Live cell imaging approaches reveal actin cytoskeleton-induced self-association of the actin-bundling protein WLIM1

Abstract

Crosslinking of actin filaments into bundles is critical for the assembly/stabilization of specific cytoskeletal structures. Relatively little is known about the molecular mechanisms underlying actin bundle formation. The two LIM domain-containing (LIM) proteins define a novel and evolutionary-conserved family of actin bundlers 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, the disruption of the actin cytoskeleton significantly impaired NtWLIM1 self-association. In addition, biochemical analyses support that F-actin facilitates the switch of purified recombinant NtWLIM1 from a monomeric to a di/oligomeric state. Based on our data we propose a model in which actin binding promotes the formation/stabilization of NtWLIM1 complexes, which in turn might drive the crosslinking of actin filaments.