The pH sensibility of actin-bundling LIM proteins is governed by the acidic properties of their C-terminal domain.

Actin-bundling Arabidopsis LIM proteins are subdivided into two subfamilies differing in their pH sensitivity. Widely-expressed WLIMs are active under low and high physiologically-relevant pH conditions, whereas pollen-enriched PLIMs are inactivated by pH values above 6.8. By a domain swapping approach we identified the C-terminal (Ct) domain of PLIMs as the domain responsible for pH responsiveness. Remarkably, this domain conferred pH sensitivity to LIM proteins, when provided "in trans" (i.e., as a single, independent, peptide), indicating that it operates through the interaction with another domain. An acidic 6xc-Myc peptide functionally mimicked the Ct domain of PLIMs and efficiently inhibited LIM actin bundling activity under high pH conditions. Together, our data suggest a model where PLIMs are regulated by an intermolecular interaction between their acidic Ct domain and another, yet unidentified, domain.

Live cell imaging reveals actin-cytoskeleton-induced self-association of the actin-bundling protein WLIM1.

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.

A LIM domain protein from tobacco involved in actin-bundling and histone gene transcription.

The two LIM domain-containing proteins from plants (LIMs) typically exhibit a dual cytoplasmic-nuclear distribution, suggesting that, in addition to their previously described roles in actin cytoskeleton organization, they participate in nuclear processes. Using a south-western blot-based screen aimed at identifying factors that bind to plant histone gene promoters, we isolated a positive clone containing the tobacco LIM protein WLIM2 (NtWLIM2) cDNA. Using both green fluorescent protein (GFP) fusion- and immunology-based strategies, we provide clear evidence that NtWLIM2 localizes to the actin cytoskeleton, the nucleus, and the nucleolus. Interestingly, the disruption of the actin cytoskeleton by latrunculin B significantly increases NtWLIM2 nuclear fraction, pinpointing a possible novel cytoskeletal-nuclear crosstalk. Biochemical and electron microscopy experiments reveal the ability of NtWLIM2 to directly bind to actin filaments and to crosslink the latter into thick actin bundles. Electrophoretic mobility shift assays show that NtWLIM2 specifically binds to the conserved octameric cis-elements (Oct) of the Arabidopsis histone H4A748 gene promoter and that this binding largely relies on both LIM domains. Importantly, reporter-based experiments conducted in Arabidopsis and tobacco protoplasts confirm the ability of NtWLIM2 to bind to and activate the H4A748 gene promoter in live cells. Expression studies indicate the constitutive presence of NtWLIM2 mRNA and NtWLIM2 protein during tobacco BY-2 cell proliferation and cell cycle progression, suggesting a role of NtWLIM2 in the activation of basal histone gene expression. Interestingly, both live cell and in vitro data support NtWLIM2 di/oligomerization. We propose that NtWLIM2 functions as an actin-stabilizing protein, which, upon cytoskeleton remodeling, shuttles to the nucleus in order to modify gene expression.

Bioinformatics as a driver, not a passenger, of translational biomedical research: Perspectives from the 6th Benelux bioinformatics conference.

The 6th Benelux Bioinformatics Conference (BBC11) held in Luxembourg on 12 and 13 December 2011 attracted around 200 participants, including internationally-renowned guest speakers and more than 100 peer-reviewed submissions from 3 continents. Researchers from the public and private sectors convened at BBC11 to discuss advances and challenges in a wide spectrum of application areas. A key theme of the conference was the contribution of bioinformatics to enable and accelerate translational and clinical research. The BBC11 stressed the need for stronger collaborating efforts across disciplines and institutions. The demonstration of the clinical relevance of systems approaches and of next-generation sequencing-based measurement technologies are among the existing opportunities for increasing impact in translational research. Translational bioinformatics will benefit from research models that strike a balance between the importance of protecting intellectual property and the need to openly access scientific and technological advances. The full conference proceedings are freely available at http://www.bbc11.lu.

Arabidopsis actin-depolymerizing factors (ADFs) 1 and 9 display antagonist activities.

We provide evidence that one of the 11 Arabidopsis actin-depolymerizing factors (ADFs), namely ADF9, does not display typical F-actin depolymerizing activity. Instead, ADF9 effectively stabilizes actin filaments in vitro and concomitantly bundles actin filaments with the highest efficiency under acidic conditions. Competition experiments show that ADF9 antagonizes ADF1 activity by reducing its ability to potentiate F-actin depolymerization. Accordingly, ectopic expression of ADF1 and ADF9 in tobacco cells has opposite effects. ADF1 severs actin filaments/bundles and promotes actin cytoskeleton disassembly, whereas ADF9 induces the formation of long bundles. Together these data reveal an additional degree of complexity in the comprehension of the biological functions of the ADF family and illustrate that antagonist activities can be displayed by seemingly equivalent actin-binding proteins.

