Regulation of cell migration and survival by focal adhesion targeting of Lasp-1

Large-scale proteomic and functional analysis of isolated pseudopodia revealed the Lim, actin, and SH3 domain protein (Lasp-1) as a novel protein necessary for cell migration, but not adhesion to, the extracellular matrix (ECM). Lasp-1 is a ubiquitously expressed actin-binding protein with a unique domain configuration containing SH3 and LIM domains, and is overexpressed in 8-12% of human breast cancers. We find that stimulation of nonmotile and quiescent cells with growth factors or ECM proteins facilitates Lasp-1 relocalization from the cell periphery to the leading edge of the pseudopodium, where it associates with nascent focal complexes and areas of actin polymerization. Interestingly, although Lasp-1 dynamics in migratory cells occur independently of c-Abl kinase activity and tyrosine phosphorylation, c-Abl activation by apoptotic agents specifically promotes phosphorylation of Lasp-1 at tyrosine 171, which is associated with the loss of Lasp-1 localization to focal adhesions and induction of cell death. Thus, Lasp-1 is a dynamic focal adhesion protein necessary for cell migration and survival in response to growth factors and ECM proteins.     

Targeted disruption of the Lasp-1 gene is linked to increases in histamine-stimulated gastric HCl secretion

Lasp-1 (LIM and SH3 domain protein 1) is a multidomain actin-binding protein that is differentially expressed within epithelial tissues and brain. In the gastric mucosa, Lasp-1 is highly expressed in the HCl-secreting parietal cell, where it is prominently localized within the F-actin-rich subcellular regions. Histamine-induced elevation of parietal cell [cAMP]i increases Lasp-1 phosphorylation, which is correlated with activation of HCl secretion. To determine whether Lasp-1 is involved in the regulation of HCl secretion in vivo, we generated a murine model with a targeted disruption of the Lasp-1 gene. Lasp-1-null mice had slightly lower body weights but developed normally and had no overt phenotypic abnormalities. Basal HCl secretion was unaffected by loss of Lasp-1, but histamine stimulation induced a more robust acid secretory response in Lasp-1-null mice compared with wild-type littermates. A similar effect of histamine was observed in isolated gastric glands on the basis of measurements of accumulation of the weak base [14C]aminopyrine. In addition, inhibition of the acid secretory response to histamine by H2 receptor blockade with ranitidine proceeded more slowly in glands from Lasp-1-null mice. These findings support the conclusion that Lasp-1 is involved in the regulation of parietal HCl secretion. We speculate that cAMP-dependent phosphorylation of Lasp-1 alters interactions with F-actin and/or endocytic proteins that interact with Lasp-1, thereby regulating the trafficking/activation of the H+, K+-ATPase (proton pump).     

Contribution of the LIM Domain and Nebulin-Repeats to the Interaction of Lasp-2 with Actin Filaments and Focal Adhesions

Lasp-2 binds to actin filaments and concentrates in the actin bundles of filopodia and lamellipodia in neural cells and focal adhesions in fibroblastic cells. Lasp-2 has three structural regions: a LIM domain, a nebulin-repeat region, and an SH3 domain; however, the region(s) responsible for its interactions with actin filaments and focal adhesions are still unclear. In this study, we revealed that the N-terminal fragment from the LIM domain to the first nebulin-repeat module (LIM-n1) retained actin-binding activity and showed a similar subcellular localization to full-length lasp-2 in neural cells. The LIM domain fragment did not interact with actin filaments or localize to actin filament bundles. In contrast, LIM-n1 showed a clear subcellular localization to filopodial actin bundles. Although truncation of the LIM domain caused the loss of F-actin binding activity and the accumulation of filopodial actin bundles, these truncated fragments localized to focal adhesions. These results suggest that lasp-2 interactions with actin filaments are mediated through the cooperation of the LIM domain and the first nebulin-repeat module in vitro and in vivo. Actin filament binding activity may be a major contributor to the subcellular localization of lasp-2 to filopodia but is not crucial for lasp-2 recruitment to focal adhesions.     

