Genetic deletion of ABP-120 alters the three-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 cross-linking of actin filaments.
Cox
D
D
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
Ridsdale
J A
JA
Condeelis
J
J
Hartwig
J
J
D008841:Actin Cytoskeleton / Q000648:ultrastructure*; D000199:Actins / Q000378:metabolism; D000546:Amanitins / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics*; D000242:Cyclic AMP / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure; D004023:Dictyostelium / Q000187:drug effects / Q000235:genetics / Q000648:ultrastructure*; D017353:Gene Deletion; D008840:Microfilament Proteins / Q000235:genetics*; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D010586:Phagocytes / Q000648:ultrastructure; D011092:Polyethylene Glycols / Q000494:pharmacology; D011554:Pseudopodia / Q000648:ultrastructure*
D000199:Actins; D000546:Amanitins; D002352:Carrier Proteins; D008840:Microfilament Proteins; C060680:abpC protein, Dictyostelium; D011092:Polyethylene Glycols; C028877:7-nitrobenz-2-oxa-1,3-diazole-phallacidin; D000242:Cyclic AMP