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EGF-like peptide-enhanced cell motility in Dictyostelium functions independently of the cAMP-mediated pathway and requires active Ca2+/calmodulin signaling
Authors:Huber Robert  O'Day Danton H
Institution:
  • a Department of Cell & Systems Biology, 25 Harbord Street, University of Toronto, Toronto, ON, Canada M5S 3G5
  • b Department of Biology, University of Toronto at Mississauga, 3359 Mississauga Road, Mississauga, ON, Canada L5L 1C6
  • Abstract:Current knowledge suggests that cell movement in the eukaryotic slime mold Dictyostelium discoideum is mediated by different signaling pathways involving a number of redundant components. Our previous research has identified a specific motility-enhancing function for epidermal growth factor-like (EGFL) repeats in Dictyostelium, specifically for the EGFL repeats of cyrA, a matricellular, calmodulin (CaM)-binding protein in Dictyostelium. Using mutants of cAMP signaling (carA, carC, gpaB, gpbA), the endogenous calcium (Ca2+) release inhibitor TMB-8, the CaM antagonist W-7, and a radial motility bioassay, we show that DdEGFL1, a synthetic peptide whose sequence is obtained from the first EGFL repeat of cyrA, functions independently of the cAMP-mediated signaling pathways to enhance cell motility through a mechanism involving Ca2+ signaling, CaM, and RasG. We show that DdEGFL1 increases the amounts of polymeric myosin II heavy chain and actin in the cytoskeleton by 24.1 ± 10.7% and 25.9 ± 2.1% respectively and demonstrate a link between Ca2+/CaM signaling and cytoskeletal dynamics. Finally, our findings suggest that carA and carC mediate a brake mechanism during chemotaxis since DdEGFL1 enhanced the movement of carA/carC cells by 844 ± 136% compared to only 106 ± 6% for parental DH1 cells. Based on our data, this signaling pathway also appears to involve the G-protein β subunit, RasC, RasGEFA, and protein kinase B. Together, our research provides insight into the functionality of EGFL repeats in Dictyostelium and the signaling pathways regulating cell movement in this model organism. It also identifies several mechanistic components of DdEGFL1-enhanced cell movement, which may ultimately provide a model system for understanding EGFL repeat function in higher organisms.
    Keywords:EGFL  Epidermal growth factor-like  CaM  Calmodulin  CaMBP  Calmodulin-binding protein  Ca2+  Calcium  MHC  Myosin II heavy chain  PKB  Protein kinase B  GEF  Guanine nucleotide exchange factor  GAP  GTPase-activating protein  PI3K  Phosphatidylinositol 3-kinase  PLA2  Phospholipase A2  cAMP  3&prime  -5&prime  -cyclic adenosine monophosphate  car  cAMP receptor  IP3  Inositol 1  4  5-trisphosphate  GDP  Guanine diphosphate  GTP  Guanine triphosphate  SDS-PAGE  Sodium dodecyl sulfate polyacrylamide gel Electrophoresis
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