Lasp anchors the Drosophila male stem cell niche and mediates spermatid individualization

Lasp family proteins contain an amino-terminal LIM domain, two actin-binding nebulin repeats and a carboxyl-terminal SH3 domain. Vertebrate Lasp-1 localizes to focal adhesions and the leading edge of migrating cells, and is required for cell migration. To assess the in vivo function of Lasp, we generated a null mutant in Drosophila Lasp. Lasp(1) is homozygous viable, but male sterile. In Lasp mutants the stem cell niche is no longer anchored to the apical tip of the testis, and actin cone migration is perturbed resulting in improper spermatid individualization. Hub cell mislocalization can by phenocopied by expressing Lasp or betaPS integrin RNAi transgenes in somatic cells, and Lasp genetically interacts with betaPS integrin, demonstrating that Lasp functions together with integrins in hub cells to anchor the stem cell niche. Finally, we show that the stem cell niche is maintained even if it is not properly localized.     

Lasp-2 expression, localization, and ligand interactions: a new Z-disc scaffolding protein

The nebulin family of actin-binding proteins plays an important role in actin filament dynamics in a variety of cells including striated muscle. We report here the identification of a new striated muscle Z-disc associated protein: lasp-2 (LIM and SH3 domain protein-2). Lasp-2 is the most recently identified member of the nebulin family. To evaluate the role of lasp-2 in striated muscle, lasp-2 gene expression and localization were studied in chick and mouse tissue, as well as in primary cultures of chick cardiac and skeletal myocytes. Lasp-2 mRNA was detected as early as chick embryonic stage 25 and lasp-2 protein was associated with developing premyofibril structures, Z-discs of mature myofibrils, focal adhesions, and intercalated discs of cultured cardiomyocytes. Expression of GFP-tagged lasp-2 deletion constructs showed that the C-terminal region of lasp-2 is important for its localization in striated muscle cells. Lasp-2 organizes actin filaments into bundles and interacts directly with the Z-disc protein alpha-actinin. These results are consistent with a function of lasp-2 as a scaffolding and actin filament organizing protein within striated muscle Z-discs.     

Chromosomal assignment and expression pattern of the murine Lasp-1 gene

The human Lasp-1 (LIM and SH3 protein) gene was previously identified by differential screening of a breast cancer-derived metastatic lymph node cDNA library. It was located on the q12–q21 region of human chromosome 17 and was shown to be amplified and overexpressed in 12% of breast tumours. Lasp-1 defines a new LIM-protein subfamily, as it associates a C-terminal Src homology 3 (SH3) domain to a N-terminal LIM motif. In this study, the isolation and characterization of the cDNA encoding the mouse Lasp-1 protein are described, and it is shown to be highly conserved with its human counterpart. In addition to the LIM and SH3 domains, both human and mouse Lasp-1 contain an actin-binding domain. The mouse gene was mapped by in situ hybridization to the 11C–11D region of chromosome 11. Northern blot analysis shows that this gene is expressed from 7.5 to 17.5 days post-coitum of mouse embryogenesis and in almost all adult tissues.     

Actin-binding Verprolin Is a Polarity Development Protein Required for the Morphogenesis and Function of the Yeast Actin Cytoskeleton

Yeast verprolin, encoded by VRP1, is implicated in cell growth, cytoskeletal organization, endocytosis and mitochondrial protein distribution and function. We show that verprolin is also required for bipolar bud-site selection. Previously we reported that additional actin suppresses the temperature-dependent growth defect caused by a mutation in VRP1. Here we show that additional actin suppresses all known defects caused by vrp1-1 and conclude that the defects relate to an abnormal cytoskeleton. Using the two-hybrid system, we show that verprolin binds actin. An actin-binding domain maps to the LKKAET hexapeptide located in the first 70 amino acids. A similar hexapeptide in other acting-binding proteins was previously shown to be necessary for actin-binding activity. The entire 70– amino acid motif is conserved in novel higher eukaryotic proteins that we predict to be actin-binding, and also in the actin-binding proteins, WASP and N-WASP. Verprolin-GFP in live cells has a cell cycle-dependent distribution similar to the actin cortical cytoskeleton. In fixed cells hemagglutinin-tagged Vrp1p often co-localizes with actin in cortical patches. However, disassembly of the actin cytoskeleton using Latrunculin-A does not alter verprolin's location, indicating that verprolin establishes and maintains its location independent of the actin cytoskeleton. Verprolin is a new member of the actin-binding protein family that serves as a polarity development protein, perhaps by anchoring actin. We speculate that the effects of verprolin upon the actin cytoskeleton might influence mitochondrial protein sorting/function via mRNA distribution.     

