Transient expression of the t-isoform of plastins/fimbrin in the stereocilia of developing auditory hair cells

The transduction of auditory signals by cochlear hair cells depends upon the integrity of hair cell stereociliary bundles. Stereocilia contain a central core of actin filaments, cross-linked by actin bundling proteins. In the cochlea, the two proteins described to date as responsible for the spatial arrangement of actin filaments in sterocilia are fimbrin and the recently discovered espin. Fimbrin (the chick homolog of human I-plastin) belongs to the plastins/fimbrin family that includes two additional isoforms of plastins, T- and L-plastin. In the present study, we used isoform specific antibodies to investigate the presence of the T- and L-isoforms of plastin/fimbrin in the adult and developing rat cochlea. We found that T-plastin, but not L-plastin, is expressed in the rat cochlea. During postnatal development of the rat organ of Corti, T-plastin can be detected in the core of stereocilia from early stages of hair cell differentiation, and its expression gradually increases in stereocilia as hair cells mature. However, as opposed to other actin-binding proteins expressed in stereocilia, T-plastin is absent from the stereocilia of mature hair cells. Such temporally restricted expression strengthens the idea of functional differences between plastins isoforms, and suggests that T-plastin could have a specific role in stereocilia formation.     

Correction of the N-terminal sequences of the human plastin isoforms by using anchored polymerase chain reaction: identification of a potential calcium-binding domain.

Plastins are a family of at least three cytoplasmic protein isoforms that are expressed differentially between cells of the hematopoietic lineages and cells of solid tissues. Expression of the L-plastin isoform appears to be restricted to replicating blood cells, and the two T-plastin isoforms appear to be restricted to replicating cells of solid tissues. However, L-plastin is induced in many human solid tumor-derived cells. We used the anchored polymerase chain reaction technique to amplify and clone the missing 5' ends of plastin mRNAs. We found that both plastin isoforms contain a potential calcium binding site near the N terminus.     

Biochemical characterization of the L-plastin-actin interaction shows a resemblance with that of alpha-actinin and allows a distinction to be made between the two actin-binding domains of the molecule

Actin interaction with L-plastin, a plastin/fimbrins isoform of the alpha-actinin family of molecules, is poorly characterized, from the biochemical point of view. Besides, molecular modeling of the T-isoform has recently provided a complete model of interaction with filamentous actin [Volkmann, N., DeRosier, D., Matsudaira, P., and Hanein, D. (2001) J. Cell Biol. 153, 947-956]. In this study, we report that recombinant L-plastin binds actin in a manner that strongly resembles that of the alpha-actinin-actin interface. The similitudes concern the absence of specificity toward the actin isoform and the inhibition of the binding by phosphoinositides. Furthermore, the participation of actin peptides 112-125 and 360-372 in the interface together with an inhibition of the rate of pyrenyl F-actin depolymerization is in favor of a lateral binding of the plastin isoform along the filament axis and strenghtens the similitudes in the way L-plastin and alpha-actinin bind to actin. We have also investigated the functional aspect and the putative equivalence of the two actin-binding domains of L-plastin toward actin binding. We demonstrate for the first time that the two recombinant fragments, expressed as single domains, have different affinities for actin. We further analyzed the difference using chemical cross-linking and F-actin depolymerization experiments assayed by fluorescence and high-speed centrifugation. The results clearly demonstrate that the two actin-binding domains of plastin display different modes of interaction with the actin filament. We discuss these results in light of the model of actin interaction proposed for T-plastin.     

Generation and Characterization of Monoclonal Antibodies That Specifically Recognize p65/L-Plastin Isoform but Not T-Plastin Isoform

The 65-kDa protein (p65) was previously identified as a phosphorylated protein in activated macrophages, and has turned out to be a member of a plastin protein family characterized by a series of Ca²⁺-, calmodulin-, and β-actin-binding domains. In mice, two isoforms, p65/L-plastin and T-plastin, have so far been identified; p65/L-plastin is expressed in hemopoietic cells and cancer cells, and T-plastin in solid tissue cells. We generated monoclonal antibodies to p65/L-plastin, examined the isoform-specificity by using recombinant (r) T-plastin, and found that the antibodies were specific for rp65/L-plastin, whereas immune sera to rp65/L-plastin showed cross-reactions to rT-plastin. One of the antibodies, p65-7B5, was demonstrated to react to native p65/L-plastin by Western blot, flow cytometric, and immunohistochemical analysis. Furthermore, p65-7B5 has made it possible to detect p65/L-plastin-expressing cells in tissues where T-plastin is abundantly expressed. These reagents and procedures should provide specific tools to investigate the role of p65/L-plastin in leukocytes.     

