Marleen Van Troys

Ins and outs of ADF/cofilin activity and regulation

The actin-binding proteins of the actin-depolymerisation factor (ADF)/cofilin family were first described more than three decades ago, but research on these proteins still occupies a front role in the actin and cell migration field. Moreover, cofilin activity is implicated in the malignant, invasive properties of cancer cells. The effects of ADF/cofilins on actin dynamics are diverse and their regulation is complex. In stimulated cells, multiple signalling pathways can be initiated resulting in different activation/deactivation switches that control ADF/cofilin activity. The output of this entire regulatory system, in combination with spatial and temporal segregation of the activation mechanisms, underlies the contribution of ADF/cofilins to various cell migration/invasion phenotypes. In this framework, we describe current views on how ADF/cofilins function in migrating and invading cells.

Listeria comet tails: the actin-based motility machinery at work

Listeria monocytogenes is a master of mimicry that uses the host cell actin system both to move within the cytoplasm of infected cells and for cell-to-cell spread. Recent studies of Listeria and similarly acting pathogens have generated leaps in our understanding of the actin-based force producing machinery. This machinery is essential for most motile properties of cells, not least for cell migration. In a minimal configuration, it consists of the Arp2/3-complex, Ena-VASP proteins, cofilin, capping protein and a nucleation-promoting factor. In this review, we discuss current models of pseudopodial protrusions and describe how the road to more complex models lies open and is already paved by recent studies using Listeria-based biomimetic motility assays.

Structural modules in actin-binding proteins: towards a new classification

The number of actin binding proteins for which (part of) the three-dimensional structure is known, is steadily increasing. This has led to a picture in which defined structural modules with actin binding capacity are shared between different actin binding proteins. A classification of these based on their common three-dimensional modules appears a logical future step and in this review we provide an initial list starting from the currently known structures. The discussed cases illustrate that a comparison of the similarities and variations within the common structural actin binding unit of different members of a particular class may ultimately provide shortcuts for defining their actin target site and for understanding their effect on actin dynamics. Within this concept, the multitude of possible interactions by an extensive, and still increasing, list of actin binding proteins becomes manageable because they can be presented as variations upon a limited number of structural themes. We discuss the possible evolutionary routes that may have produced the present array of actin binding modules.

A QUICK screen for Lrrk2 interaction partners - leucine-rich repeat kinase 2 is involved in actin cytoskeleton dynamics

Mutations in human leucine-rich repeat kinase 2 (Lrrk2), a protein of yet unknown function, are linked to Parkinsons disease caused by degeneration of midbrain dopaminergic neurons. The protein comprises several domains including a GTPase and a kinase domain both affected by several pathogenic mutations. To elucidate the molecular interaction network of endogenous Lrrk2 under stoichiometric constraints, we applied QUICK (quantitative immunoprecipitation combined with knockdown) in NIH3T3 cells. The identified interactome reveals actin isoforms as well as actin-associated proteins involved in actin filament assembly, organization, rearrangement, and maintenance, suggesting that the biological function of Lrrk2 is linked to cytoskeletal dynamics. In fact, we demonstrate Lrrk2 de novo binding to F-actin and its ability to modulate its assembly in vitro. When tested in intact cells, knockdown of Lrrk2 causes morphological alterations in NIH3T3 cells. In developing dopaminergic midbrain primary neurons, Lrrk2 knockdown results in shortened neurite processes, indicating a physiological role of Lrrk2 in cytoskeletal organization and dynamics of dopaminergic neurons. Hence, our results demonstrate that molecular interactions as well as the physiological function of Lrrk2 are closely related to the organization of the actin-based cytoskeleton, a crucial feature of neuronal development and neuron function.

