|Dr William D. Phillips||Lecturer (in-charge)||University||Nov||1992-|
|Azita Ahadizdeh||Research Assistant (0.5)||NHMRC||1994|
|Jeon Cha||BSc(Hons) student||1994|
|Hong Han||PhD student||ADCOS||1995-|
|Shihong Yang||MSc student||1995-|
|Sarah Lee||BSc(Hons) student||1995|
Current effective full-time personnel = 4.0
This Laboratory is concerned with investigating the molecular basis of synaptic connections between nerve and muscle cells. Recent developments in molecular biology are employed to study the functions and interactions of the protein molecules that make up functioning synapses. The aim is to determine the series of interactions between proteins that are responsible for the formation of a synapse. Information of this kind will be essential to future improvements in the management of neuromuscular diseases.
Interactions of postsynaptic proteins
Previous studies to clarify the roles of two skeletal muscle proteins in the formation of the postsynaptic membrane of the muscle cell showed that the 43k postsynaptic protein is expressed in the early stages of muscle differentiation and takes part in the incipient stages of postsynaptic differentiation. It induces the reorganization of acetylcholine receptors on the surface of muscle cells in culture into small membrane domains, probably by forming a physical link to the underlying skeleton of the cell. In addition, dystrophin-related protein (utrophin), may serve to stabilize these micro-clusters of acetylcholine receptor and 43k protein into large domains typical of the postsynaptic membrane of muscle cells.
In 1994 the formation of membrane domains by the 43k protein when it is introduced into fibroblasts was studied at a high resolution by confocal microscopy. These studies provided evidence that the formation of the postsynaptic membrane may involve several growth steps thatemploy first self-association of acetylcholine receptors followed by formation of larger membrane domains by 43k protein and finally the assembly of full-sized receptor clusters that involve members of the cytoskeleton such as utrophin. Furthermore, these studies revealed for the first time that the 43k protein can function to cluster another member of the family of ligand gated ion channels, the GABAA receptor, a major signalling protein in the central nervous system.
Receptors for the brain inhibitory neurotransmitter, gamma-aminobutyric acid are clustered by the acetylcholine receptor-associated 43k protein. Immunofluorescent staining revealed for the first time the ability of membrane domains formed by 43k (right hand panel) to organize a member of another class of ligand gated ion channels, the GABAA receptor (Left hand panel).
The latter result suggests that the 43k protein recognizes some common features of the two receptor types, and raises the important question of whether 43k or some homologue may be involved in synapse formation in the brain (see figure).
Work was begun on a new approach to studying the interactions of post-synaptic proteins. Elsewhere, the yeast two-hybrid system has been used to great effect to identify interactions between intracellular proteins in a range of systems and species. The Laboratory has begun to use this system to detect and define interactions between proteins that populate the cytoplasmic face of the postsynaptic membrane. It is hoped that this new and sensitive assay system will provide not only fine-grained information about structural interactions but also clues as to the mechanisms by which receptor density in the postsynaptic membrane may be regulated.
Differentiation of presynaptic membrane
A new area of research in 1994 related to the differentiation of the presynaptic membrane. The release of transmitter substances during synaptic signalling is believed to occur at circumscribed 'active zones' on the presynaptic membrane. Proteins such as syntaxin are thought to identify these active zones as sites for targeting of synaptic membrane vesicles. Little is currently known about how these membrane domains may become organized. To begin to study the process by which the presynaptic active zones are formed, the Laboratory raised antibodies against recombinant syntaxin. When expressed in fibroblasts, cloned syntaxin associated with the plasma membrane just as it does in neurons. Syntaxin was found to be localized at the neuromuscular junction, consistent with its hypothesized role in organizing transmitter secretion in these membrane domains.
The laboratory is located in rooms 277 and 278 of the Anderson Stuart Building. Dr Phillips' office is room 270c. The laboratory has recently been equipped with facilities for molecular cloning and DNA sequencing, sterile eukaryotic cell culture and autoradiography. The equipment includes apparatus for agarose and poly-acrylamide gel electrophoresis, electroblotting, electroporation of bacteria, a thermal cycler, bench- and micro-centrifuges, thermostatic baths, laminar air flow work cabinet, CO2 cell culture incubator, liquid nitrogen storage vessel, fine balances, pH meter, magnetic stirrer/hotplate, refrigerators, freezers, computers and electronic data storage facilities.
(Earlier publications arose from work at Washington Univ. School of Medicine, St. Louis, USA while those from Phillips et al. (1993) onwards were completed in Sydney after appointment in Nov. 1992)
MOST RECENT TOTAL ANNUAL CITATIONS (for 1993): 33
|ARC||Molecular manipulation of the
|NHMRC||The localization of acetylcholine
receptors on the postsynaptic membrane
of skeletal muscle
|NHMRC||Syncytial integration of autonomic
Total for 1993: $87,240
Total for 1994: $83,804
|Med 2||Pharm 1||Total|
|Practical classes (no.)||21||(7)||-||12||(4)||-||33|
Total formal contact teaching time = 87 h