title: CyrA, a matricellular protein that modulates cell motility in Dictyostelium discoideum.
abstract: CyrA, an extracellular matrix (slime sheath), calmodulin (CaM)-binding protein in Dictyostelium discoideum, possesses four tandem EGF-like repeats in its C-terminus and is proteolytically cleaved during asexual development. A previous study reported the expression and localization of CyrA cleavage products CyrA-C45 and CyrA-C40. In this study, an N-terminal antibody was produced that detected the full-length 63kDa protein (CyrA-C63). Western blot analyses showed that the intracellular expression of CyrA-C63 peaked between 12 and 16h of development, consistent with the time that cells are developing into a motile, multicellular slug. CyrA immunolocalization and CyrA-GFP showed that the protein localized to the endoplasmic reticulum, particularly its perinuclear component. CyrA-C63 secretion began shortly after the onset of starvation peaking between 8 and 16h of development. A pharmacological analysis showed that CyrA-C63 secretion was dependent on intracellular Ca(2+) release and active CaM, PI3K, and PLA2. CyrA-C63 bound to CaM both intra- and extracellularly and both proteins were detected in the slime sheath deposited by migrating slugs. In keeping with its purported function, CyrA-GFP over-expression enhanced cAMP-mediated chemotaxis and CyrA-C45 was detected in vinculin B (VinB)-GFP immunoprecipitates, thus providing a link between the increase in chemotaxis and a specific cytoskeletal component. Finally, DdEGFL1-FITC was detected on the membranes of cells capped with concanavalin A suggesting that a receptor exists for this peptide sequence. Together with previous studies, the data presented here suggests that CyrA is a bona fide matricellular protein in D. discoideum.
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON, Canada M5S 3G5. robert.huber@utoronto.ca
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Huber
authors.forename: Andres
authors.initials: A
authors.lastname: Suarez
authors.forename: Danton H
authors.initials: DH
authors.lastname: O'Day
mesh_terms: D000918:Antibody Specificity; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002633:Chemotaxis; D003208:Concanavalin A / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D004023:Dictyostelium / Q000502:physiology*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D047468:Immunoprecipitation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D015800:Protozoan Proteins / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D020539:Sequence Analysis, Protein; D015398:Signal Transduction; D013997:Time Factors; D016596:Vinculin / Q000378:metabolism
chemical_list: D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D016326:Extracellular Matrix Proteins; D015800:Protozoan Proteins; D011956:Receptors, Cell Surface; D003208:Concanavalin A; D016596:Vinculin; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP; D019869:Phosphatidylinositol 3-Kinases; D002118:Calcium