Molecular basis for dissimilar nuclear trafficking of the actin-bundling protein isoforms T- and L-plastin

T- and L-plastin are highly similar actin-bundling proteins implicated in the regulation of cell morphology, lamellipodium protrusion, bacterial invasion and tumor progression. We show that T-plastin localizes predominantly to the cytoplasm, whereas L-plastin distributes between nucleus and cytoplasm in HeLa or Cos cells. T-plastin shows nuclear accumulation upon incubation of cells with the CRM1 antagonist leptomycin B (LMB). We identified a Rev-like nuclear export sequence (NES) in T-plastin that is able to export an otherwise nuclear protein in an LMB-dependent manner. Deletion of the NES promotes nuclear accumulation of T-plastin. Mutation of residues L17, F21 or L26 in the T-plastin NES inhibits nuclear efflux. L-plastin harbors a less conserved NES and lacks the F21 T-plastin residue. Insertion of a Phe residue in the L-plastin NES specifically enhances its export activity. These findings explain why both isoforms exhibit specific distribution patterns in eukaryotic cells.     

Interaction of activated Rab5 with actin-bundling proteins, L- and T-plastin and its relevance to endocytic functions in mammalian cells

Rab5 is a GTP-binding protein that is crucial for endocytic machinery functions. We previously identified L-plastin as a binding protein for Rab5, using an affinity column with constitutively active Rab5. L- and T-plastin are isoforms of a plastin protein family belonging to actin-bundling proteins that are implicated in the regulation of cell morphology, lamellipodium protrusion, bacterial invasion and tumor progression. However, the physiological relevance of Rab5 binding to plastin has remained unclear. Here, we show that L- and T-plastin interacted only with activated Rab5 and that they co-localized with Rab5 on the plasma membrane and endosome. Rab5 activity was also higher in both L- and T-plastin over-expressing Cos-1 cells. Furthermore, expression of L- and T-plastin increased the rate of fluid-phase endocytosis. These findings imply that the Rab5 is either activated or the activity is sustained by interaction with plastin, and that this interaction influences endocytic activity.     

Generation and characterization of monoclonal antibodies that specifically recognize p65/L-plastin isoform but not T-plastin isoform

The 65-kDa protein (p65) was previously identified as a phosphorylated protein in activated macrophages, and has turned out to be a member of a plastin protein family characterized by a series of Ca(2+)-, calmodulin-, and beta-actin-binding domains. In mice, two isoforms, p65/L-plastin and T-plastin, have so far been identified; p65/L-plastin is expressed in hemopoietic cells and cancer cells, and T-plastin in solid tissue cells. We generated monoclonal antibodies to p65/L-plastin, examined the isoform-specificity by using recombinant (r) T-plastin, and found that the antibodies were specific for rp65/L-plastin, whereas immune sera to rp65/L-plastin showed cross-reactions to rT-plastin. One of the antibodies, p65-7B5, was demonstrated to react to native p65/L-plastin by Western blot, flow cytometric, and immunohistochemical analysis. Furthermore, p65-7B5 has made it possible to detect p65/L-plastin-expressing cells in tissues where T-plastin is abundantly expressed. These reagents and procedures should provide specific tools to investigate the role of p65/L-plastin in leukocytes.     

