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.

VLN2Regulates Plant Architecture by Affecting Microfilament Dynamics and Polar Auxin Transport in Rice

“…Zhang et al, 2011b;Bao et al, 2012;van der Honing et al, 2012;Qu et al, 2013), firmly support the notion that plant villins function as a major MF bundling factor in vivo. Our results showed that the bundling frequency was 0.503 310 25 events/µm 2 /s in root epidermal cells (Table 1), which is lower than that in Arabidopsis pollen tubes (2.3 3 10 24 events/ µm 2 /s; Zheng et al, 2013) and hypocotyl epidermal cells (6.9 3 10 25 events/mm 2 /s; Hoffmann et al, 2014), suggesting a possible association between bundling frequency and a cell's growing status. Unexpectedly, inspection of MF dynamics revealed that severing frequency is significantly increased in vln2.…”

The plant LIM proteins: unlocking the hidden attractions

“…Deletion of any of the LIM domain reduces affinity to actin, but does not abolish it completely (Thomas et al 2007a). The concrete model of actin bundling has been noticed recently (Hoffmann et al 2014). NtWLIM1 interacts locally with adjoining actin filaments and triggered them to zipper into tight bundles.…”

“…Transcriptional regulation (PAL box binding)/lignin biosynthesis Kawaoka et al (2000) NtWLIM1 Actin dynamics Thomas et al (2006Thomas et al ( , 2007a; Hoffmann et al (2014) …”

“…Additionally, binding of LIM domain with actin filament also makes them more competent for dimerization. During this process, they allow neighbouring actin filaments to come closer and craft bundles (Hoffmann et al 2014).…”

“…The first such 2LIM was described in Helianthus (Baltz et al 1992) and was found associated with pollen development and growth. The 2LIM was also observed as key regulators of actin dynamics (Thomas et al 2007a;Hoffmann et al 2014), a feature associated with its function in pollen and fibre development. Though the nucleic acid binding activity was speculated and observed early (Baltz and Evrard 1996), the substantial evidence of LIM-triggered transcriptional regulation came only after the work of Kawaoka et al (2000) and Moes et al (2013).…”