminished. In summary, these data demonstrate that normal structural and functional differentiation of Drosophila AZs requires concerted action of Brp and Syt.
KW  - neuromuscular junction
KW  - Bruchpilot
KW  - synaptic delay
KW  - dSTORM
KW  - synaptotagmin
KW  - presynaptic differentiation
KW  - neurotransmitter release
KW  - active zone
KW  - synaptic transmission
KW  - fluorescent probes
Y1  - 2015
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-148988
VL  - 9
IS  - 29
ER  - 
TY  - JOUR
A1  - Wäldchen, Sina
A1  - Lehmann, Julian
A1  - Klein, Teresa
A1  - van de Linde, Sebastian
A1  - Sauer, Markus
T1  - Light-induced cell damage in live-cell super-resolution microscopy
JF  - Scientific Reports
N2  - Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20-24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of similar to 1 kW cm\(^{-2}\) at 640 nm for several minutes, the maximum dose at 405 nm is only similar to 50 J cm\(^{-2}\), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.
KW  - optical reconstruction microscopy
KW  - tag fusion proteins
KW  - localization microscopy
KW  - photodynamic therapy
KW  - diffraction limit
KW  - illumination microscopy
KW  - structured illumination
KW  - fluorescent probes
KW  - in vitro
KW  - dynamics
Y1  - 2015
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-145207
VL  - 5
IS  - 15348
ER  -