Cytoskeletal rearrangements and the functional role of T-plastin during entry of Shigella flexneri into HeLa cells

Shigella flexneri is an enteroinvasive bacterium which causes bacillary dysentery in humans. A major feature of its pathogenic potential is the capacity to invade epithelial cells. Shigella entry into epithelial cells is considered a parasite-induced internalization process requiring polymerization of actin. Here we describe the cytoskeletal rearrangements during S. flexneri invasion of HeLa cells. After an initial contact of the bacterium with the cell surface, distinct nucleation zones of heavy chain actin polymerization appear in close proximity to the contact site underneath the parasite with long filaments being polymerized. These structures then push cellular protrusions that rise beside the entering bacterium, being sustained by tightly bundled long actin filaments organized in parallel orientation with their positive ends pointing to the cytoplasmic membrane. Finally, the cellular projections coalesce above the bacterial body, leading to its internalization. In addition, we found the actin-bundling protein plastin to be concentrated in these protrusions. Since plastin is known to bundle actin filaments in parallel orientation, colocalization of parallel actin filaments and plastin in the cellular protrusions strongly suggested a functional role of this protein in the architecture of parasite-induced cellular projections. Using transfection experiments, we show the differential recruitment of the two plastin isoforms (T- and L-) into Shigella entry zones. By transient expression of a truncated T-plastin which is deprived of one of its actin-binding sites, we also demonstrate the functional role of T-plastin in Shigella entry into HeLa cells.     

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.     

Identification of I-plastin, a human fimbrin isoform expressed in intestine and kidney.

The complete cDNA sequence of human intestine-specific plastin (I-plastin) was determined from a clone derived by PCR. It consists of a 97-bp 5' untranslated region, a 1,887-bp coding region, and a 1,655-bp 3' untranslated region. The coding region predicts a 629-residue polypeptide whose sequence displays 86, 75, and 73% identities with chicken intestine fimbrin, human T-plastin, and human L-plastin, respectively. Recombinant I-plastin cross-linked actin filaments into bundles in the absence but not in the presence of calcium. The I-plastin gene was mapped by PCR to human chromosome 3; the L- and T-plastin genes were previously mapped to chromosomes 13 and X, respectively. I-plastin mRNA was detected in the small intestine, colon, and kidneys; relatively lower levels of expression were detected in the lungs and stomach. In contrast, L-plastin expression was restricted to the spleen and other lymph node-containing organs, while T-plastin was expressed in a variety of organs, including muscle, brain, uterus, and esophagus. In contrast to the situation for the intestine, high levels of L- and T-plastin mRNAs were detected in Caco-2, a human colon-derived cell line. Immunofluorescence microscopy detected I-plastin in the brush border of the small intestine and colon. These results identify I-plastin as the human homolog of chicken intestine fimbrin and as a third plastin isoform in humans.     

Human T cell L-plastin bundles actin filaments in a calcium-dependent manner.

The amino acid sequences deduced from cDNA analyses revealed that human leucocyte L-plastin phosphorylated in response to interleukin 1, 2 closely resembles a chicken intestinal microvilli protein, fimbrin, that bundles actin filaments [de Arruda et al. (1990) J. Cell Biol. 111, 1069-1079]. In the present work, it was observed that unphosphorylated L-plastin isolated from human T cells bundled F-actin just as fimbrin does. L-Plastin acted on T cell beta-actin, but hardly acted on muscle alpha-actin or chicken gizzard gamma-actin, whereas fimbrin bundled muscle alpha-actin. Unlike fimbrin, L-plastin's actin-bundling action was strictly calcium-dependent: the bundles were formed at pCa 7, but not at pCa 6. Under suitable conditions, approximately one molecule of L-plastin bound to 8 molecules of actin monomer in the actin filament.     

Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane-cytoskeletal linker

Ezrin, a widespread protein present in actin-containing cell-surface structures, is a substrate of some protein tyrosine kinases. Based on its primary and secondary structure similarities with talin and band 4.1 it has been suggested that this protein could play a role in linking the cytoskeleton to the plasma membrane (Gould, K.L., A. Bretscher, F.S. Esch, and T. Hunter. 1989. EMBO (Eur. Mol. Biol. Organ.), J. 8:4133-4142; Turunen, O., R. Winqvist, R. Pakkanen, K.-H. Grzeschik, T. Wahlstrom, and A. Vaheri. 1989. J. Biol. Chem. 264:16727- 16732). To test this hypothesis, we transiently expressed the complete human ezrin cDNA, or truncated cDNAs encoding the amino- and carboxy- terminal domains of the protein, in CV-1 cells. Protein epitope tagging was used to unambiguously determine the subcellular distribution of the protein encoded by the transfected cDNA. We show that this protein is concentrated underneath the dorsal plasma membrane in all actin- containing structures and is partially detergent insoluble. The amino- terminal domain displays the same localization but is readily extractable by nonionic detergent. The carboxy-terminal domain colocalizes with microvillar actin filaments as well as with stress fibers and remains associated with actin filaments after detergent extraction, and with disorganized actin structures after cytochalasin D treatment. Our results clearly demonstrate that ezrin interacts with membrane-associated components via its amino-terminal domain, and with the cytoskeleton via its carboxy-terminal domain. The amino-terminal domain could include the main determinant that restricts the entire protein to the cortical cytoskeleton in contact with the dorsal plasma membrane and its specialized microdomains such as microvilli, microspikes and lamellipodia.     

Fascin, an Actin-bundling Protein, Induces Membrane Protrusions and Increases Cell Motility of Epithelial Cells

Fascin is an actin-bundling protein that is found in membrane ruffles, microspikes, and stress fibers. The expression of fascin is greatly increased in many transformed cells, as well as in specialized normal cells including neuronal cells and antigen-presenting dendritic cells. A morphological characteristic common to these cells expressing high levels of fascin is the development of many membrane protrusions in which fascin is predominantly present. To examine whether fascin contributes to the alterations in microfilament organization at the cell periphery, we have expressed fascin in LLC-PK1 epithelial cells to levels as high as those found in transformed cells and in specialized normal cells. Expression of fascin results in large changes in morphology, the actin cytoskeleton, and cell motility: fascin-transfected cells form an increased number of longer and thicker microvilli on apical surfaces, extend lamellipodia-like structures at basolateral surfaces, and show disorganization of cell–cell contacts. Cell migration activity is increased by 8–17 times when assayed by modified Boyden chamber. Microinjection of a fascin protein into LLC-PK1 cells causes similar morphological alterations including the induction of lamellipodia at basolateral surfaces and formation of an increased number of microvilli on apical surfaces. Furthermore, microinjection of fascin into REF-52 cells, normal fibroblasts, induces the formation of many lamellipodia at all regions of cell periphery. These results together suggest that fascin is directly responsible for membrane protrusions through reorganization of the microfilament cytoskeleton at the cell periphery.     

Actin with tumor-related mutation is antimorphic in Drosophila muscle: two distinct modes of myofibrillar disruption by antimorphic actins

A mutant beta-actin with an amino acid substitution from Gly-245 to Asp has been shown to be related to tumorigenic transformation of a human fibroblast cell line (Leavitt, J. et al. (1987) Mol. Cell. Biol. 7, 2467-2476). To examine the effects of this mutation, we artificially introduced the same amino acid change into the Act88F actin gene of Drosophila melanogaster. The gene (Act88FGD245) was inserted in the Drosophila genome to make transgenic adult flies which synthesize the mutant actin in the indirect flight muscles. The mutant actin was found to be antimorphic with regard to flight and also to cause myofibrillar disruption in transformants even in the presence of two normal alleles. It was initially incorporated into myofibrils and later induced their degeneration from center to periphery. This mode of myofibrillar disruption is distinct from that of previously reported Act88F mutations, where defects are found only in the peripheral region of myofibrils. This indicates that actin functions are altered differently in the two classes of antimorphic mutations.     

Interaction of a Dictyostelium member of the plastin/fimbrin family with actin filaments and actin-myosin complexes.

