The role of the actin-binding domain in the intracellular localization of ABP-120 and alpha-actinin in Dictyostelium discoideum
Date of Completion
Biology, Molecular|Biology, Cell
ABP-120 and non-muscle α-actinin are members of a family of F-actin cross-linkers that are grouped based on the high degree of amino acid similarity of their actin-binding domains. Although these two proteins have been extensively characterized, little is known about whether they bind to different kinds of F-actin structures and what regulates their binding. GFP fusion proteins were constructed consisting of either the entire cross-linking protein or its actin-binding domain alone. The localization of these fluorescent proteins was examined in living cells under a variety of conditions known to involve actin. The localization patterns of ABP-120 (GFP120) and α-Actinin (GFPα-A) were overlapping but distinct. GFP120 localized to the cell cortex as well as to new pseudopods and to the cortex of polarized cells, but was observed to localize more to the rear of the cell during cAMP and under agarose folate chemotaxis. GFPα-A was enriched in new pseudopods and to the F-actin filled leading edge of polarized cells, but was absent from the general cell cortex. During under agarose folate chemotaxis GFPα-A overwhelmingly localized to the front of the cell. Although both proteins appear to be involved in macropinocytosis, association time with the internalized macropinosome differed. ^ In phagocytosis of yeast, both proteins localize to the phagocytic cup but differ in localization to the internalized particle. During capping, α-actinin associates with the cap, while ABP-120 is excluded. Surprisingly, the localization of the actin-binding domain GFP fusion protein precisely reflected that of their respective intact GFP fusion protein. When expressed in a cell line lacking both ABP-120 and α-actinin, not only did the probes maintain their localization observed in wild type cells, the defective development program was rescued in cells expressing complete GFP fusion protein probes, suggesting the functionality of the fusion protein probes. These observations strongly suggest a regulatory function present in the actin-binding domains of these proteins, which imparts specificity of binding to F-actin structures. ^
Washington, Raymond William, "The role of the actin-binding domain in the intracellular localization of ABP-120 and alpha-actinin in Dictyostelium discoideum" (2006). Doctoral Dissertations. AAI3217052.