Nanoscopical analysis reveals an orderly arrangement of the presynaptic scaffold protein Bassoon at the Golgi-apparatus

Bassoon is a core scaffold protein of the presynaptic active zone. In brain synapses, the C-terminus of Bassoon is oriented toward the plasma membrane and its N-terminus oriented towards synaptic vesicles. At the Golgi-apparatus Bassoon is thought to assemble active zone precursor structures, but whether it is arranged in an orderly fashion is unknown. Understanding the topology of this large scaffold protein is important for models of active zone biogenesis. Using stimulated emission depletion nanoscopy in cultured hippocampal neurons, we found that an N-terminal intramolecular tag of recombinant Bassoon, but not C-terminal tag, colocalized with markers of the trans-Golgi network (TGN). The N-terminus of Bassoon was located between 48 nm and 69 nm away from TGN38, while its C-terminus was located between 100 nm and 115 nm away from TGN38. Sequences within the first 95 amino acids of Bassoon were required for this arrangement. Our results indicate that at the Golgi-apparatus Bassoon is oriented with its N-terminus towards and its C-terminus away from the trans-Golgi network membrane. Moreover, they suggest that Bassoon is an extended molecule at the trans-Golgi network with the distance between amino acids 97 and 3938 estimated to be between 46 and 52 nm. Our data are consistent with a model, in which the N-terminus of Bassoon binds to the membranes of the trans-Golgi network, while the C-terminus associates with active zone components, thus reflecting the topographic arrangement characteristic of synapses also at the Golgi-apparatu

Structurally and functionally unique complexins at retinal ribbon synapses

Ribbon synapses in retinal sensory neurons maintain large pools of readily releasable synaptic vesicles. This allows them to release several hundreds of vesicles per second at every presynaptic release site. The molecular components that cause this high transmitter release efficiency of ribbon synapses are unknown. In the present study, we identified and characterized two novel vertebrate complexins (CPXs), CPXs III and IV, that are the only CPX isoforms present in retinal ribbon synapses. CPXs III and IV are COOH-terminally farnesylated, and, like CPXs I and II, bind to SNAP receptor complexes. CPXs III and IV can functionally replace CPXs I and II, and their COOH-terminal farnesylation regulates their synaptic targeting and modulatory function in transmitter release. The novel CPXs III and IV may contribute to the unique release efficacy of retinal sensory neurons

Neuronal Profilin Isoforms Are Addressed by Different Signalling Pathways

Profilins are prominent regulators of actin dynamics. While most mammalian cells express only one profilin, two isoforms, PFN1 and PFN2a are present in the CNS. To challenge the hypothesis that the expression of two profilin isoforms is linked to the complex shape of neurons and to the activity-dependent structural plasticity, we analysed how PFN1 and PFN2a respond to changes of neuronal activity. Simultaneous labelling of rodent embryonic neurons with isoform-specific monoclonal antibodies revealed both isoforms in the same synapse. Immunoelectron microscopy on brain sections demonstrated both profilins in synapses of the mature rodent cortex, hippocampus and cerebellum. Both isoforms were significantly more abundant in postsynaptic than in presynaptic structures. Immunofluorescence showed PFN2a associated with gephyrin clusters of the postsynaptic active zone in inhibitory synapses of embryonic neurons. When cultures were stimulated in order to change their activity level, active synapses that were identified by the uptake of synaptotagmin antibodies, displayed significantly higher amounts of both isoforms than non-stimulated controls. Specific inhibition of NMDA receptors by the antagonist APV in cultured rat hippocampal neurons resulted in a decrease of PFN2a but left PFN1 unaffected. Stimulation by the brain derived neurotrophic factor (BDNF), on the other hand, led to a significant increase in both synaptic PFN1 and PFN2a. Analogous results were obtained for neuronal nuclei: both isoforms were localized in the same nucleus, and their levels rose significantly in response to KCl stimulation, whereas BDNF caused here a higher increase in PFN1 than in PFN2a. Our results strongly support the notion of an isoform specific role for profilins as regulators of actin dynamics in different signalling pathways, in excitatory as well as in inhibitory synapses. Furthermore, they suggest a functional role for both profilins in neuronal nuclei

