@article{BeckEhmannAndlaueretal.2015,
  author    = {Beck, Katherina and Ehmann, Nadine and Andlauer, Till F. M. and Ljaschenko, Dmitrij and Strecker, Katrin and Fischer, Matthias and Kittel, Robert J. and Raabe, Thomas},
  title     = {Loss of the Coffin-Lowry syndrome-associated gene RSK2 alters ERK activity, synaptic function and axonal transport in Drosophila motoneurons},
  series = {Disease Models \& Mechanisms},
  volume    = {8},
  journal   = {Disease Models \& Mechanisms},
  doi       = {10.1242/dmm.021246},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-145185},
  pages     = {1389-1400},
  year      = {2015},
  abstract  = {Plastic changes in synaptic properties are considered as fundamental for adaptive behaviors. Extracellular-signal-regulated kinase (ERK)-mediated signaling has been implicated in regulation of synaptic plasticity. Ribosomal S6 kinase 2 (RSK2) acts as a regulator and downstream effector of ERK. In the brain, RSK2 is predominantly expressed in regions required for learning and memory. Loss-of-function mutations in human RSK2 cause Coffin-Lowry syndrome, which is characterized by severe mental retardation and low IQ scores in affected males. Knockout of RSK2 in mice or the RSK ortholog in Drosophila results in a variety of learning and memory defects. However, overall brain structure in these animals is not affected, leaving open the question of the pathophysiological consequences. Using the fly neuromuscular system as a model for excitatory glutamatergic synapses, we show that removal of RSK function causes distinct defects in motoneurons and at the neuromuscular junction. Based on histochemical and electrophysiological analyses, we conclude that RSK is required for normal synaptic morphology and function. Furthermore, loss of RSK function interferes with ERK signaling at different levels. Elevated ERK activity was evident in the somata of motoneurons, whereas decreased ERK activity was observed in axons and the presynapse. In addition, we uncovered a novel function of RSK in anterograde axonal transport. Our results emphasize the importance of fine-tuning ERK activity in neuronal processes underlying higher brain functions. In this context, RSK acts as a modulator of ERK signaling.},
  language  = {en}
}
@article{MrestaniLichterSirenetal.2023,
  author    = {Mrestani, Achmed and Lichter, Katharina and Sir{\´e}n, Anna-Leena and Heckmann, Manfred and Paul, Mila M. and Pauli, Martin},
  title     = {Single-molecule localization microscopy of presynaptic active zones in Drosophila melanogaster after rapid cryofixation},
  series = {International Journal of Molecular Sciences},
  volume    = {24},
  journal   = {International Journal of Molecular Sciences},
  number    = {3},
  issn      = {1422-0067},
  doi       = {10.3390/ijms24032128},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-304904},
  year      = {2023},
  abstract  = {Single-molecule localization microscopy (SMLM) greatly advances structural studies of diverse biological tissues. For example, presynaptic active zone (AZ) nanotopology is resolved in increasing detail. Immunofluorescence imaging of AZ proteins usually relies on epitope preservation using aldehyde-based immunocompetent fixation. Cryofixation techniques, such as high-pressure freezing (HPF) and freeze substitution (FS), are widely used for ultrastructural studies of presynaptic architecture in electron microscopy (EM). HPF/FS demonstrated nearer-to-native preservation of AZ ultrastructure, e.g., by facilitating single filamentous structures. Here, we present a protocol combining the advantages of HPF/FS and direct stochastic optical reconstruction microscopy (dSTORM) to quantify nanotopology of the AZ scaffold protein Bruchpilot (Brp) at neuromuscular junctions (NMJs) of Drosophila melanogaster. Using this standardized model, we tested for preservation of Brp clusters in different FS protocols compared to classical aldehyde fixation. In HPF/FS samples, presynaptic boutons were structurally well preserved with ~22\% smaller Brp clusters that allowed quantification of subcluster topology. In summary, we established a standardized near-to-native preparation and immunohistochemistry protocol for SMLM analyses of AZ protein clusters in a defined model synapse. Our protocol could be adapted to study protein arrangements at single-molecule resolution in other intact tissue preparations.},
  language  = {en}
}
@article{PaulPauliEhmannetal.2015,
  author    = {Paul, Mila M. and Pauli, Martin and Ehmann, Nadine and Hallermann, Stefan and Sauer, Markus and Kittel, Robert J. and Heckmann, Manfred},
  title     = {Bruchpilot and Synaptotagmin collaborate to drive rapid glutamate release and active zone differentiation},
  series = {Frontiers in Cellular Neuroscience},
  volume    = {9},
  journal   = {Frontiers in Cellular Neuroscience},
  number    = {29},
  doi       = {10.3389/fncel.2015.00029},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-148988},
  year      = {2015},
  abstract  = {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.},
  language  = {en}
}