INTERACTIONS OF MICROTUBULE-ASSOCIATED PROTEIN MAP2 WITH UNPOLYMERIZED AND POLYMERIZED TUBULIN AND ACTIN USING A 96-WELL MICROTITER PLATE SOLID-PHASE IMMUNOASSAY

A solid-phase immunoassay is used to study the protein-protein interac tions between microtubule-associated protein MAP2 and the cytoskeletal proteins tubulin and actin. The assay can be performed on 96-well mic rotiter plates and can be used to study the interactions with both sub unit proteins and their respective polymers, microtubules and microfil aments. The microtiter format allows a large number of samples to be p rocessed, and a number of conditions can be varied. In this solid-phas e immunoassay MAP2 bound to microtubules/microfilaments and tubulin di mers/G-actin in a concentration-dependent manner. However, the bound M AP2 was not dissociated from the filaments even at high NaCl concentra tions, while simultaneous addition of NaCl diminished MAP2 binding to these proteins. MgCl2 was 1 order of magnitude more efficient in decre asing MAP2 binding compared with NaCl, suggesting that MAP2 may act by ''screening'' the electrostatic repulsion between tubulin dimers. The role of MAP2 in cross-linking microfilaments and microtubules was als o examined. Microtubule/tubulin-bound MAP2 showed a diminished ability to bind to both microfilaments and G-actin, while microfilament/G-act in-bound MAP2 was able to bind efficiently to both microtubules and tu bulin dimers. These differences in MAP2 behavior, depending on the ini tial binding partner, may be physiologically important in the cellular coordination of filament distribution. Although the solid-phase assay has been used to study MAP2 interactions, it is felt that the assay c ould be generally applied to other MAPs.