GENETIC DELETION OF ABP-120 ALTERS THE 3-DIMENSIONAL ORGANIZATION OF ACTIN-FILAMENTS IN DICTYOSTELIUM PSEUDOPODS

This study extends the observations on the defects in pseudopod formation of ABP-120(+) and ABP-120(-) cells by a detailed morphological and biochemical analysis of the actin based cytoskeleton. Both ABP-120(+) and ABP-120(-) cells polymerize the same amount of F-actin in response to stimulation with cAMP However, unlike ABP-120(+) cells, ABP-120(-) cells do not incorporate actin into the Triton X-100-insoluble cytoskeleton at 30-50 s, the time when ABP-120 is incorporated into the cytoskeleton and when pseudopods are extended after cAMP stimulation in wild-type cells. By confocal and electron microscopy, pseudopods extended by ABP-120(-) cells are not as large or thick as those produced by ABP-120(+) cells and in the electron microscope, an altered filament network is found in pseudopods of ABP-120(+) cells when compared to pseudopods of ABP-120(-) cells. The actin filaments found in areas of pseudopods in ABP-120(+) cells either before or after stimulation were long, straight, and arranged into space filling orthogonal networks. Protrusions of ABP-120(-) cells are less three-dimensional, denser, and filled with multiple foci of aggregated filaments consistent with collapse of the filament network due to the absence of ABP-120-mediated cross-linking activity. The different organization of actin filaments may account for the diminished size of protrusions observed in living and fixed ABP- 120(-) cells compared to ABP-120(+) cells and is consistent with the role of ABP-120 in regulating pseudopod extension through its crosslinking of actin filaments.