Gelsolin Family Research Articles

Distinct Roles of Four Gelsolin-like Domains of Caenorhabditis elegans Gelsolin-like Protein-1 in Actin Filament Severing, Barbed End Capping, and Phosphoinositide Binding

Caenorhabditis elegans gelsolin-like protein-1 (GSNL-1) is a new member of the gelsolin family of actin regulatory proteins [Klaavuniemi, T., Yamashiro, S., and Ono, S. (2008) J. Biol. Chem. 283, 26071-26080]. It is an unconventional gelsolin-related protein with four gelsolin-like (G) domains (G1-G4), unlike typical gelsolin-related proteins with three or six G domains. GSNL-1 severs actin filaments and caps the barbed end in a calcium-dependent manner similar to that of gelsolin. In contrast, GSNL-1 has properties different from those of gelsolin in that it remains bound to F-actin and does not nucleate actin polymerization. To understand the mechanism by which GSNL-1 regulates actin dynamics, we investigated the domain-function relationship of GSNL-1 by analyzing activities of truncated forms of GSNL-1. G1 and the linker between G1 and G2 were sufficient for actin filament severing, whereas G1 and G2 were required for barbed end capping. The actin severing activity of GSNL-1 was inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), and a PIP2-sensitive domain was mapped to G1 and G2. At least two actin-binding sites were detected: a calcium-dependent G-actin-binding site in G1 and a calcium-independent G- and F-actin-binding site in G3 and G4. These results reveal both conserved and different utilization of G domains between C. elegans GSNL-1 and mammalian gelsolin for actin regulatory functions.

Flii Control: Balancing Migration and Adhesion

Wound healing in the skin requires a compromise between adhesion and migration. Both processes include modulation of the cytoskeleton, cell-surface receptors, and receptor ligands., In this issue, Kopecki et al. demonstrate that overexpression of Flii, an actin-remodeling protein, impedes wound healing but inhibits hemidesmosome formation. In contrast, Flii deficiency results in enhanced wound healing while promoting hemidesmosome assembly. We discuss potential mechanisms that could explain how this unique gelsolin family member might regulate both stable keratinocyte adhesion and motility.

Flightless I Regulates Hemidesmosome Formation and Integrin-Mediated Cellular Adhesion and Migration during Wound Repair

Flightless I (Flii), a highly conserved member of the gelsolin family of actin-remodelling proteins associates with actin structures and is involved in cellular motility and adhesion. Our previous studies have shown that Flii is an important negative regulator of wound repair. Here, we show that Flii affects hemidesmosome formation and integrin-mediated keratinocyte adhesion and migration. Impaired hemidesmosome formation and sparse arrangements of keratin cytoskeleton tonofilaments and actin cytoskeleton anchoring fibrils were observed in Flii(Tg/+) and Flii(Tg/Tg) mice with their skin being significantly more fragile than Flii(+/-) and WT mice. Flii(+/-) primary keratinocytes showed increased adhesion on laminin and collagen I than WT and Flii(Tg/Tg) primary keratinocytes. Decreased expression of CD151 and laminin-binding integrins alpha3, beta1, alpha6 and beta4 were observed in Flii overexpressing wounds, which could contribute to the impaired wound re-epithelialization observed in these mice. Flii interacts with proteins directly linked to the cytoplasmic domain of integrin receptors suggesting that it may be a mechanical link between ligand-bound integrin receptors and the actin cytoskeleton driving adhesion-signaling pathways. Therefore Flii may regulate wound repair through its effect on hemidesmosome formation and integrin-mediated cellular adhesion and migration.