Lasp-1 regulates podosome function

Eukaryotic cells form a variety of adhesive structures to connect with their environment and to regulate cell motility. In contrast to classical focal adhesions, podosomes, highly dynamic structures of different cell types, are actively engaged in matrix remodelling and degradation. Podosomes are composed of an actin-rich core region surrounded by a ring-like structure containing signalling molecules, motor proteins as well as cytoskeleton-associated proteins. Lasp-1 is a ubiquitously expressed, actin-binding protein that is known to regulate cytoskeleton architecture and cell migration. This multidomain protein is predominantely present at focal adhesions, however, a second pool of Lasp-1 molecules is also found at lamellipodia and vesicle-like microdomains in the cytosol.In this report, we show that Lasp-1 is a novel component and regulator of podosomes. Immunofluorescence studies reveal a localization of Lasp-1 in the podosome ring structure, where it colocalizes with zyxin and vinculin. Life cell imaging experiments demonstrate that Lasp-1 is recruited in early steps of podosome assembly. A siRNA-mediated Lasp-1 knockdown in human macrophages affects podosome dynamics as well as their matrix degradation capacity. In summary, our data indicate that Lasp-1 is a novel component of podosomes and is involved in the regulation of podosomal function.     

Fimbrin is a homologue of the cytoplasmic phosphoprotein plastin and has domains homologous with calmodulin and actin gelation proteins

Fimbrin is an actin-bundling protein found in intestinal microvilli, hair cell stereocilia, and fibroblast filopodia. The complete protein sequence (630 residues) of chicken intestine fimbrin has been determined from two full-length cDNA clones. The sequence encodes a small amino-terminal domain (115 residues) that is homologous with two calcium-binding sites of calmodulin and a large carboxy-terminal domain (500 residues) consisting of a fourfold-repeated 125-residue sequence. This repeat is homologous with the actin-binding domain of alpha-actinin and the amino-terminal domains of dystrophin, actin-gelation protein, and beta-spectrin. The presence of this duplicated domain in fimbrin links actin bundling proteins and gelation proteins into a common family of actin cross-linking proteins. Fimbrin is also homologous in sequence with human L-plastin and T-plastin. L-plastin is found in only normal or transformed leukocytes where it becomes phosphorylated in response to IL 1 or phorbol myristate acetate. T-plastin is found in cells of solid tissues where it does not become phosphorylated. Neoplastic cells derived from solid tissues express both isoforms. The differences in expression, sequence, and phosphorylation suggest possible functional differences between fimbrin isoforms.     

Mammalian Abp1, a signal-responsive F-actin-binding protein, links the actin cytoskeleton to endocytosis via the GTPase dynamin

The actin cytoskeleton has been implicated in endocytosis, yet few molecular links to the endocytic machinery have been established. Here we show that the mammalian F-actin-binding protein Abp1 (SH3P7/HIP-55) can functionally link the actin cytoskeleton to dynamin, a GTPase that functions in endocytosis. Abp1 binds directly to dynamin in vitro through its SH3 domain. Coimmunoprecipitation and colocalization studies demonstrated the in vivo relevance of this interaction. In neurons, mammalian Abp1 and dynamin colocalized at actin-rich sites proximal to the cell body during synaptogenesis. In fibroblasts, mAbp1 appeared at dynamin-rich sites of endocytosis upon growth factor stimulation. To test whether Abp1 functions in endocytosis, we overexpressed several Abp1 constructs in Cos-7 cells and assayed receptor-mediated endocytosis. While overexpression of Abp1's actin-binding modules did not interfere with endocytosis, overexpression of the SH3 domain led to a potent block of transferrin uptake. This implicates the Abp1/dynamin interaction in endocytic function. The endocytosis block was rescued by cooverexpression of dynamin. Since the addition of the actin-binding modules of Abp1 to the SH3 domain construct also fully restored endocytosis, Abp1 may support endocytosis by combining its SH3 domain interactions with cytoskeletal functions in response to signaling cascades converging on this linker protein.     

