RobustCircuit Project 1

Project 1 focuses on robust synapse formation resulting from imprecise molecular active zone ‘seeding’ events.

Imprecision: In Drosophila larval motoneurons, the recruitment of the molecular components to initiate active zone assembly is noisy, based on preliminary data showing these ‘seeding’ events to be spatiotemporally unpredictable and often reversible.

Robustness: Mature motoneuron synapses comprise a variable number of boutons and active zones in these boutons, yet the active zone number per pre-postsynaptic membrane contact is tightly controlled and the resulting electrophysiological properties are precise and robust to morphological variability. The active zone number and active zone average size are precisely matched to muscle size in order to ensure proper excitation and contraction.

Hypothesis: Molecular ‘seeding’ events occur stochastically, followed by selection through local and system-wide cues and subsequent stabilization.

Project Summary

The addition of new synaptic connections is a process fundamental to the development and function of synaptic circuits. However, how a developing neuron controls the number, placement and strength of individual synaptic release sites is poorly understood. In particular, the dynamics and control points that ultimately steer synapse assembly are unknown and challenging to study in most in vivo systems. The developing Drosophila larval neuromuscular junction offers a model where the probabilistic dynamics of synapse assembly can be studied live and in real time over the course of several hours. We have pioneered intravital imaging of synapse assembly at the molecular and subcellular levels and previously shown that the outcome of this developmental process is a stoichiometrically well-defined pre-postsynaptic protein architecture, precondition for robust functionality. Synaptic function is robust to the developmental variability of neuromuscular morphology and synaptic arrangement. During development, Liprin-a clusters kick-off (“seed”) the assembly process of nascent active zones, while components specific for mature active zones follow considerably later. Notably, Liprin-a clusters display a highly fluctuating behavior characterized by probabilistic stabilization or disassembly. In P1, we seek to understand how the probabilistic nature of synapse assembly leads to functionally robust NMJ structure and function. We hypothesize that Liprin-a clusters are subject to several testable influences that determine the fate of each synaptic seeding event individually, including the dynamics and availability of proteins required for synaptic assembly as well as cell-non-autonomous trans-synaptic signals required for the stabilization of nascent synapses. In preparation for RobustCircuit, we have identified genetic inroads to manipulate and test molecular mechanisms controlling nascent synapse dynamics. Based on our analysis of the synapse seeding process, P1 is devised to establish a model for the development of functional robustness based on dynamic and probabilistic decision-making processes at the level of molecular synapse assembly.

References

1. Gotz*, T.W.B., D. Puchkov, V. Lysiuk, J. Lutzkendorf, A.G. Nikonenko, C. Quentin, M. Lehmann, S.J. Sigrist, and A.G. Petzoldt. 2021. Rab2 regulates presynaptic precursor vesicle biogenesis at the trans-Golgi. J Cell Biol. 220(5): e202006040.
2. Ramesh*, N., M.J.F. Escher, M.M. Mampell, M.A. Bohme, T.W.B. Gotz, P. Goel, T. Matkovic, A.G. Petzoldt, D. Dickman, and S.J. Sigrist. 2021. Antagonistic interactions between two Neuroligins coordinate pre- and postsynaptic assembly. Curr Biol. 31(8):1711-1725.
3. Pooryasin*, A., M. Maglione, M. Schubert, T. Matkovic-Rachid, S.M. Hasheminasab, U. Pech, A. Fiala, T. Mielke, and S.J. Sigrist. 2021. Unc13A and Unc13B contribute to the decoding of distinct sensory information in Drosophila. Nat Commun. 12:1932.
4. Huang*, S., C. Piao, C.B. Beuschel, T. Gotz, and S.J. Sigrist. (2020). Presynaptic Active Zone Plasticity Encodes Sleep Need in Drosophila. Curr Biol. 30(6):1077-1091.
5. Petzoldt*, A.G., T.W.B. Gotz*, J.H. Driller*, J. Lutzkendorf*, S. Reddy-Alla, T. Matkovic-Rachid, S. Liu, E. Knoche, S. Mertel, V. Ugorets, M. Lehmann, N. Ramesh, C.B. Beuschel, B. Kuropka, C. Freund, U. Stelzl, B. Loll, F. Liu, M.C. Wahl, and S.J. Sigrist. (2020). RIM-binding protein couples synaptic vesicle recruitment to release sites. J Cell Biol. 219(7): e201902059.
6. Vukoja*, A., U. Rey*, A.G. Petzoldt*, C. Ott, D. Vollweiter, C. Quentin, D. Puchkov, E. Reynolds, M. Lehmann, S. Hohensee, S. Rosa, R. Lipowsky, S.J. Sigrist, and V. Haucke. (2018). Presynaptic Biogenesis Requires Axonal Transport of Lysosome-Related Vesicles. Neuron. 99:1216-1232 e1217.
7. Petzoldt, A.G*., Y.H. Lee*, O. Khorramshahi, E. Reynolds, A.J. Plested, H. Herzel, and S.J. Sigrist. 2014. Gating characteristics control glutamate receptor distribution and trafficking in vivo. Curr Biol. 24:2059-2065.