The Cyclase-associated Protein CAP as Regulator of Cell Polarity and cAMP Signaling inDictyostelium

Abstract

Cyclase-associated protein (CAP) is an evolutionarily conserved regulator of the G-actin/F-actin ratio and, in yeast, is involved in regulating the adenylyl cyclase activity. We show that cell polarization, F-actin organization, and phototaxis are altered in a Dictyostelium CAP knockout mutant. Furthermore, in complementation assays we determined the roles of the individual domains in signaling and regulation of the actin cytoskeleton. We studied in detail the adenylyl cyclase activity and found that the mutant cells have normal levels of the aggregation phase-specific adenylyl cyclase and that receptor-mediated activation is intact. However, cAMP relay that is responsible for the generation of propagating cAMP waves that control the chemotactic aggregation of starving Dictyostelium cells was altered, and the cAMP-induced cGMP production was significantly reduced. The data suggest an interaction of CAP with adenylyl cyclase in Dictyostelium and an influence on signaling pathways directly as well as through its function as a regulatory component of the cytoskeleton.

Ablation of cyclase-associated protein 2 (CAP2) leads to cardiomyopathy

“…WH2 motifs are intrinsically disordered, adopting an a-helical structure only upon binding to actin [11]. CAP in lower eukaryotes as well as mouse CAP1 are involved in cell polarity, motility, and receptor-mediated endocytosis [12][13][14]. Additionally, mammalian CAP1 was shown to be a proapoptotic protein [15].…”

Filamin-regulated F-actin Assembly Is Essential for Morphogenesis and Controls Phototaxis in Dictyostelium

“…Immunofluorescence methods are described in Ref. 12, and the nuclei were stained with 4,6-diamidino-2-phenylindole. Monoclonal antibodies recognizing ddFLN are described in Brink et al (21), the GFP-specific monoclonal antibody K3-184-2 is described in Ref.…”

“…Monoclonal antibodies recognizing ddFLN are described in Brink et al (21), the GFP-specific monoclonal antibody K3-184-2 is described in Ref. 12, and GFP-tagged actin for expression in D. discoideum was generated as in Ref. 45.…”

“…Escalante et al (9) reported that the BTB protein MigA is necessary for chemotaxis at the single cell level and for slug migration at the multicellular level. Several cytoskeleton-associated proteins such as GRP 125 (10) or villidin (11) and proteins involved in signal transduction and regulation of the cytoskeleton like CAP (12) are also involved in controlling phototactic slug behavior. Furthermore, cells lacking sphingosine-1-phosphate lyase, an enzyme that functions in fatty acid metabolism, showed extremely limited directional migration, and the slugs developed directly at the site of aggregation (13).…”

Depletion of Nesprin-2 is associated with an embryonic lethal phenotype in mice

“…Highly preabsorbed and affinity purified Alexa 488 and Alexa 568 conjugated secondary antibodies (Molecular Probes) were used in indirect immunofluorescence. 4,6-diamino-2-phenylindone (DAPI, Sigma) was used to visualize DNA [15,21,41,42]. Appropriate controls with secondary antibodies only were performed.…”