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The presynaptic protein Bruchpilot is locally synthesized to support synapse maturation but is not required for AZ-specific plasticity

Manhart, Linda

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Abstract

The nervous system is a highly interconnected network, where diverse partners are connected via varying numbers of synapses. Activity-induced synaptic plasticity is an important measure enabling adaptions to environmental cues, learning and memory or homeostatic regulations of network activity. Drosophila melanogaster is a powerful model organism for investigating the underlying mechanisms of synaptic plasticity, particularly at the neuromuscular junction. Here, identified synapses are accessible to genetic manipulations, intra-vital imaging and electrophysiological recordings. However, the majority of investigations regarding synapse plasticity focused on late larval stages. Therefore, we know very little about developmental mechanisms that establish and maintain synaptic strength during normal development. Thus, our understanding of the mechanisms that regulate the maturation of individual presynaptic active zones (AZs) and the emergence of release heterogeneity is still scarce. To address the developmental mechanisms underlying the heterogeneity of AZ release probability, I developed novel techniques to visualize incorporation and degradation rates of the AZ scaffold proteins Brp and Rbp by timed induction of endogenous tagging. Together with fluorescence recovery after photobleaching experiments in collaboration, I was able to determine that Brp proteins remain in individual AZs with a half-life of approximately 24 hours. Subsequently, they are targeted for degradation, since Brp proteins are not redistributed between AZs. By tracking incorporation rates into established AZs, I demonstrated that strong AZs might not be maintained long-term, but are dynamically adjusted throughout animal life. Therefore, the heterogeneity of AZ release probabilities does not solely arise through differential AZ birth, but is accompanied by AZ-specific mechanisms. To address if the preferential supply of individual AZs is mediated via local protein synthesis (LPS), I performed FISH experiments and determined the localization of brp and rbp mRNA. While Rbp proteins are exclusively translated in the soma, brp transcripts are present in distal segments of motorneuron axons. Therefore, Brp levels are supplied through both, long-distance transport of pre-assembled AZ building blocks and LPS. Further, I showed that the brp 3’UTR is required for axonal transcript localization and needed for the regulation of mRNA stability thereby enabling adjustments of protein amounts. Moreover, I demonstrated that LPS is not essential for both, developmental AZ heterogeneity and activity- dependent synaptic plasticity. However, LPS likely supports AZ maintenance and enables protein remodeling independent of otherwise co-transported proteins. Taken together, the presynaptic protein Bruchpilot is preferentially localized to individual AZs, thereby contributing to developmental AZ heterogeneity. Its local synthesis supports synapse maturation, but is not required for AZ-specific plasticity.

Item Type: Dissertation
Supervisor: Evers, Dr. Jan Felix
Place of Publication: Heidelberg
Date of thesis defense: 13 December 2019
Date Deposited: 17 Dec 2019 13:01
Date: 2020
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
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