SEQUENCES OF ACTIN IMPLICATED IN THE POLYMERIZATION PROCESS - A SIMPLIFIED MATHEMATICAL APPROACH TO PROBE THE ROLE OF THESE SEGMENTS

Regulation of actin polymerization and depolymerization is essential f or the functions of actin in non-muscle cells and is mediated by a lar ge number of heterologous actin-binding proteins which questions their true impact on the polymerization process. As a model, we report here the modulating effect of monospecific antibody fragments (Fab) as in vitro effecters on actin polymerization kinetics. Polymerization curve s were obtained through fluorescence measurements. They were fitted us ing analytical equations derived from classical models describing the actin polymerization process with the aim of identifying kinetic steps potentially altered by the effecters. The study was limited to three short segments bore by the 300-328 sequence which is located in actin subdomain 3 and implicated in one of the monomer-monomer interfaces. W e observed that antibodies which inhibited actin polymerization reacte d with both G-and F-actins, modulated both nucleation and elongation s teps, enhanced actin monomer dissociation from the filament and appare ntly did not act as capping or sequestering proteins. Among the antibo dy populations specific for a restricted and selected sequence in subd omain 3 of actin (sequence 300-326), only those directed to epitopes l ocated near Met 305 and 325 were effective. In contrast, antibodies di rected towards the a-helix located between the two preceding epitopes had no effect. All the results analyzed here emphasize the important r ole of some discrete regions and their conformational state in regulat ion of the interconversion between monomeric and polymeric actins whic h could be controlled in different ways by the various actin-binding p roteins.