Zyxin interacts with the SH3 domains of the cytoskeletal proteins LIM-nebulette and Lasp-1

Zyxin is a versatile component of focal adhesions in eukaryotic cells. Here we describe a novel binding partner of zyxin, which we have named LIM-nebulette. LIM-nebulette is an alternative splice variant of the sarcomeric protein nebulette, which, in contrast to nebulette, is expressed in non-muscle cells. It displays a modular structure with an N-terminal LIM domain, three nebulin-like repeats, and a C-terminal SH3 domain and shows high similarity to another cytoskeletal protein, Lasp-1 (LIM and SH3 protein-1). Co-precipitation studies and results obtained with the two-hybrid system demonstrate that LIM-nebulette and Lasp-1 interact specifically with zyxin. Moreover, the SH3 domain from LIM-nebulette is both necessary and sufficient for zyxin binding. The SH3 domains from Lasp-1 and nebulin can also interact with zyxin, but the SH3 domains from more distantly related proteins such as vinexin and sorting nexin 9 do not. On the other hand, the binding site in zyxin is situated at the extreme N terminus as shown by site-directed mutagenesis. LIM-nebulette and Lasp-1 use the same linear binding motif. This motif shows some similarity to a class II binding site but does not contain the classical PXXP sequence. LIM-nebulette reveals a subcellular distribution at focal adhesions similar to Lasp-1. Thus, LIM-nebulette, Lasp-1, and zyxin may play an important role in the organization of focal adhesions.     

Synthetic actin-binding domains reveal compositional constraints for function

The actin-binding domains of many proteins consist of a canonical type 1/type 2 arrangement of the structurally conserved calponin homology domain. Using the actin-binding domain of alpha-actinin-1 as a scaffold we have generated synthetic actin-binding domains by altering position and composition of the calponin homology domains. We show that the presence of two calponin homology domains alone and in the context of an actin-binding domain is not sufficient for actin-binding, and that both single and homotypic type 2 calponin homology domain tandems fail to bind to actin in vitro and in transfected cells. In contrast, single and tandem type 1 calponin homology domain arrays bind actin directly but result in defective turnover rates on actin filaments, and in aberrant actin bundling when introduced into the full-length alpha-actinin molecule. An actin-binding domain harboring the calponin homology domains in an inverted position, however, functions both in isolation and in the context of the dimeric alpha-actinin molecule. Our data demonstrate that the dynamics and specificity of actin-binding via actin-binding domains requires both the filament binding properties of the type 1, and regulation by type 2 calponin homology domains, and appear independent of their position.     

GFP-FABD2 fusion construct allows in vivo visualization of the dynamic actin cytoskeleton in all cells of Arabidopsis seedlings

In vivo visualization of filamentous actin in all cells of Arabidopsis thaliana seedlings is essential for understanding the numerous roles of the actin cytoskeleton in diverse processes of cell differentiation. A previously introduced reporter construct based on the actin-binding domain of mouse talin proved to be useful for unravelling some of these aspects in cell layers close to the organ surface. However, cells more deeply embedded, especially stelar cells active in polar transport of auxin, show either diffuse or no fluorescence at all due to the lack of expression of the fusion protein. The same problem is encountered in the root meristem. Recently introduced actin reporters based on fusions between A. thaliana fimbrin 1 and GFP gave brilliant results in organs from the root differentiation zone upwards to the leaves, however failed to depict the filamentous actin cytoskeleton in the transition zone of the root, in the apical meristem and the root cap. To overcome these problems, we have prepared new transgenic lines for the visualization of F-actin in vivo. We report here that a construct consisting of GFP fused to the C-terminal half of A. thaliana fimbrin 1 reveals dynamic arrays of F-actin in all cells of stably transformed A. thaliana seedlings.     