Caenorhabditis elegans Gelsolin-like Protein 1 Is a Novel Actin Filament-severing Protein with Four Gelsolin-like Repeats

The gelsolin family of proteins is a major class of actin regulatory proteins that sever, cap, and nucleate actin filaments in a calcium-dependent manner and are involved in various cellular processes. Typically, gelsolin-related proteins have three or six repeats of gelsolin-like (G) domain, and each domain plays a distinct role in severing, capping, and nucleation. The Caenorhabditis elegans gelsolin-like protein-1 (gsnl-1) gene encodes an unconventional gelsolin-related protein with four G domains. Sequence alignment suggests that GSNL-1 lacks two G domains that are equivalent to fourth and fifth G domains of gelsolin. In vitro, GSNL-1 severed actin filaments and capped the barbed end in a calcium-dependent manner. However, unlike gelsolin, GSNL-1 remained bound to the side of F-actin with a submicromolar affinity and did not nucleate actin polymerization, although it bound to G-actin with high affinity. These results indicate that GSNL-1 is a novel member of the gelsolin family of actin regulatory proteins and provide new insight into functional diversity and evolution of gelsolin-related proteins.

Flightless-I, a gelsolin family member and transcriptional regulator, preferentially binds directly to activated cytosolic CaMK-II

In order to evaluate links between Ca2+/calmodulin (CaM)-dependent protein kinase type II (CaMK-II) and cell cycle progression, CaMK-II binding partners were sought in proliferating cells by epitope-tag tandem mass spectrometry. One protein identified was the gelsolin family member, flightless-I (Fli-I). Fli-I is not a CaMK-II substrate, but binds directly and preferentially to constitutively active (T287D) CaMK-II over inactive CaMK-II. Fli-I gradually enters the nucleus upon CaMK-II inhibition and is retained in the cytosol by T287D CaMK-II. CaMK-II inhibition and Fli-I overexpression suppress transcription of β-catenin dependent transcriptional reporters, whereas Fli-I suppression enhances their transcription. These findings support a novel mechanism whereby cytosolic CaMK-II influences β-catenin dependent gene expression through Fli-I. Structured summaryMINT-6603828:delta CaMK-II (uniprotkb:Q6PHZ2) binds (MI:0407) to Flightless-I (uniprotkb:Q9JJ28) by anti bait coimmunoprecipitation (MI:0006)MINT-6603847:delta CaMK-II (uniprotkb:Q6PHZ2) phosphorylates (MI:0217) MAP2 (uniprotkb:P20357) by protein kinase assay (MI:0424)MINT-6603768:beta CaMK-II (uniprotkb:P28652) physically interacts (MI:0218) with Flightless-I (uniprotkb:Q9JJ28) by anti bait coimmunoprecipitation (MI:0006)MINT-6603759, MINT-6603776, MINT-6603786:delta CaMK-II (uniprotkb:Q6PHZ2) physically interacts (MI:0218) with Flightless-I (uniprotkb:Q9JJ28) by anti bait coimmunoprecipitation (MI:0006)MINT-6603724:CaMK-II delta (uniprotkb:Q6PHZ2) physically interacts (MI:0218) with beta 5tubulin (uniprotkb:P99024), beta 2c tubulin (uniprotkb:P68372), alpha 1a tubulin (uniprotkb:P68369), b-Actin (uniprotkb:P60710), Tropomodulin-3 (uniprotkb:Q9JHJ0), Flightless-I (uniprotkb:Q9JJ28), FLAP1 (uniprotkb:Q3UZ39) and FLAP2 (uniprotkb:Q91WK0) by anti tag coimmunoprecipitation (MI:0007).

The Flightless I Homolog, fli-1, Regulates Anterior/Posterior Polarity, Asymmetric Cell Division and Ovulation During Caenorhabditis elegans Development

Flightless I (Fli I) is an evolutionarily conserved member of the gelsolin family, containing actin-binding and severing activity in vitro. The physiological function of Fli I during animal development remains largely undefined. In this study, we reveal a key role of the Caenorhabditis elegans Fli I homolog, fli-1, in specifying asymmetric cell division and in establishing anterior-posterior polarity in the zygote. The fli-1 gene also regulates the cytokinesis of somatic cells and the development of germline and interacts with the phosphoinositol-signaling pathway in the regulation of ovulation. The fli-1 reporter gene shows that the localization of FLI-1 coincides with actin-rich regions and that the actin cytoskeleton is impaired in many tissues in the fli-1 mutants. Furthermore, the function of fli-1 in C. elegans can be functionally substituted by the Drosophila Fli I. Our studies demonstrate that fli-1 plays an important role in regulating the actin-dependent events during C. elegans development.

