Ultrastructure and regulation of adhesion at a genetically tractable model synapse

Lead Research Organisation: University of Manchester
Department Name: Life Sciences

Abstract

Behaviour, learning, memory and cognition are all outward manifestations of the complex neuronal networks that make up the nervous system. Neuronal networks are composed of nerve cells which display long projections forming reproducible points of contact with other nerve, muscle or gland cells. Electrical messages pass along these processes and are transmitted to other cells at the points of contact. Synapses are specialised structures at these contacts which carry out the information transfer. Since they can manipulate or filter information during the transfer process, synapses are key regulators of information flow in neuronal networks, essential for nervous system function and behaviour. Inappropriate function of synapses has dire consequences such as epileptic attacks, mental retardation or death. To execute their function appropriately, a number of structural features have to be correctly established when synapses form during nervous system development. They have to be established at the right places (i.e. at contacts between appropriate cells); synaptic components have to assemble precisely opposite on the signal-sending and -receiving sides of the contact; at each neuronal contact synapses have to assemble in appropriate numbers so that information transfer occurs at a strength adequate for its position in the neuronal network. So called adhesion molecules contribute essentially to all these developmental processes. Adhesion molecules sit on the surface of cells and stick to molecules on other surfaces or of material deposited in the surrounding of cells. Thus, adhesion molecules hold cells together and in certain body locations. The same is true for intercellular adhesion between neurons and their target cells at synapses. Accordingly, a number of synaptic adhesion molecules have been described, but their regulation over time and interdependencies between different classes of adhesion molecules leading to proper synapse development are hardly understood. Here we propose to focus studies of synaptic adhesion on a well known synaptic contact between nerve and muscle cells in a genetic model organism, the fruitfly Drosophila. Drosophila provides numerous advantages speeding up research into genes and their products (such as adhesion molecules). Since genes are often preserved during evolution and are similar between species as distant as humans and the fruitfly, our research will have implications for synapse-related medically relevant research. This project will make extensive use of highly sophisticated imaging techniques (electron microscopy) which allows to view cellular specialisations at extremely high resolution. Here, we will look into the detailed structures formed by adhesion molecules holding neuromuscular contacts of the fruitfly together. From such observations we can deduce, what kind of molecules are involved and whether they differ between different areas of the synaptic contact. In parallel, we will analyse potential defects of synapses in flies with inherited diseases (mutations) affecting adhesion molecules. Together with the structural descriptions, this will help us to pinpoint those molecules required at this particular synaptic contact and determine how they are arranged to carry out their function. Finally, we will try to understand how these molecules function during development in the stepwise process from the early neuromuscular contact to the final appearance and size of the functional synaptic contact.

Technical Summary

Synapses are key regulators of information flow in neuronal networks. During development, synaptic contacts form in a position-specific manner, take on reproducible shapes, sizes and structures. This development crucially depends on spatial and temporal regulation of adhesion, but little is known about the details of this regulation. We will address mechanisms of genetic adhesion capitalising on 1) the genetically tractable Drosophila neuromuscular junction (NMJ), 2) a sound foundation of existing data, and 3) the outstanding experience of the applicants on Drosophila synapses, and on confocal and ultrastructrual analysis techniques. We will investigate the interplay of different forms of adhesion (basement membrane attachments, synaptic and extra-synaptic neuromuscular contacts), all of which have been demonstrated to be essential for NMJ formation or maintenance. Focussing on the mature, late embryonic NMJ, we will employ our expertise on electron tomography to describe the structure of these adhesive contacts. We will demonstrate similarities and dissimilarities between the three adhesion forms with respect to the content of cleft-spanning elements and extracellular matrix components. Such data will allow us to make predictions about the number and kind of different adhesion systems to be expected. In parallel, we will test at least 13 candidate genes for their potential involvement in NMJ adhesion and structure using a lamininA-deficient background, in which detachment phenotypes are more likely to occur. This procedure will pinpoint required adhesion molecules, which can be mapped onto the structural descriptions obtained by ET. Finally, we will look into developmental dynamics of adhesion by analysing normal and mutant NMJs at earlier stages of development using ultrastructural 3D reconstructions. This way we will distinguish early from late requirements of adhesion molecules and understand how mutant phenotypes of the mature NMJ develop over time.
 
Description • We were able to confirm the role of ECM in synaptic adhesion at the Drosophila NMJ and were able to pinpoint this as an indirect role mediated through basement membrane adhesion.

• We established a list of 285 adhesion factors through in silico searches, and carried out detailed ultrastructural analyses of 18 genotypes selected through in silico and literature searches; none of these factors showed an obvious detachment phenotype.

• We carried out combinations of loss of functions of adhesion factors to an unprecedented degree, including complete deletion of classical cadherins, flamingo-type cadherins and neurexins at the NMJ. We did not find any enhanced detachment - leading to the key statement that synaptic adhesion is redundant to such a degree that future studies at any synapse are highly unlikely to ever produce a detachment phenotype.
Exploitation Route It clearly demonstrates the enormous functional redundancy of synaptic adhesion and demonstrates important limitations for any kind of work into this topic also by others and in other models. It is consistent with the fact that no de-adhesion phenotypes have been observed at other synapse models.
Sectors Other

URL https://www.escholar.manchester.ac.uk/uk-ac-man-scw:158385
 
Description Equipment, biomedical resources or technology development grant
Amount £286,793 (GBP)
Funding ID 087742/Z/08/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2009 
End 08/2012
 
Description PhD studentship scheme
Amount £100,000 (GBP)
Funding ID BB/D526561/1 - Robin Beaven 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2008 
End 09/2012
 
Description Value in People Award Scheme
Amount £14,945 (GBP)
Funding ID 092403/Z/10/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2010 
End 03/2011
 
Description Value in People Award Scheme
Amount £14,816 (GBP)
Funding ID 087820/Z/08/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2009 
End 08/2010
 
Description Value in People Award Scheme
Amount £10,143 (GBP)
Funding ID 092403/Z/10/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2010 
End 12/2010