MOLECULAR NEUROSCIENCE LABORATORY

William D. Phillips

Improving therapies for neuromuscular disorders demands a more fundamental understanding of the precise molecular interactions that go towards build up a functioning synapse. The aim of this laboratory is to employ techniques of molecular biology and cell biology to study the function of proteins involved in the formation and physiological function of synapses.

RESEARCH in 1993

During 1993, collaborative work was completed and published that helped to clarify the roles of two skeletal muscle proteins in the formation of the postsynaptic membrane of the muscle cell. These studies 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. The 43k postsynaptic protein was then shown to induce 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. The latter study also showed that another protein, dystrophin-related protein, may serve to stabilize these micro-clusters of acetylcholine receptor and 43k protein into large domains typical of the postsynaptic membrane of muscle cells.

Large domains formed on the plasma membrane of fibroblasts in culture by the 43k protein. In panel A, imunofluorescent staining reveals the distribution of 43k protein in the membrane. Panel B shows the distribution of acetylcholine receptors that have become immobilized and physically linked to the cytoskeleton. In muscle cells the immobilization of receptors at the synapse is essential for synaptic function. The mechanism by which 43k forms these membrane domains is currently being investigated.

RESEARCH PLANNED for 1994

Analysis of the mechanism of 43k-mediated postsynaptic membrane differentiat-ion will continue with site-directed mutagenesis being used to identify parts of the polypeptide sequence that may be responsible for the ability of 43k protein to interact with the plasma membrane, the cytoskeleton and the acetylcholine receptor. Using this approach the Laboratory will seek to unravel the series of molecular interactions that leads to the aggregation of the acetylcholine receptors in the postsynaptic membrane. Double label immunofluorescence followed by confocal microscopy will then be used for the first time to produce a fine resolution quantitative description of the interaction of the recombinant synaptic proteins at the level of the lipid bilayer. In 1994 the laboratory will also begin to investigate the differentiation of the presynaptic transmitter release site membrane. In particular, the cell biology of the presynaptic membrane protein, syntaxin, will be investigated. Syntaxin has been implicated in targeting of synaptic vesicles to the inner face of the presynaptic membrane and has been shown to interact with other important presynaptic proteins in ways that have yet to be clearly defined. Antibodies to recombinant syntaxin A have already been raised in the Laboratory and will be used to study the spatial distribution of the protein at mature and developing synapses. The interaction of the recombinant syntaxin protein with the plasma membrane will be studied by introducing it into fibroblast cell lines.

PERSONNEL

Dr William D. Phillips Lecturer (in-charge) University Nov. 1992-

Azita Ahadizadeh Research Assistant (0.5) NHMRC 1994-

PhD student (part-time)

Jeon Cha BSc(Hons) student 1994

Current effective full-time personnel = 3.0

FACILITIES

The laboratory is located in rooms 277 and 278 of the Anderson Stuart Building. The office of Dr Phillips in 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 polyacrylamide gel electrophoresis and electroblotting and electroporation of DNA and proteins, 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 and computers.

FUNDING in 1993 and 1994

ARC Molecular manipulation of the Phillips WD 1993 $10,000

postsynaptic membrane 1994 $5,000

Ramaciotti Molecular manipulation of the Phillips WD 1993 $15,000

postsynaptic membrane

NHMRC The localisation of acetylcholine Phillips WD 1994 $47,000

membrane receptors on the

postsynaptic of skeletal muscle

NHMRC Syncytial integration of autonomic Bennett MR 1994 $35,000

transmission Phillips WD

Gibson W

Total for 1993: $25,000

Total for 1994: $87,000

TEACHING in 1993

BMedSc 3/Science 3

Lectures: 6, new, on cellular physiology

Practical classes: 1, of 3 h, repeated, on neuromuscular transmission

Tutorials: 7, each of 2 h, involving research paper discussions

Examination: short essay questions in a written exam plus a longer essay written in the student's own time

Dentistry 2, Science 2 and Science Auxiliary

These different students were taught concurrently

Lectures: 7, new, on cellular neurophysiology

Examination: exam consisting of multiple choice questions and/or short answer questions.

Medicine 2

Tutorials: 1, repeated 6 times, on nerve electrophysiology

Practical class: 1, of 3 h, on electrophysiology repeated four times

Pharmacy 1

Lectures: 6, new, on nervous system and neurophysiology

Examination: multiple choice questions (of the 5 alternatives, one correct variety).

Total distribution (hours of formal teaching)

Sci 3 Dent/Sc2ScAux Med2 Pha1 Total

Lectures 6 7 - 6 19

Practical classes (no.) 20(9) - 12(4) - 32

Tutorials - - 6 - 6

Total formal contact teaching time = 57 h.

OTHER ACTIVITIES in 1993

Seminars and invited talks

Australian Neuroscience Society Annual Meeting, Melbourne (month?)

Dept of Anatomy, Univ. of N.S.W. (month?)

Children's Medical Research Institute, Westmead (month?)

Anderson Stuart Seminar Series (month?)

Australian Neuroscience Colloquium, Sydney (month?)

Organizer for:

Anderson Stuart Seminar Series (1993-present)

Poster session for the 1994 Australian Neuroscience Society Scientific Meeting (month?)

5-YEAR RESEARCH PUBLICATIONS

(The following arose from work at The University of Sydney [1989] and Washington University School of Medicine, St. Louis, USA [1991-92]; publication marked * was completed in Sydney after appointment)

JOURNAL ARTICLES

1989

Phillips WD, Bennett MR (1989) The distribution of intracellular acetylcholine receptors and nuclei in developing avian fast-twitch muscle fibres during synapse elimination. Journal of Neurocytology, 18, 241-255

1991

Phillips WD, Kopta C, Blount P, Gardner PD, Steinbach JH, Merlie JP (1991) ACh receptor-rich membrane domains organized in fibroblasts by recombinant 43-kilodalton protein. Science, 251, 568-570

Phillips WD, Maimone M, Merlie JP (1991) Mutagenesis of the 43-kD postsynaptic protein defines domains involved in plasma membrane targeting and AChR clustering. Journal of Cell Biology, 115, 1713-1724

1992

Phillips WD, Merlie JP (1992) Recombinant neuromuscular synapses. Bioessays, 14, 671-679

1993

Noakes PG, Phillips WD, Hanley TA, Sanes JR, Merlie JP (1993) 43k protein and acetylcholine receptors co-localize during the initial stages of neuromuscular synapse formation in vivo. Developmental Biology, 155, 275-280

Phillips WD, Noakes PG, Roberds SL, Campbell KP, and Merlie JP (1993) Clustering and immobilization of acetylcholine receptors by the 43-kD protein: a possible role for dystrophin-related protein (DRP). Journal of Cell Biology, 123, 729-740 *

CONFERENCE ABSTRACT AND PRESENTATION in 1993

Phillips WD, Merlie JP (1993) 43k, a molecular organiser of the postsynaptic membrane. Proceedings of the Australian Neuroscience Society, 4, 71. City? (month?)