Stoichiometric Biology of the Synapse

Stoichiometric Biology of the Synapse

by Benjamin Wilhelm
Doctoral thesis
Date of Examination:2013-04-12
Date of issue:2013-05-07
Advisor:Prof. Dr. Silvio Rizzoli
Referee:Prof. Dr. Erwin Neher
Referee:Prof. Dr. Michael Hörner
Referee:Prof. Dr. Stefan Hell
Referee:Prof. Dr. Ralf Heinrich
Referee:Dr. Stefan Hallermann
Persistent Address: http://dx.doi.org/10.53846/goediss-3814

 

 

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Abstract

English

Synaptic transmission at chemical synapses relies on the fusion of neurotransmitter-loaded vesicles with the plasma membrane of the pre-synaptic terminal (exocytosis). The synaptic vesicle material is then retrieved from the plasma membrane (endocytosis) and new vesicles are formed, completing what has been termed synaptic vesicle recycling. Both exo- and endocytosis are tightly regulated processes, involving a plethora of specific proteins. Much is already known about the nature of these proteins and about their interplay during vesicle recycling. However, this is not sufficient for a global understanding of synaptic function. Two critical lines of evidence are still missing: we lack quantitative information on protein numbers in the synapse and we also have limited data on their locations. In other words, the molecular anatomy of the synapse is still unknown. Here I addressed this problem by integrating several quantitative biochemistry and microscopy approaches. First, I determined the physical parameters (size, shape and organelle composition) of synapses isolated from rat brain (synaptosomes), using three-dimensional reconstructions of ultrathin electron microscopy sections. Second, I performed quantitative immunoblots to determine absolute copy numbers for 59 major proteins involved in synaptic vesicle exo- and endocytosis. Third, I determined the spatial organization of the proteins by imaging them using stimulated emission depletion (STED) microscopy, with a lateral precision of at least 40-50 nm. The information obtained from all of these assays was used to generate a three-dimensional graphical model of the pre-synaptic terminal, placing synaptic proteins in the appropriate locations, at their determined copy numbers. My findings enable us for the first time to draw conclusions on how the spatio-temporal availability of proteins determines the functional regulation of the synapse. For example, my results suggest that the availability of cystein-string-protein (CSP) and Complexin controls exocytosis and that the availability of the Clathrin light chain governs endocytosis. Overall, my data imply that synaptic function is primarily regulated by the abundance of specific proteins, rather than by the function of control mechanisms. This type of regulation is much simpler than many models proposed in the past. For example, no negative feedback loops are needed to limit synaptic processes – the limited availability of key components is sufficient for their control. Finally, since most synaptic mechanisms have closely related counterparts in other cellular areas, I suggest that the type of regulation I observed is not restricted to the synapse, but is likely applicable to the entire cell.
Keywords: Synapse; Quantitative Biology; Molecular Architecture; STED Microscopy
 
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