Adaption of a material model and development of a stochastic failure criterion for ceramic matrix composite structures

Ceramic matrix composites (CMCs) show high thermal resistance combined with damage tolerance and non-brittle failure. Therefore, they are suitable for mechanically loaded components at high temperatures. For dimensioning of components, an adaption of existing simulation and calculation methods for CMC materials is required. The material investigated in this work is a wound oxide CMC with a defined fiber orientation and a highly porous matrix. Due to manufacturing conditions, randomly distributed macroscopic pores are present in the composite. Since failure is initiated by these pores, a statistical investigation of strength is performed based on the weakest link theory. Several series of tensile tests were performed to reveal the relation between stress and strain and obtain their ultimate values. The application of two statistical criteria showed that fracture strains are well represented by Weibull distributions. The tensile tests were compared with results of finite element simulations, using the simulation software ANSYS. An anisotropic Weibull criterion was set up and adapted to the results of the tensile tests. Hereby, the anisotropic nonlinear deformation behavior as well as the scatter of failure strain could be reproduced by numerical simulations. The adapted material model and failure criterion were validated by further experiments and exemplified by application in a reliability analysis of a notional flame tube

Development of a Stochastic Simulation Approach for High Temperature Components made of WHIPOX (oxide/oxide CMC)

Ceramic matrix composites (CMCs) show high thermal resistance combined with damage tolerance and non-brittle failure. Therefore they are suitable for mechanically loaded components under high temperatures. The introduction of oxide CMCs as material for combustion chambers of stationary gas turbines is a subject of current research. For the dimensioning of components, the adaption of existing simulation and calculation methods to CMC materials is required. This includes the development of a failure criterion ensuring high reliability of components against failure under consideration of the scatter in material strength. The material investigated in this work is WHIPOX, a wound oxide/oxide CMC with a defined fiber orientation and highly porous matrix. A number of tensile tests, performed at different temperature levels, were evaluated. The temperature-dependent deformation parameters of a modified orthotropic material model were determined from the experimental results and integrated in finite element simulations. Additionally, a failure criterion is required for component design. Due to manufacturing conditions, macroscopic pores with random orientation, location and size are present in the composite. Since failure is assumed to be caused by these pores, a statistical investigation of strength is performed. Therefore, an anisotropic Weibull criterion was set up and adapted to the results of the tensile tests. Thus the non-linear deformation behavior as well as the specimen’s scatter in strength could be reproduced by finite element simulation and post processing. The adapted material model and failure criterion were validated by further experiments, e.g. three point bending tests, which showed good agreement between simulated and experimental results. Moreover the failure probability of a thermally loaded combustion chamber was investigated to demonstrate the applicability in industrial context

Adaption of a Material Model and Development of a Stochastic Failure Criterion for Ceramic Matrix Composite Structures

Ceramic matrix composites (CMCs) show high thermal resistance combined with damage tolerance and non-brittle failure. Therefore they are suitable for mechanically loaded components under high temperatures. The introduction of oxide CMCs as material for combustion chambers of stationary gas turbines is a subject of current research. For the dimensioning of components, the adaption of existing simulation and calculation methods to CMC materials is required. This includes the development of a failure criterion ensuring high reliability of components against failure under consideration of the scatter in material strength. The material investigated in this work is a wound oxide CMC with a defined fiber orientation and highly porous matrix. Due to manufacturing conditions, macroscopic pores with random orientation, location and size are present in the composite. Since failure is assumed to be caused by these pores, a statistical investigation of strength is performed, following the Weibull Theory. A number of tensile tests were evaluated and compared with results of finite element simulations (ANSYS). By application of an optimization tool (optiSlang), the parameters of different orthotropic material models were determined. An anisotropic Weibull criterion was set up and adapted to the results of the tensile tests. Thus the non-linear deformation behavior as well as the specimen’s scatter in strength could be reproduced. The adapted material model and failure criterion were validated by further experiments, e.g. three point bending tests, which showed good agreement between simulated and experimental results. Moreover the failure probability of a thermally loaded combustion chamber was investigated to demonstrate the applicability in industrial context