Tobacco WLIM1 Is a Novel F-Actin Binding Protein Involved in Actin Cytoskeleton Remodeling

We used confocal microscopy and in vitro analyses to show that Nicotiana tabacum WLIM1, a LIM domain protein related to animal Cys-rich proteins, is a novel actin binding protein in plants. Green fluorescent protein (GFP)–tagged WLIM1 protein accumulated in the nucleus and cytoplasm of tobacco BY2 cells. It associated predominantly with actin cytoskeleton, as demonstrated by colabeling and treatment with actin-depolymerizing latrunculin B. High-speed cosedimentation assays revealed the ability of WLIM1 to bind directly to actin filaments with high affinity. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed a highly dynamic in vivo interaction of WLIM1-GFP with actin filaments. Expression of WLIM1-GFP in BY2 cells significantly delayed depolymerization of the actin cytoskeleton induced by latrunculin B treatment. WLIM1 also stabilized actin filaments in vitro. Importantly, expression of WLIM1-GFP in Nicotiana benthamiana leaves induces significant changes in actin cytoskeleton organization, specifically, fewer and thicker actin bundles than in control cells, suggesting that WLIM1 functions as an actin bundling protein. This hypothesis was confirmed by low-speed cosedimentation assays and direct observation of F-actin bundles that formed in vitro in the presence of WLIM1. Taken together, these data identify WLIM1 as a novel actin binding protein that increases actin cytoskeleton stability by promoting bundling of actin filaments.

Actin-filament cross-linking protein T-plastin increases Arp2/3-mediated actin-based movement.

Increasing evidence suggests that actin cross-linking or bundling proteins might not only structure the cortical actin cytoskeleton but also control actin dynamics. Here, we analyse the effects of T-plastin/T-fimbrin, a representative member of an important actin-filament cross-linking protein by combining a quantitative biomimetic motility assay with biochemical and cell-based approaches. Beads coated with the VCA domain of the Wiskott/Aldrich-syndrome protein (WASP) recruit the actin-nucleating Arp2/3 complex, polymerize actin at their surface and undergo movement when placed in cell-free extracts. T-Plastin increased the velocity of VCA beads 1.5 times, stabilized actin comets and concomitantly displaced cofilin, an actin-depolymerizing protein. T-Plastin also decreased the F-actin disassembly rate and inhibited cofilin-mediated depolymerization of actin filaments in vitro. Importantly, a bundling-incompetent variant comprising the first actin-binding domain (ABD1) had similar effects. In cells, this domain induced the formation of long actin cables to which other actin-regulating proteins were recruited. Altogether, these results favor a mechanism in which binding of ABD1 controls actin turnover independently of cross-link formation. In vivo, this activity might contribute to the assembly and maintenance of the actin cytoskeleton of plasma-membrane protrusions.

Solution structures of the C-terminal headpiece subdomains of human villin and advillin, evaluation of headpiece F-actin-binding requirements.

Headpiece (HP) is a 76‐residue F‐actin‐binding module at the C terminus of many cytoskeletal proteins. Its 35‐residue C‐terminal subdomain is one of the smallest known motifs capable of autonomously adopting a stable, folded structure in the absence of any disulfide bridges, metal ligands, or unnatural amino acids. We report the three‐dimensional solution structures of the C‐terminal headpiece subdomains of human villin (HVcHP) and human advillin (HAcHP), determined by two‐dimensional 1H‐NMR. They represent the second and third structures of such C‐terminal headpiece subdomains to be elucidated so far. A comparison with the structure of the chicken villin C‐terminal subdomain reveals a high structural conservation. Both C‐terminal subdomains bind specifically to F‐actin. Mutagenesis is used to demonstrate the involvement of Trp 64 in the F‐actin‐binding surface. The latter residue is part of a conserved structural feature, in which the surface‐exposed indole ring is stacked on the proline and lysine side chain embedded in a PXWK sequence motif. On the basis of the structural and mutational data concerning Trp 64 reported here, the results of a cysteine‐scanning mutagenesis study of full headpiece, and a phage display mutational study of the 69–74 fragment, we propose a modification of the model, elaborated by Vardar and coworkers, for the binding of headpiece to F‐actin.

