Frühe Assemblierungsphase der aktiven Zone in Drosophila

The dissection of presynaptic assembly processes has proven difficult in the past. Apart from genetic redundancies, this likely reflects a highly cooperative and regulated nature of synapse assembly, complicating the straightforward deduction of linear molecular models. This study genetically dissects defined event hierarchies and assembly intermediates and complements these with biochemical, electrophysiological, ultrastructural and in vivo protein trafficking data. Analysis of chemically-induced alleles of the active zone (AZ) protein BRP, positions it as a direct building block of the electron dense cytomatrix at the AZ (CAZ). An unbiased proteomics screen reveals that the RhoGAP DSyd-1 physically interacts with BRP and localizes to AZs. Moreover, DSyd-1 closely co-localizes with a further AZ protein, DLiprin-á. Using transposon-based genetics two dsyd-1 deficient alleles were synthesized. dsyd-1 mutant animals elicit impaired locomotive behavior and a reduced life span, while NMJ synapse numbers and evoked synaptic currents are reduced. Furthermore, AZ morphology appears abnormal in dsyd-1 deficient animals with the CAZ often appearing misshapen. Floating electron dense material is, moreover, found at ectopic positions. In vivo imaging reveals that DSyd-1 and DLiprin-á accumulate early during synapse development, preceding postsynaptic glutamate receptor accumulation, as well as presynaptically localized BRP. Analysis of dliprin-á; dsyd-1 double mutants indicates that overgrown BRP accumulations found in dsyd-1 mutants are dependent on the presence of dliprin-á. In fact, defining a hierarchy of the two proteins, DLiprin-á localization is largely impaired in dsyd-1 mutant animals, while DSyd-1 localizes normally in mutants for dliprin-á. Unlike BRP, DLiprin-á and DSyd-1 clusters appear to be able to decompose again, indicating that early AZ assembly is still reversible. Thus, AZ assembly can be divided into an early, reversible step at nascent site and a later, largely irreversible maturation phase. DLiprin-á mobility is largely elevated in dsyd-1 mutants, specifically indicating a clamping function of DSyd-1, which possibly shapes the transition between nascent and maturing synapses. Presynaptic DSyd-1 is further shown to regulate postsynaptic receptor field composition, increasing the amount of slow desensitizing IIA subunit-containing glutamate receptor complexes. This process is independent of DLiprin-á. Following phenotypic similarities, Drosophila Neurexin is proposed as a further DSyd-1 substrate. Indeed, postsynaptic Neuroligin1, a potential DNrx interactor, is identified as localizing towards the edge of postsynaptic densities here. Mutants in dnlg1 exhibit aberrant NMJ morphology with increased sizes of postsynaptic densities, impaired neurotransmission and boutons lacking postsynaptic receptor fields. Such orphan boutons are also occasionally found in dsyd-1 mutants. Moreover, DNlg1 immunoreactivity is drastically reduced in the absence of DSyd-1. It thus appears likely that presynaptic DSyd-1 regulates the levels of postsynaptic DNlg1, potentially via presynaptic DNrx. In a proteomics approach, the GTPase Dynamin is uncovered as a potential interactor of BRP. This physical interaction is confirmed in vitro. The interaction platform is fine-mapped to an N-terminal 30 aa of BRP and the GTPase effector domain along with a domain towards the very C-term of Dynamin. This interaction might link the BRP-based CAZ to the SV exo/endo-cycle