Duplicated gelsolin family genes in zebrafish: a novel scinderin-like gene (scinla) encodes the major corneal crystallin.

We have previously identified a gelsolin-like protein (C/L-gelsolin) as a corneal crystallin in zebrafish. Here we show by phylogenetic analysis that there are at least six genes encoding gelsolin-like proteins based on their gelsolin domains in zebrafish: gsna and gsnb group with the vertebrate gelsolin gene, scina and scinb group with the scinderin (adseverin) gene, and scinla (C/L-gelsolin) and scinlb are novel scinderin-like genes. RT-PCR showed that scinla, scinlb, and gsnb are preferentially expressed in the adult cornea whereas gsna is expressed to a similar extent in cornea, lens, brain, and heart; scina and scinb expression were detectable only in whole zebrafish and not in these adult tissues. Quantitative RT-PCR and 2-dimensional polyacrylamide gel electrophoresis followed by MALDI/TOF mass spectroscopy confirmed high expression of beta-actin and scinla, moderate expression of scinlb, and very low expression of gsna and gsnb in the cornea. Finally, transgenic zebrafish carrying a green fluorescent protein reporter transgene driven by a 4 kb scinla promoter fragment showed expression in the cornea, snout, dorsal fin, and tail fin of 3-day-old zebrafish larvae. Our data suggest that scinla and scinlb are diverged paralogs of the vertebrate scinderin gene and show that scinla encodes the zebrafish corneal crystallin previously called C/L-gelsolin.

Downregulation of gelsolin family proteins counteracts cancer cell invasion in vitro

Gelsolin and CapG are both actin binding proteins that modulate a variety of physiological processes by interacting differently with the actin cytoskeleton. Several studies suggest that overexpression of these proteins promotes invasion in vitro. In this study we explored the contribution of these proteins in human cancer cell invasion and motility. We show that down regulation of CapG or gelsolin in several types of cancer cells, including MDA-MB 231 and PC-3 cells, significantly reduces the invasive and motile properties of cells, as well as cell aggregation. These results point to a role for CapG and gelsolin as tumor activator.

Flightless I: An actin-remodelling protein and an important negative regulator of wound repair

Flightless I (FliI) is a member of the gelsolin family of actin-remodelling proteins, and has been identified as having two functional protein family domains: a leucine rich repeat (LRR) domain and a gelsolin-like domain. This unique structure allows FliI to act as an actin-remodelling protein as well as a nuclear receptor co-activator with ability to interact with various other proteins important in cellular signaling. The actin cytoskeleton is an integral component of all cells and the effect of FliI protein on actin remodelling is a vital part of cellular motility, contraction and adhesion. The product of the FliI gene is expected to provide a vital link between the molecules of yet unidentified signal transduction pathways and the actin cytoskeleton. Exact signaling pathways and mechanisms underpinning FliI effects in wound healing are yet to be fully identified however strong research evidence clearly identifies this molecule as a possible new therapeutic target whose manipulation may greatly improve wound healing and could lead to potential innovative medical applications.

Structural basis and evolutionary origin of actin filament capping by twinfilin

Dynamic reorganization of the actin cytoskeleton is essential for motile and morphological processes in all eukaryotic cells. One highly conserved protein that regulates actin dynamics is twinfilin, which both sequesters actin monomers and caps actin filament barbed ends. Twinfilin is composed of two ADF/cofilin-like domains, Twf-N and Twf-C. Here, we reveal by systematic domain-swapping/inactivation analysis that the two functional ADF-H domains of twinfilin are required for barbed-end capping and that Twf-C plays a critical role in this process. However, these domains are not functionally equivalent. NMR-structure and mutagenesis analyses, together with biochemical and motility assays showed that Twf-C, in addition to its binding to G-actin, interacts with the sides of actin filaments like ADF/cofilins, whereas Twf-N binds only G-actin. Our results indicate that during filament barbed-end capping, Twf-N interacts with the terminal actin subunit, whereas Twf-C binds between two adjacent subunits at the side of the filament. Thus, the domain requirement for actin filament capping by twinfilin is remarkably similar to that of gelsolin family proteins, suggesting the existence of a general barbed-end capping mechanism. Furthermore, we demonstrate that a synthetic protein consisting of duplicated ADF/cofilin domains caps actin filament barbed ends, providing evidence that the barbed-end capping activity of twinfilin arose through a duplication of an ancient ADF/cofilin-like domain.

