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Analysis of protein interactions of a cytoplasmic dynein mutant which causes motor neuron death.

Reference	BB/D012309/1
Principal Investigator / Supervisor	Professor Majid Hafezparast
Co-Investigators / Co-Supervisors
Institution	University of Sussex
Department	Sch of Life Sciences
Funding type	Research
Value (£)	211,117
Status	Completed
Type	Research Grant
Start date	12/06/2006
End date	11/06/2009
Duration	36 months

Abstract

Motor neurons are highly specialised cells that control muscle movement. These neurons are dependent on the axonal transport system for communications between the cell body and axonal termini and disruption in this pathway could have severe effects on their survival. Cytoplasmic dynein is a microtubule associated motor protein complex involved in, among other cellular functions, transport of membranous and proteins from axons towards the cell body (retrograde transport). Using a positional cloning approach, I identified a point mutation that changes phenylalanine 580 to tyrosine in the N-terminal region of the heavy chain subunit (DNCHC1) of cytoplasmic dynein in a mutant mouse strain called legs at odd angles (Loa). In collaboration with others, I showed that this mutation causes motor neuron degeneration and impairs nerve growth and branching, but the underlying molecular mechanisms that lead to these defects are not known. The Loa mutation is in a region of DNCHC1 responsible for binding with other accessory proteins of the cytoplasmic dynein complex, including dynein intermediate chains (ICs). I propose that this mutation compromises the binding of one or more of these accessory proteins to DNCHC1, leading to the neurodegeneration and neurodevelopmental defects in Loa mice. The aim of this project is therefore to identify the protein(s) whose interaction with DNCHC1 is affected as a result of the Loa mutation. In order to address this we will use a targeted proteomics approach performing primarily overexpression studies on wild type and mutant DNCHC1 in cell culture, followed by co-immunoprecipitation and TAP-tag affinity purification of their dynein complexes. We will then use mass spectroscopy to identify the protein(s) whose interaction with the heavy chain has been disrupted in Loa.

Summary

Cytoplasmic dynein is a large protein complex consisting of several subunits. This protein is a molecular motor involved in a range of cellular processes including chromosome segregation during cell division and transport of organelles and trophic factors. Recently we identified a mutation in the heavy chain subunit of cytoplasmic dynein in a mouse mutant called legs at odd angles (Loa). This mutation causes death of a group of neurones called motor neurones, which control muscle movement [Hafezparast et al 2003, Science, 300:808]. The molecular mechanisms of motor neurone death caused by the mutation in dynein is not known. We hypothesise that the Loa mutation interrupts protein-protein interactions within the dynein complex. In order to test this hypothesis, we wish to utilise the Loa mouse as a model system and use a set of genetic, biochemical, and cell biological tools to determine these mechanisms. The data generated in this study will further our understanding of some aspects of axonal transport and its role in motor neuron maintenance and survival.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Neuroscience and Behaviour
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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