Analogous F-actin Binding by Cofilin and Gelsolin Segment 2 Substantiates Their Structural Relationship

Cofilin is representative for a family of low molecular weight actin filament binding and depolymerizing proteins. Recently the three-dimensional structure of yeast cofilin and of the cofilin homologs destrin and actophorin were resolved, and a striking similarity to segments of gelsolin and related proteins was observed (Hatanaka, H., Ogura, K., Moriyama, K., Ichikawa, S., Yahara, I., and Inagaka, F. (1996) Cell 85, 1047–1055; Fedorov, A. A., Lappalainen, P., Fedorov, E. V., Drubin, D. G., and Almo, S. C. (1997) Nat. Struct. Biol. 4, 366–369; Leonard, S. A., Gittis, A. G., Petrella, E. C., Pollard, T. D., and Lattman, E. E. (1997) Nat. Struct. Biol. 4, 369–373). Using peptide mimetics, we show that the actin binding site stretches over the entire cofilin α-helix 112–128. In addition, we demonstrate that cofilin and its actin binding peptide compete with gelsolin segments 2–3 for binding to actin filaments. Based on these competition data, we propose that cofilin and segment 2 of gelsolin use a common structural topology to bind to actin and probably share a similar target site on the filament. This adds a functional dimension to their reported structural homology, and this F-actin binding mode provides a basis to further enlighten the effect of members of the cofilin family on actin filament dynamics.

The LIM domains of WLIM1 define a new class of actin bundling modules

Actin filament bundling, i.e. the formation of actin cables, is an important process that relies on proteins able to directly bind and cross-link subunits of adjacent actin filaments. Animal cysteine-rich proteins and their plant counterparts are two LIM domain-containing proteins that were recently suggested to define a new family of actin cytoskeleton regulators involved in actin filament bundling. We here identified the LIM domains as responsible for F-actin binding and bundling activities of the tobacco WLIM1. The deletion of one of the two LIM domains reduced significantly, but did not entirely abolish, the ability of WLIM1 to bind actin filaments. Individual LIM domains were found to interact directly with actin filaments, although with a reduced affinity compared with the native protein. Variants lacking the C-terminal or the inter-LIM domain were only weakly affected in their F-actin stabilizing and bundling activities and trigger the formation of thick cables containing tightly packed actin filaments as does the native protein. In contrast, the deletion of one of the two LIM domains negatively impacted both activities and resulted in the formation of thinner and wavier cables. In conclusion, we demonstrate that the LIM domains of WLIM1 are new autonomous actin binding and bundling modules that cooperate to confer WLIM1 high actin binding and bundling activities.

Unbalancing the phosphatidylinositol-4,5-bisphosphate-cofilin interaction impairs cell steering.

Cofilin is a key player in actin dynamics during cell migration. Its activity is regulated by (de)phosphorylation, pH, and binding to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2)]. Here, we here use a human cofilin-1 (D122K) mutant with increased binding affinity for PI(4,5)P(2) and slower release from the plasma membrane to study the role of the PI(4,5)P(2)-cofilin interaction in migrating cells. In fibroblasts in a background of endogenous cofilin, D122K cofilin expression negatively affects cell turning frequency. In carcinoma cells with down-regulated endogenous cofilin, D122K cofilin neither rescues the drastic morphological defects nor restores the effects in cell turning capacity, unlike what has been reported for wild-type cofilin. In cofilin knockdown cells, D122K cofilin expression promotes outgrowth of an existing lamellipod in response to epidermal growth factor (EGF) but does not result in initiation of new lamellipodia. This indicates that, next to phospho- and pH regulation, the normal release kinetics of cofilin from PI(4,5)P(2) is crucial as a local activation switch for lamellipodia initiation and as a signal for migrating cells to change direction in response to external stimuli. Our results demonstrate that the PI(4,5)P(2) regulatory mechanism, that is governed by EGF-dependent phospholipase C activation, is a determinant for the spatial and temporal control of cofilin activation required for lamellipodia initiation.