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High molecular weight protein MAP 2 promoting microtubule assembly in vitro is associated with microtubules in cells
Abstract:
Brain high molecular weight (HMW) protein promoting microtubule assembly in vitro and identical to MAP 2 (one of the proteins which copurify with tubulin through microtubule assembly-disassembly cycles), is shown to be associated with microtubules in interphase and mitotic cells. This HMW protein was purified earlier (Kuznetsov et al., 1978), directly from bovine brain without previous obtaining total microtubule protein. Now we have obtained a monospecific antibody against it. Identity of the HMW protein with MAP 2 is inferred from SDS-electrophoresis and immunological tests; its intracellular localization is determined by indirect immunofluorescent staining of cultured bovine tracheal epithelium. the anti-MAP 2 antibody stains the same structures in the cells as the tubulin antibody: it stains the fibrillar network in interphase cells, mitotic spindle, and the stem body. No fibrillar structures in the cells treated with colchicine or vinblastine were stained with the antibody against MAP 2. Anti-MAP 2 also stains tubulin-containing paracrystals which have been formed in the vinblastine-treated cells. Therefore HMW protein MAP 2 which promotes tubulin polymerization in vitro is associated with microtubules in vivo.
Insights
High molecular weight (HMW) protein, identified as Microtubule-Associated Protein 2 (MAP 2), promotes microtubule assembly. This MAP 2 protein is closely associated with cellular microtubules in both interphase and mitotic cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Microtubules are essential cytoskeletal components involved in various cellular processes.
- Microtubule-Associated Protein 2 (MAP 2) is a high molecular weight (HMW) protein known to copurify with tubulin.
- The in vivo association of MAP 2 with microtubules requires further investigation.
Purpose of the Study:
- To confirm the identity of a purified bovine brain HMW protein with MAP 2.
- To investigate the intracellular localization and association of MAP 2 with microtubules in vivo.
- To elucidate the role of MAP 2 in microtubule dynamics.
Main Methods:
- Purification of HMW protein from bovine brain.
- Generation of a monospecific antibody against the HMW protein.
- SDS-electrophoresis and immunological tests for protein identification.
- Indirect immunofluorescent staining of cultured bovine tracheal epithelial cells.
- Treatment of cells with colchicine and vinblastine to disrupt microtubules.
Main Results:
- The purified HMW protein was confirmed to be identical to MAP 2 through SDS-electrophoresis and immunological assays.
- Anti-MAP 2 antibody staining patterns mirrored tubulin antibody staining in interphase and mitotic cells.
- MAP 2 was localized to the fibrillar network in interphase cells, the mitotic spindle, and the stem body.
- Disruption of microtubules with colchicine or vinblastine abolished MAP 2 staining of fibrillar structures.
- Anti-MAP 2 antibody also stained tubulin-containing paracrystals in vinblastine-treated cells.
Conclusions:
- The HMW protein MAP 2 is intrinsically associated with cellular microtubules in vivo.
- MAP 2 plays a significant role in stabilizing microtubules and influencing their assembly.
- These findings enhance our understanding of microtubule organization and function within the cell.