title: Live cell imaging reveals actin-cytoskeleton-induced self-association of the actin-bundling protein WLIM1.
abstract: Crosslinking of actin filaments into bundles is essential for the assembly and stabilization of specific cytoskeletal structures. However, relatively little is known about the molecular mechanisms underlying actin bundle formation. The two LIM-domain-containing proteins define a novel and evolutionarily conserved family of actin-bundling proteins whose actin-binding and -crosslinking activities primarily rely on their LIM domains. Using TIRF microscopy, we describe real-time formation of actin bundles induced by tobacco NtWLIM1 in vitro. We show that NtWLIM1 binds to single filaments and subsequently promotes their interaction and zippering into tight bundles of mixed polarity. NtWLIM1-induced bundles grew by both elongation of internal filaments and addition of preformed fragments at their extremities. Importantly, these data are highly consistent with the modes of bundle formation and growth observed in transgenic Arabidopsis plants expressing a GFP-fused Arabidopsis AtWLIM1 protein. Using two complementary live cell imaging approaches, a close relationship between NtWLIM1 subcellular localization and self-association was established. Indeed, both BiFC and FLIM-FRET data revealed that, although unstable NtWLIM1 complexes can sporadically form in the cytosol, stable complexes concentrate along the actin cytoskeleton. Remarkably, disruption of the actin cytoskeleton significantly impaired self-association of NtWLIM1. In addition, biochemical analyses support the idea that F-actin facilitates the switch of purified recombinant NtWLIM1 from a monomeric to a di- or oligomeric state. On the basis of our data, we propose a model in which actin binding promotes the formation and stabilization of NtWLIM1 complexes, which in turn might drive the crosslinking of actin filaments.
authors.affiliation: Centre de Recherche Public-Santé, 84 Val Fleuri, L-1526 Luxembourg, Luxembourg.
authors.forename: Céline
authors.initials: C
authors.lastname: Hoffmann
authors.forename: Danièle
authors.initials: D
authors.lastname: Moes
authors.forename: Monika
authors.initials: M
authors.lastname: Dieterle
authors.forename: Katrin
authors.initials: K
authors.lastname: Neumann
authors.forename: Flora
authors.initials: F
authors.lastname: Moreau
authors.forename: Angela
authors.initials: A
authors.lastname: Tavares Furtado
authors.forename: Dominique
authors.initials: D
authors.lastname: Dumas
authors.forename: André
authors.initials: A
authors.lastname: Steinmetz
authors.forename: Clément
authors.initials: C
authors.lastname: Thomas
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure*; D017360:Arabidopsis / Q000378:metabolism / Q000648:ultrastructure*; D001665:Binding Sites; D002460:Cell Line; D049452:Green Fluorescent Proteins; D060588:LIM Domain Proteins / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000378:metabolism / Q000648:ultrastructure; D011485:Protein Binding; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
chemical_list: D060588:LIM Domain Proteins; D049452:Green Fluorescent Proteins
keywords: Actin dynamics; Arabidopsis; BiFC; FLIM-FRET; LIM proteins; TIRF microscopy; Tobacco BY-2 cells