A CapG gain-of-function mutant reveals critical structural and functional determinants for actin filament severing

CapG is the only member of the gelsolin family unable to sever actin filaments. Changing amino acids 84-91 (severing domain) and 124-137 (WH2-containing segment) simultaneously to the sequences of gelsolin results in a mutant, CapG-sev, capable of severing actin filaments. The gain of severing function does not alter actin filament capping, but is accompanied by a higher affinity for monomeric actin, and the capacity to bind and sequester two actin monomers. Analysis of CapG-sev crystal structure suggests a more loosely folded inactive conformation than gelsolin, with a shorter S1-S2 latch. Calcium binding to S1 opens this latch and S1 becomes separated from a closely interfaced S2-S3 complex by an extended arm consisting of amino acids 118-137. Modeling with F-actin predicts that the length of this WH2-containing arm is critical for severing function, and the addition of a single amino acid (alanine or histidine) eliminates CapG-sev severing activity, confirming this prediction. We conclude that efficient severing utilizes two actin monomer-binding sites, and that the length of the WH2-containing segment is a critical functional determinant for severing.

Structural Analysis of an Echinococcus granulosus Actin-Fragmenting Protein by Small-Angle X-Ray Scattering Studies and Molecular Modeling

The Echinococcus granulosus actin filament-fragmenting protein (EgAFFP) is a three domain member of the gelsolin family of proteins, which is antigenic to human hosts. These proteins, formed by three or six conserved domains, are involved in the dynamic rearrangements of the cytoskeleton, being responsible for severing and capping actin filaments and promoting nucleation of actin monomers. Various structures of six domain gelsolin-related proteins have been investigated, but little information on the structure of three domain members is available. In this work, the solution structure of the three domain EgAFFP has been investigated through small-angle x-ray scattering (SAXS) studies. EgAFFP exhibits an elongated molecular shape. The radius of gyration and the maximum dimension obtained by SAXS were, respectively, 2.52 ± 0.01 nm and 8.00 ± 1.00 nm, both in the absence and presence of Ca 2+. Two different molecular homology models were built for EgAFFP, but only one was validated through SAXS studies. The predicted structure for EgAFFP consists of three repeats of a central β-sheet sandwiched between one short and one long α-helix. Possible implications of the structure of EgAFFP upon actin binding are discussed.

Evolution of the gelsolin family of actin-binding proteins as novel transcriptional coactivators

The gelsolin gene family encodes a number of higher eukaryotic actin-binding proteins that are thought to function in the cytoplasm by severing, capping, nucleating or bundling actin filaments. Recent evidence, however, suggests that several members of the gelsolin family may have adopted unexpected nuclear functions including a role in regulating transcription. In particular, flightless I, supervillin and gelsolin itself have roles as coactivators for nuclear receptors, despite the fact that their divergence appears to predate the evolutionary appearance of nuclear receptors. Flightless I has been shown to bind both actin and the actin-related BAF53a protein, which are subunits of SWI/SNF-like chromatin remodelling complexes. The primary sequences of some actin-related proteins such as BAF53a exhibit conservation of residues that, in actin itself, are known to interact with gelsolin-related proteins. In summary, there is a growing body of evidence supporting a biological role in the nucleus for actin, Arps and actin-binding proteins and, in particular, the gelsolin family of actin-binding proteins.

