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Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks
Published on: December 24, 2016
Localization of tau and other proteins of isolated marginal bands
Abstract:
To determine which proteins were associated with and intrinsic to the marginal band (MB) of microtubules (MTs), we studied protein components of MBs isolated from nucleated erythrocytes by differential detergent solubilization of the membrane skeleton (MS). MBs isolated from dogfish erythrocytes contained major proteins in the tubulin M(r) range. A high molecular weight protein of approximately 290 kD that bound antibody to syncolin and to heat-stable brain MAPs was present in the whole cytoskeleton. However, most of it was solubilized by the MB isolation medium, together with the MS. Dogfish erythrocyte cytoskeletons and isolated MBs were examined with polyclonal and monoclonal antibodies against mammalian brain tau and chicken erythrocyte tau. As shown by immunofluorescence and immunoblotting, these antibodies bound to proteins in the 50 to 67 kD range, located along the length of isolated MBs. Two-dimensional SDS-PAGE revealed isolated MB proteins of pI approximately 6.8 in the same molecular weight range, as well as alpha- and beta-tubulin with pI approximately 5.4. Subtilisin or high-salt treatment of isolated MBs resulted in unbundling of MTs, indicating involvement of MAPs. MBs isolated from chicken erythrocyte cytoskeletons also contained tau as shown by anti-mammalian brain tau immunofluorescence. Both chicken and dogfish isolated MBs also bound phalloidin, but the binding was usually discontinuous and, for any given MB, matched the pattern of anti-syncolin binding. Both syncolin and F-actin were part of the MS remnant remaining after MT disassembly, supporting their assignment to a specialized MS region at the MB/MS interface. In contrast, tau protein appears to be intrinsic to the MB, where it may have an MT stabilizing and bundling function.
Insights
This study identifies tau protein as intrinsic to the marginal band (MB) of microtubules in erythrocytes, suggesting a role in stabilizing and bundling these structures. Other proteins like syncolin and F-actin are associated with the membrane skeleton.
Area of Science:
- Cell Biology
- Cytoskeleton Research
- Protein Biochemistry
Background:
- The marginal band (MB) of microtubules (MTs) is a crucial cytoskeletal structure in nucleated erythrocytes.
- Understanding the protein composition of the MB is essential for elucidating its function.
- Previous studies have focused on tubulin, but other associated proteins remain less characterized.
Purpose of the Study:
- To identify proteins associated with and intrinsic to the marginal band (MB) of microtubules (MTs).
- To investigate the role of microtubule-associated proteins (MAPs) in MB structure and stability.
- To differentiate between MB-intrinsic proteins and those associated with the membrane skeleton (MS).
Main Methods:
- Differential detergent solubilization of membrane skeleton (MS) from nucleated erythrocytes.
- Isolation and biochemical analysis of MBs from dogfish and chicken erythrocytes.
- Immunofluorescence and immunoblotting using antibodies against tau, syncolin, and F-actin.
- Two-dimensional SDS-PAGE to analyze protein composition.
- Enzymatic (Subtilisin) and high-salt treatments to assess protein involvement.
Main Results:
- MBs contain major proteins in the tubulin molecular weight (M(r)) range.
- Tau protein (50-67 kD) was identified along the length of isolated MBs in both dogfish and chicken erythrocytes, indicating it is intrinsic.
- Syncolin and F-actin were localized to the membrane skeleton (MS) remnant, not the MB itself.
- Microtubule-associated proteins (MAPs) are involved in MB structure, as indicated by unbundling after treatment.
Conclusions:
- Tau protein is an intrinsic component of the erythrocyte marginal band (MB).
- Tau protein likely functions in stabilizing and bundling microtubules within the MB.
- Syncolin and F-actin are associated with the membrane skeleton (MS) at the MB/MS interface, not the MB itself.