Swiprosin-1 Is a Novel Actin Bundling Protein That Regulates Cell Spreading and Migration

Protein functions are often revealed by their localization to specialized cellular sites. Recent reports demonstrated that swiprosin-1 is found together with actin and actin-binding proteins in the cytoskeleton fraction of human mast cells and NK-like cells. However, direct evidence of whether swiprosin-1 regulates actin dynamics is currently lacking. We found that swiprosin-1 localizes to microvilli-like membrane protrusions and lamellipodia and exhibits actin-binding activity. Overexpression of swiprosin-1 enhanced lamellipodia formation and cell spreading. In contrast, swiprosin-1 knockdown showed reduced cell spreading and migration. Swiprosin-1 induced actin bundling in the presence of Ca²⁺, and deletion of the EF-hand motifs partially reduced bundling activity. Swiprosin-1 dimerized in the presence of Ca²⁺ via its coiled-coil domain, and a lysine (Lys)-rich region in the coiled-coil domain was essential for regulation of actin bundling. Consistent with these observations, mutations of the EF-hand motifs and coiled-coil region significantly reduced cell spreading and lamellipodia formation. We provide new evidence of how swiprosin-1 influences cytoskeleton reorganization and cell spreading.     

Binding of filamentous actin to anthrax toxin receptor 1 decreases its association with protective antigen

ANTXR1 is a type I membrane protein that binds the protective antigen (PA) component of anthrax toxin. The cytosolic domain of ANTXR1 has a novel actin-binding region that influences the interaction of the ectodomain with PA. Here, we have investigated features of the cytosolic domain of ANTXR1 that reduce the association of the receptor with PA. We mutated a stretch of conserved acidic amino acids adjacent to the actin-binding region and found that the mutation increased the affinity for monomeric actin in vitro. ANTXR1 bearing this mutation exhibited increased association with the cytoskeleton and bound less PA compared to the wild-type receptor, confirming the inverse correlation between the two interactions. To determine whether binding of actin is sufficient to regulate the ectodomain, we replaced the actin-binding region of ANTXR1 with that from the yeast protein abp140 and with the WH2 domain of WAVE2. Although both of these domains bound monomeric actin in vitro, only the sequence from abp140 reduced binding of PA to a hybrid receptor. The actin binding regions of ANTXR1 and abp140, but not the WH2 domain, colocalized with actin stress fibers, which suggested that filamentous actin regulates ANTXR1. Consistent with this notion, disruption of actin filaments using latrunculin A increased the amount of PA bound to cells. This work provides evidence that cytoskeletal dynamics regulate ANTXR1 function.     

The carboxy terminus of AFAP-110 modulates direct interactions with actin filaments and regulates its ability to alter actin filament integrity and induce lamellipodia formation

The actin filament-associated protein AFAP-110 is an SH2/SH3 binding partner for Src. AFAP-110 contains several protein-binding motifs in its amino terminus and has been hypothesized to function as an adaptor molecule that could link signaling proteins to actin filaments. Recent studies using deletional mutagenesis demonstrated that AFAP-110 can alter actin filament integrity in SV40 transformed Cos-1 cells. Thus, AFAP-110 may be positioned to modulate the effects of Src upon actin filaments. In this report, we sought to determine whether (a) AFAP-110 could interact with actin filaments directly and (b) deletion mutants could affect actin filament integrity and cell shape in untransformed fibroblast cells. The data demonstrate that the carboxy terminus of AFAP-110 is both necessary and sufficient for actin filament association, in vivo and in vitro. Analysis of the carboxy terminus revealed a mean 40% similarity with other known actin-binding motifs, indicating a mechanism for binding to actin filaments. AFAP-110 can also induce lamellipodia formation. Contiguous with the alpha-helical, actin-binding motif is an alpha-helical, leucine zipper motif. Deletion of the leucine zipper motif (AFAP(Deltalzip)) followed by cellular expression enabled AFAP(Deltalzip) to alter actin filament integrity and cell shape in untransformed cells as evidenced by the induction of lamellipodia formation. We hypothesize that AFAP-110 may be an important signaling protein that can directly modulate changes in actin filament integrity and induce lamellipodia formation.     

Interactions of elongation factor 1alpha with F-actin and beta-actin mRNA: implications for anchoring mRNA in cell protrusions

