Chapter 11 Isolation of the Actin Cytoskeleton from Amoeboid Cells of Dictyostelium

Abstract

This chapter describes methods for the isolation of the actin cytoskeleton of Dictyostelium using sea urchin eggs. Those studies involved the isolation of a low-speed sedimentable actin fraction from unfertilized and fertilized sea urchin eggs to study changes in assembly states of the actin on fertilization. A number of similar procedures for isolation of cytoskeletal preparations from Dictyostelium are developed by other investigators to study cytoskeleton-associated proteins. The use of isolated cortices for ultrastructural localization of proteins with gold probes is described. A detergent-insoluble membrane fragment associated with the filamentous actin matrix isolated from Dictyostelium cells has been used to identify a group of integral membrane proteins that have the capacity to bind actin. A pool of folate receptors on vegetative cells that are associated with the detergent-insoluble cytoskeleton is identified. The association of the receptor with the cytoskeleton is maintained for up to 12 hours of development. It is shown that the amount of myosin in a Triton-resistant low-speed pellet of developed Dictyostelium cells increases significantly as the cell responds to the chemoattractant cyclic adenosine monophosphate (AMP). It is also shown that the kinase activity responsible for a chemoattractant-elicited increase in myosin heavy-chain phosphorylation is in the detergent insoluble pellet. Data presented suggest that the changes in the composition and organization of the isolated cytoskeleton can reflect physiological activities of the cell. The isolated cytoskeleton should be useful as a starting material for studying the modulation of proteins that are regulated during motile events.