The Flightless I protein and the gelsolin family in nuclear hormone receptor-mediated signalling

The Drosophila melanogaster flightless I protein and its homologues in higher eukaryotes (FliI) are conserved members of the gelsolin family of actin-binding proteins. Members of the gelsolin family generally contain three or six copies of a 125-amino-acid residue gelsolin-related repeating unit, and may contain additional domains including the C-terminal villin-related 'headpiece' or N-terminal extensions such as the leucine-rich repeat of the FliI protein. Numerous studies including work done with mouse knockouts for gelsolin, villin and CapG support a role for the family in cytoskeletal actin dynamics. In both fruitfly and mouse, the FliI protein is also essential for early development. Recent studies indicate that supervillin, gelsolin and FliI are involved in intracellular signalling via nuclear hormone receptors including the androgen, oestrogen and thyroid hormone receptors. This unexpected role in signalling has opened a new area in research on the gelsolin family and is providing important new insights into the mechanisms of gene regulation via nuclear receptors.

The gelsolin family of actin regulatory proteins: modular structures, versatile functions

This issue of FEBS Letters includes two manuscripts describing structural studies of gelsolin, the best-characterized member of a superfamily of actin binding proteins that sever, cap, and in some cases nucleate and bundle actin filaments. The manuscripts by Narayan et al. and Irobi et al. provide snapshots of gelsolin domains activated by calcium and in complex with the actin monomer, revealing new insights into the remarkable actin regulatory activities of this versatile protein. These studies build upon nearly a quarter of a century of research on gelsolin’s effects on actin dynamics and its role in normal and diseased cells. In the following minireview, we summarize the structural studies that have provided insights into gelsolin’s severing and capping activities and look to the future of work on this remarkable molecule.

The role of F‐actin cytoskeleton‐associated gelsolin in the guinea pig capacitation and acrosome reaction

The acrosomal reaction (AR) is a regulated sperm exocytotic process that involves fusion of the plasma membrane (PM) with the outer acrosomal membrane (OAM). Our group has described F-actin cytoskeletons associated to these membranes. It has been proposed that in regulated exocytosis, a cortical cytoskeleton acts as a barrier that obstructs membrane fusion, and must be disassembled for exocytosis to occur. Actin-severing proteins from the gelsolin family have been considered to break this barrier. The present study attempted to determine if gelsolin has a function in guinea pig sperm capacitation and AR. By indirect immunofluorescence (IIF), gelsolin was detected in the apical and postacrosomal regions of the head and in the flagellum in both capacitated and non-capacitated guinea pig spermatozoa. By Western blotting, gelsolin was detected in isolated PM and OAM of non-capacitated spermatozoa. Gelsolin and actin were detected in a mixture of PM-OAM obtained by sonication, and both proteins were absent in membranes of capacitated spermatozoa. Inhibition of three different pathways of PIP2 hydrolysis during capacitation did not cancel gelsolin loss from membranes. Gelsolin was detected by Western blotting associated to membrane cytoskeletons obtained after phalloidin F-actin stabilization and Triton-X treatment; additionally, by immunoprecipitation, it was shown that gelsolin is associated with actin. By electron microscopy we observed that skeletons disassemble during capacitation, but phalloidin prevents disassembly. A three-dimensional skeleton was observed that apparently joins PM with OAM. Exogenous gelsolin stimulates AR assayed in a permeabilized spermatozoa model. Results suggest that gelsolin disassembles F-actin cytoskeletons during capacitation, promoting AR.

Cap G, a Gelsolin Family Protein Modulating Protective Effects of Unidirectional Shear Stress

