title: Stoichiometry of Nck-dependent actin polymerization in living cells.
abstract: Regulation of actin dynamics through the Nck/N-WASp (neural Wiskott-Aldrich syndrome protein)/Arp2/3 pathway is essential for organogenesis, cell invasiveness, and pathogen infection. Although many of the proteins involved in this pathway are known, the detailed mechanism by which it functions remains undetermined. To examine the signaling mechanism, we used a two-pronged strategy involving computational modeling and quantitative experimentation. We developed predictions for Nck-dependent actin polymerization using the Virtual Cell software system. In addition, we used antibody-induced aggregation of membrane-targeted Nck SH3 domains to test these predictions and to determine how the number of molecules in Nck aggregates and the density of aggregates affected localized actin polymerization in living cells. Our results indicate that the density of Nck molecules in aggregates is a critical determinant of actin polymerization. Furthermore, results from both computational simulations and experimentation support a model in which the Nck/N-WASp/Arp2/3 stoichiometry is 4:2:1. These results provide new insight into activities involving localized actin polymerization, including tumor cell invasion, microbial pathogenesis, and T cell activation.
authors.affiliation: Department of Genetics and Developmental Biology, Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, and Richard D Berlin Center for Cell Analysis & Modeling, University of Connecticut Health Center, Farmington, CT 06030, USA.
authors.forename: Jonathon A
authors.initials: JA
authors.lastname: Ditlev
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Michalski
authors.forename: Greg
authors.initials: G
authors.lastname: Huber
authors.forename: Gonzalo M
authors.initials: GM
authors.lastname: Rivera
authors.forename: William A
authors.initials: WA
authors.lastname: Mohler
authors.forename: Leslie M
authors.initials: LM
authors.lastname: Loew
authors.forename: Bruce J
authors.initials: BJ
authors.lastname: Mayer
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D002470:Cell Survival; D003198:Computer Simulation; D057809:HEK293 Cells; D006801:Humans; D015513:Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D058105:Polymerization; D015398:Signal Transduction; D018909:src Homology Domains
chemical_list: D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; C078332:Nck protein; D015513:Oncogene Proteins