title: Importance of Interaction between Integrin and Actin Cytoskeleton in Suspension Adaptation of CHO cells.
abstract: The biopharmaceutical production process relies upon mammalian cell technology where single cells proliferate in suspension in a chemically defined synthetic environment. This environment lacks exogenous growth factors, usually contributing to proliferation of fibroblastic cell types such as Chinese hamster ovary (CHO) cells. Use of CHO cells for production hence requires a lengthy 'adaptation' process to select clones capable of proliferation as single cells in suspension. The underlying molecular changes permitting proliferation in suspension are not known. Comparison of the non-suspension-adapted clone CHO-AD and a suspension-adapted propriety cell line CHO-SA by flow cytometric analysis revealed a highly variable bi-modal expression pattern for cell-to-cell contact proteins in contrast to the expression pattern seen for integrins. Those have a uni-modal expression on suspension and adherent cells. Integrins showed a conformation distinguished by regularly distributed clusters forming a sphere on the cell membrane of suspension-adapted cells. Actin cytoskeleton analysis revealed reorganisation from the typical fibrillar morphology found in adherent cells to an enforced spherical subcortical actin sheath in suspension cells. The uni-modal expression and specific clustering of integrins could be confirmed for CHO-S, another suspension cell line. Cytochalasin D treatment resulted in breakdown of the actin sheath and the sphere-like integrin conformation demonstrating the link between integrins and actin in suspension-adapted CHO cells. The data demonstrates the importance of signalling changes, leading to an integrin rearrangement on the cell surface, and the necessity of the reinforcement of the actin cytoskeleton for proliferation in suspension conditions.
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK. c.walther@sheffield.ac.uk.|Chemical and Biological Engineering, University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield, S1 3JD, UK. c.walther@sheffield.ac.uk.
authors.forename: Christa G
authors.identifier: http://orcid.org/0000-0002-8962-3102
authors.initials: CG
authors.lastname: Walther
authors.affiliation: Department of Applied Sciences and Health, Coventry University, James Starley Building, Coventry, CV1 5FB, UK.|Chemical and Biological Engineering, University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield, S1 3JD, UK.
authors.forename: Robert
authors.initials: R
authors.lastname: Whitfield
authors.affiliation: Chemical and Biological Engineering, University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield, S1 3JD, UK.
authors.forename: David C
authors.initials: DC
authors.lastname: James
mesh_terms: D008841:Actin Cytoskeleton / Q000235:genetics*; D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion / Q000235:genetics; D018929:Cell Culture Techniques / Q000379:methods*; D002462:Cell Membrane / Q000235:genetics; D049109:Cell Proliferation / Q000235:genetics*; D006224:Cricetinae; D003412:Cricetulus; D005109:Extracellular Matrix / Q000235:genetics; D016023:Integrins / Q000096:biosynthesis* / Q000235:genetics; D059010:Single-Cell Analysis
chemical_list: D016023:Integrins
keywords: Actin cytoskeleton; Cell surface proteins; Cell-to-extracellular matrix interaction; Chinese hamster ovary cells; Integrin signalling; Suspension adaptation