Atherosclerosis is a progressive and complex pathophysiological process occurring in large arteries. Although it is of multifactorial origin, the disease develops at preferential sites along the vasculature in regions experiencing specific hemodynamic conditions that are predisposed to endothelial dysfunction. The exact mechanisms allowing endothelial cells to discriminate between plaque-free and plaque-prone flows remain to be explored. To investigate such mechanisms, we performed a proteomic analysis on endothelial cells exposed in vitro to these two-flow patterns. A few spots on the two-dimensional gel had an intensity that was differentially regulated by plaque-free versus plaque-prone flows. One of them was further investigated and identified as macrophage-capping protein (Cap G), a member of the gelsolin protein superfamily. A 2-fold increase of Cap G protein and a 5-fold increase of Cap G mRNA were observed in cells exposed to a plaque-free flow as compared with static cultures. This increase was not observed in cells exposed to plaque-prone flow. Plaque-free flow induced a corresponding increase in nuclear and cytoskeletal-associated Cap G. Finally, overexpression of Cap G in transfection assays increased the motility potential of endothelial cells. These observations together with the known functions of Cap G suggest that Cap G may contribute to the protective effect exerted by plaque-free flow on endothelial cells. On the contrary, in cells exposed to a plaque-prone flow, no induction of Cap G expression could be observed.

Fliih, a gelsolin-related cytoskeletal regulator essential for early mammalian embryonic development.

The Drosophila melanogaster flightless I gene is required for normal cellularization of the syncytial blastoderm. Highly conserved homologues of flightless I are present in Caenorhabditis elegans, mouse, and human. We have disrupted the mouse homologue Fliih by homologous recombination in embryonic stem cells. Heterozygous Fliih mutant mice develop normally, although the level of Fliih protein is reduced. Cultured homozygous Fliih mutant blastocysts hatch, attach, and form an outgrowing trophoblast cell layer, but egg cylinder formation fails and the embryos degenerate. Similarly, Fliih mutant embryos initiate implantation in vivo but then rapidly degenerate. We have constructed a transgenic mouse carrying the complete human FLII gene and shown that the FLII transgene is capable of rescuing the embryonic lethality of the homozygous targeted Fliih mutation. These results confirm the specific inactivation of the Fliih gene and establish that the human FLII gene and its gene product are functional in the mouse. The Fliih mouse mutant phenotype is much more severe than in the case of the related gelsolin family members gelsolin, villin, and CapG, where the homozygous mutant mice are viable and fertile but display alterations in cytoskeletal actin regulation.

Der f 16: a novel gelsolin-related molecule identified as an allergen from the house dust mite, Dermatophagoides farinae

Allergen from the house dust mite ( Dermatophagoides sp.) is a major trigger factor of allergic disorders, and its characterization is crucial for the development of specific diagnosis or immunotherapy. Here we report the identification of a novel dust mite ( Dermatophagoides farinae) antigen whose primary structure belongs to the gelsolin family, a group of actin cytoskeleton-regulatory proteins. Isolated mite cDNA, termed Der f 16, encodes 480 amino acids comprising a four-repeated gelsolin-like segmental structure, which is not seen in conventional gelsolin family members. Enzyme immunoassay indicated that recombinant Der f 16 protein, prepared using an Escherichia coli expression system, bound IgE from mite-allergic patients at 47% (8/17) frequency. This is the first evidence that the gelsolin family represents a new class of allergen recognizable by atopic patient IgE.

The ADF/cofilin family: actin-remodeling proteins.

The ADF/cofilins are a family of actin-binding proteins expressed in all eukaryotic cells so far examined. Members of this family remodel the actin cytoskeleton, for example during cytokinesis, when the actin-rich contractile ring shrinks as it contracts through the interaction of ADF/cofilins with both monomeric and filamentous actin. The depolymerizing activity is twofold: ADF/cofilins sever actin filaments and also increase the rate at which monomers leave the filament's pointed end. The three-dimensional structure of ADF/cofilins is similar to a fold in members of the gelsolin family of actin-binding proteins in which this fold is typically repeated three or six times; although both families bind polyphosphoinositide lipids and actin in a pH-dependent manner, they share no obvious sequence similarity. Plants and animals have multiple ADF/cofilin genes, belonging in vertebrates to two types, ADF and cofilins. Other eukaryotes (such as yeast, Acanthamoeba and slime moulds) have a single ADF/cofilin gene. Phylogenetic analysis of the ADF/cofilins reveals that, with few exceptions, their relationships reflect conventional views of the relationships between the major groups of organisms.