The targeting of mRNA and local protein synthesis is important for the generation and maintenance of cell polarity. As part of the translational machinery as well as an actin/microtubule-binding protein, elongation factor 1alpha (EF1alpha) is a candidate linker between the protein translation apparatus and the cytoskeleton. We demonstrate in this work that EF1alpha colocalizes with beta-actin mRNA and F-actin in protrusions of chicken embryo fibroblasts and binds directly to F-actin and beta-actin mRNA simultaneously in vitro in actin cosedimentation and enzyme-linked immunosorbent assays. To investigate the role of EF1alpha in mRNA targeting, we mapped the two actin-binding sites on EF1alpha at high resolution and defined one site at the N-terminal 49 residues of domain I and the other at the C-terminal 54 residues of domain III. In vitro actin-binding assays and localization in vivo of recombinant full-length EF1alpha and its various truncates demonstrated that the C terminus of domain III was the dominant actin-binding site both in vitro and in vivo. We propose that the EF1alpha-F-actin complex is the scaffold that is important for beta-actin mRNA anchoring. Disruption of this complex would lead to delocalization of the mRNA. This hypothesis was tested by using two dominant negative polypeptides: the actin-binding domain III of EF1alpha and the EF1alpha-binding site of yeast Bni1p, a protein that inhibits EF1alpha binding to F-actin and also is required for yeast mRNA localization. We demonstrate that either domain III of EF1alpha or the EF1alpha-binding site of Bni1p inhibits EF1alpha binding to beta-actin mRNA in vitro and causes delocalization of beta-actin mRNA in chicken embryo fibroblasts. Taken together, these results implicate EF1alpha in the anchoring of beta-actin mRNA to the protrusion in crawling cells.     

Sla1p is a functionally modular component of the yeast cortical actin cytoskeleton required for correct localization of both Rho1p-GTPase and Sla2p, a protein with talin homology

SLA1 was identified previously in budding yeast in a genetic screen for mutations that caused a requirement for the actin-binding protein Abp1p and was shown to be required for normal cortical actin patch structure and organization. Here, we show that Sla1p, like Abp1p, localizes to cortical actin patches. Furthermore, Sla1p is required for the correct localization of Sla2p, an actin-binding protein with homology to talin implicated in endocytosis, and the Rho1p-GTPase, which is associated with the cell wall biosynthesis enzyme beta-1,3-glucan synthase. Mislocalization of Rho1p in sla1 null cells is consistent with our observation that these cells possess aberrantly thick cell walls. Expression of mutant forms of Sla1p in which specific domains were deleted showed that the phenotypes associated with the full deletion are functionally separable. In particular, a region of Sla1p encompassing the third SH3 domain is important for growth at high temperatures, for the organization of cortical actin patches, and for nucleated actin assembly in a permeabilized yeast cell assay. The apparent redundancy between Sla1p and Abp1p resides in the C-terminal repeat region of Sla1p. A homologue of SLA1 was identified in Schizosaccharomyces pombe. Despite relatively low overall sequence homology, this gene was able to rescue the temperature sensitivity associated with a deletion of SLA1 in Saccharomyces cerevisiae.     

Myosin heavy-chain kinase A from Dictyostelium possesses a novel actin-binding domain that cross-links actin filaments

Myosin heavy-chain kinase A (MHCK A) catalyses the disassembly of myosin II filaments in Dictyostelium cells via myosin II heavy-chain phosphorylation. MHCK A possesses a 'coiled-coil'-enriched domain that mediates the oligomerization, cellular localization and actin-binding activities of the kinase. F-actin (filamentous actin) binding by the coiled-coil domain leads to a 40-fold increase in MHCK A activity. In the present study we examined the actin-binding characteristics of the coiled-coil domain as a means of identifying mechanisms by which MHCK A-mediated disassembly of myosin II filaments can be regulated in the cell. Co-sedimentation assays revealed that the coiled-coil domain of MHCK A binds co-operatively to F-actin with an apparent K(D) of approx. 0.5 muM and a stoichiometry of approx. 5:1 [actin/C(1-498)]. Further analyses indicate that the coiled-coil domain binds along the length of the actin filament and possesses at least two actin-binding regions. Quite surprisingly, we found that the coiled-coil domain cross-links actin filaments into bundles, indicating that MHCK A can affect the cytoskeleton in two important ways: (1) by driving myosin II-filament disassembly via myosin II heavy-chain phosphorylation, and (2) by cross-linking/bundling actin filaments. This discovery, along with other supporting data, suggests a model in which MHCK A-mediated bundling of actin filaments plays a central role in the recruitment and activation of the kinase at specific sites in the cell. Ultimately this provides a means for achieving the robust and highly localized disruption of myosin II filaments that facilitates polarized changes in cell shape during processes such as chemotaxis, cytokinesis and multicellular development.