title: Green fluorescent protein-mTalin causes defects in actin organization and cell expansion in Arabidopsis and inhibits actin depolymerizing factor's actin depolymerizing activity in vitro.
abstract: Expression of green fluorescent protein (GFP) linked to an actin binding domain is a commonly used method for live cell imaging of the actin cytoskeleton. One of these chimeric proteins is GFP-mTalin (GFP fused to the actin binding domain of mouse talin). Although it has been demonstrated that GFP-mTalin colocalizes with the actin cytoskeleton, its effect on actin dynamics and cell expansion has not been studied in detail. We created Arabidopsis (Arabidopsis thaliana) plants harboring alcohol inducible GFP-mTalin constructs to assess the effect of GFP-mTalin expression in vivo. We focused on the growing root hair as this is a model cell for studying cell expansion and root hair tip growth that requires a highly dynamic and polar actin cytoskeleton. We show that alcohol inducible expression of GFP-mTalin in root hairs causes severe defects in actin organization, resulting in either the termination of growth, cell death, and/or changes in cell shape. Fluorescence recovery after photobleaching experiments demonstrate that the interaction of GFP-mTalin and actin filaments is highly dynamic. To assess how GFP-mTalin affects actin dynamics we performed cosedimentation assays of GFP-mTalin with actin on its own or in the presence of the actin modulating protein, actin depolymerizing factor. We show that that GFP-mTalin does not affect actin polymerization but that it does inhibit the actin depolymerizing activity of actin depolymerizing factor. These observations demonstrate that GFP-mTalin can affect cell expansion, actin organization, and the interaction of actin binding proteins with actin.
authors.affiliation: The Integrative Cell Biology Laboratory, School of Biological and Biomedical Sciences, University of Durham, Durham, DH1 3LE, United Kingdom.
authors.forename: Tijs
authors.initials: T
authors.lastname: Ketelaar
authors.forename: Richard G
authors.initials: RG
authors.lastname: Anthony
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Hussey
mesh_terms: D051339:Actin Depolymerizing Factors; D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D017360:Arabidopsis / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D049108:Cell Enlargement; D003599:Cytoskeleton / Q000737:chemistry / Q000378:metabolism; D051345:Destrin; D018506:Gene Expression Regulation, Plant; D049452:Green Fluorescent Proteins; D008840:Microfilament Proteins / Q000378:metabolism*; D018517:Plant Roots / Q000254:growth & development / Q000648:ultrastructure; D030821:Plants, Genetically Modified; D016608:Talin / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
chemical_list: D051339:Actin Depolymerizing Factors; D000199:Actins; D051345:Destrin; D008840:Microfilament Proteins; D016608:Talin; C496077:Tln1 protein, mouse; D049452:Green Fluorescent Proteins