Arabidopsis LIM proteins: a family of actin bundlers with distinct expression patterns and modes of regulation.

Recently, a number of two LIM-domain containing proteins (LIMs) have been reported to trigger the formation of actin bundles, a major higher-order cytoskeletal assembly. Here, we analyzed the six Arabidopsis thaliana LIM proteins. Promoter-ß-glucuronidase reporter studies revealed that WLIM1, WLIM2a, and WLIM2b are widely expressed, whereas PLIM2a, PLIM2b, and PLIM2c are predominantly expressed in pollen. LIM-green fluorescent protein (GFP) fusions all decorated the actin cytoskeleton and increased actin bundle thickness in transgenic plants and in vitro, although with different affinities and efficiencies. Remarkably, the activities of WLIMs were calcium and pH independent, whereas those of PLIMs were inhibited by high pH and, in the case of PLIM2c, by high [Ca(2+)]. Domain analysis showed that the C-terminal domain is key for the responsiveness of PLIM2c to pH and calcium. Regulation of LIM by pH was further analyzed in vivo by tracking GFP-WLIM1 and GFP-PLIM2c during intracellular pH modifications. Cytoplasmic alkalinization specifically promoted release of GFP-PLIM2c but not GFP-WLIM1, from filamentous actin. Consistent with these data, GFP-PLIM2c decorated long actin bundles in the pollen tube shank, a region of relatively low pH. Together, our data support a prominent role of Arabidopsis LIM proteins in the regulation of actin cytoskeleton organization and dynamics in sporophytic tissues and pollen.

Actin bundling in plants.

Tight regulation of plant actin cytoskeleton organization and dynamics is crucial for numerous cellular processes including cell division, expansion and intracellular trafficking. Among the various actin regulatory proteins, actin-bundling proteins trigger the formation of bundles composed of several parallel actin filaments closely packed together. Actin bundles are present in virtually all plant cells, but their biological roles have rarely been addressed directly. However, decades of research in the plant cytoskeleton field yielded a bulk of data from which an overall picture of the functions supplied by actin bundles in plant cells emerges. Although plants lack several equivalents of animal actin-bundling proteins, they do possess major bundler classes including fimbrins, villins and formins. The existence of additional players is not excluded as exemplified by the recent characterization of plant LIM proteins, which trigger the formation of actin bundles both in vitro and in vivo. This apparent functional redundancy likely reflects the need for plant cells to engineer different types of bundles that act at different sub-cellular locations and exhibit specific function-related properties. By surveying information regarding the properties of plant actin bundles and their associated bundling proteins, the present review aims at clarifying why and how plants make actin bundles.

Regulators of PP2C phosphatase activity function as abscisic acid sensors.

The plant hormone abscisic acid (ABA) acts as a developmental signal and as an integrator of environmental cues such as drought and cold. Key players in ABA signal transduction include the type 2C protein phosphatases (PP2Cs) ABI1 and ABI2, which act by negatively regulating ABA responses. In this study, we identify interactors of ABI1 and ABI2 which we have named regulatory components of ABA receptor (RCARs). In Arabidopsis, RCARs belong to a family with 14 members that share structural similarity with class 10 pathogen-related proteins. RCAR1 was shown to bind ABA, to mediate ABA-dependent inactivation of ABI1 or ABI2 in vitro, and to antagonize PP2C action in planta. Other RCARs also mediated ABA-dependent regulation of ABI1 and ABI2, consistent with a combinatorial assembly of receptor complexes.

Nuclear localization of the mutant protein phosphatase abi1 is required for insensitivity towards ABA responses in Arabidopsis.

ABI1, a protein phosphatase 2C, is a key component of ABA signal transduction in Arabidopsis that regulates numerous ABA responses, such as stomatal closure, seed germination and inhibition of vegetative growth. The abi1-1 mutation, so far the only characterized dominant allele for ABI1, impairs ABA responsitivity in both seeds and vegetative tissues. The site of action of ABI1 is unknown. We show that there is an essential requirement for nuclear localization of abi1 to confer insensitivity towards ABA responses. Transient analyses in protoplasts revealed a strict dependence of wild-type ABI1 and mutant abi1 on a functional nuclear localization sequence (NLS) for regulating ABA-dependent gene expression. Arabidopsis lines with ectopic expression of various abi1 forms corroborated the necessity of a functional NLS to control ABA sensitivity. Disruption of the NLS function in abi1 rescued ABA-controlled gene transcription to wild-type levels, but also attenuated abi1-conferred insensitivity towards ABA during seed germination, root growth and stomatal movement. The mutation in the PP2C resulted in a preferential accumulation of the protein in the nucleus. Application of a proteosomal inhibitor led to both a preferential nuclear accumulation of ABI1 and an enhancement of PP2C-dependent inhibitory action on the ABA response. Thus, abi1-1 acts as a hypermorphic allele, and ABI1 reprograms sensitivity towards ABA in the nucleus.