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A novel component of the axonal cortical cytoskeleton, A60, defined by a monoclonal antibody
1Biological Laboratory, University of Kent, Canterbury, UK.
Insights
A novel Mr 60,000 protein, designated A60, has been identified in the axonal cytoskeleton. This protein is a peripheral membrane component primarily located in axons and distinct from known neurofilament proteins.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- The axonal cytoskeleton plays a crucial role in neuronal structure and function.
- Novel proteins associated with the axonal cortex are continually being discovered.
- Understanding these components is key to deciphering neuronal organization.
Purpose of the Study:
- To describe a novel Mr 60,000 protein recognized by the monoclonal antibody DR1.
- To characterize the localization and properties of this protein within the axon.
- To determine its relationship with known cytoskeletal elements, particularly neurofilaments.
Main Methods:
- Production of monoclonal antibody DR1 using bovine brain membrane extracts.
- Immunofluorescence microscopy to determine protein localization.
- Subcellular fractionation and biochemical extraction to define protein properties.
- Copurification studies with neurofilaments.
Main Results:
- A Mr 60,000 protein (A60) was identified and localized primarily to the axonal cortex, lining the axolemma.
- A60 behaves as a cytoskeleton-associated peripheral membrane protein, requiring harsh conditions for solubilization.
- The protein is more abundant in the central nervous system than in peripheral nerves.
- A60 copurifies with neurofilaments but does not react with anti-intermediate filament antibodies, suggesting it's not an intermediate filament protein.
Conclusions:
- A60 is a previously unrecognized component of the axonal cortical cytoskeleton.
- Its distinct biochemical properties and association with neurofilaments suggest a unique role in axonal structure.
- Further research into A60 could reveal new insights into axonal organization and neurodegenerative diseases.
Abstract:
A Mr 60,000 protein of the axonal cortical cytoplasm, which is recognized by a novel monoclonal antibody, is described. The antibody, DR1, was produced by immunizing mice with a soluble extract of bovine brain membranes that is enriched in known membrane cytoskeletal proteins. DR1 recognizes a Mr 60,000 protein in this extract. Immunofluorescence and subcellular fractionation reveal that the protein is primarily located in axons, where it appears to form a thick lining to the axolemma. Operationally, this Mr 60,000 protein is defined as a cytoskeleton-associated peripheral membrane protein. It is solubilized from brain membranes only under harsh conditions (0.1 M-NaOH), but not with KI (0.8 M) or Triton X-100 (1%). It is present at higher levels in the central nervous system than in peripheral nerves that have been examined. The Mr 60,000 protein copurifies with neurofilaments through cycles of assembly and disassembly. It does not appear to react with the anti-IFA antibody, suggesting that it is not a member of the intermediate filament class of proteins. This Mr 60,000 protein, which we designate A60, is distinct from other known neurofilament-associated proteins, including the Mr 60,000 protein alpha-internexin and the Mr 58,000 intermediate filament protein peripherin. A60 is suggested as being a previously unrecognized component of the axonal cortical cytoskeleton.
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