A protein purified from cytoskeletal fractions of Dictyostelium discoideum proved to be a member of the fimbrin/plastin family of actin-bundling proteins. Like other family members, this Ca(2+)-inhibited 67-kDa protein contains two EF hands followed by two actin-binding sites of the alpha-actinin/beta-spectrin type. Dd plastin interacted selectively with actin isoforms: it bound to D. discoideum actin and to beta/gamma-actin from bovine spleen but not to alpha-actin from rabbit skeletal muscle. Immunofluorescence labeling of growth phase cells showed accumulation of Dd plastin in cortical structures associated with cell surface extensions. In the elongated, streaming cells of the early aggregation stage, Dd plastin was enriched in the front regions. To examine how the bundled actin filaments behave in myosin II-driven motility, complexes of F-actin and Dd plastin were bound to immobilized heavy meromyosin, and motility was started by photoactivating caged ATP. Actin filaments were immediately propelled out of bundles or even larger aggregates and moved on the myosin as separate filaments. This result shows that myosin can disperse an actin network when it acts as a motor and sheds light on the dynamics of protein-protein interactions in the cortex of a motile cell where myosin II and Dd plastin are simultaneously present.     

Differential localization of tropomyosin isoforms in cultured nonmuscle cells

We have previously shown that chicken embryo fibroblast (CEF) cells and human bladder carcinoma (EJ) cells contain multiple isoforms of tropomyosin, identified as a, b, 1, 2, and 3 in CEF cells and 1, 2, 3, 4, and 5 in human EJ cells by one-dimensional SDS-PAGE (Lin, J. J.-C., D. M. Helfman, S. H. Hughes, and C.-S. Chou. 1985. J. Cell Biol. 100: 692-703; and Lin, J. J.-C., S. Yamashiro-Matsumura, and F. Matsumura. 1984. Cancer Cells 1:57-65). Both isoform 3 (TM-3) of CEF and isoforms 4,5 (TM-4,-5) of human EJ cells are the minor isoforms found respectively in normal chicken and human cells. They have a lower apparent molecular mass and show a weaker affinity to actin filaments when compared to the higher molecular mass isoforms. Using individual tropomyosin isoforms immobilized on nitrocellulose papers and sequential absorption of polyclonal antiserum on these papers, we have prepared antibodies specific to CEF TM-3 and to CEF TM-1,-2. In addition, two of our antitropomyosin mAbs, CG beta 6 and CG3, have now been demonstrated by Western blots, immunoprecipitation, and two-dimensional gel analysis to have specificities to human EJ TM-3 and TM-5, respectively. By using these isoform-specific reagents, we are able to compare the intracellular localizations of the lower and higher molecular mass isoforms in both CEF and human EJ cells. We have found that both lower and higher molecular mass isoforms of tropomyosin are localized along stress fibers of cells, as one would expect. However, the lower molecular mass isoforms are also distributed in regions near ruffling membranes. Further evidence for this different localization of different tropomyosin isoforms comes from double-label immunofluorescence microscopy on the same CEF cells with affinity-purified antibody against TM-3, and monoclonal CG beta 6 antibody against TM-a, -b, -1, and -2 of CEF tropomyosin. The presence of the lower molecular mass isoform of tropomyosin in ruffling membranes may indicate a novel way for the nonmuscle cell to control the stability and organization of microfilaments, and to regulate the cell motility.     

Actin in the ciliated protozoan Climacostomum virens: purification by DNAse I affinity chromatography, electrophoretic characterization, and immunological analysis.

The anti-actin monoclonal antibody (mab) JLA20 (Lin: Proc. Natl. Acad. Sci. U.S.A. 78:2335-2339, 1981) labels a 43 kD protein on Western blots of Climacostomum cell extracts; this protein does not react with an anti-alpha-smooth muscle actin mab (Skalli et al.: J. Cell Biol. 103:2787-2796, 1986) nor with an anti-alpha-sarcomeric actin mab (Skalli et al.: Am. J. Pathol. 130:515-531, 1988). This protein binds to DNAse I and can be purified by DNAse I affinity chromatography. The affinity-purified actin also reacts with mab JLA20. Two-dimensional gel analysis reveals that Climacostomum actin focuses as three spots which are more basic than the mammalian actin isoforms. After addition of KCl, the affinity-purified actin polymerizes into filaments as shown by electron microscopy after negative staining.     

Plastin 1 Binds to Keratin and Is Required for Terminal Web Assembly in the Intestinal Epithelium

Plastin 1 (I-plastin, fimbrin) along with villin and espin is a prominent actin-bundling protein of the intestinal brush border microvilli. We demonstrate here that plastin 1 accumulates in the terminal web and interacts with keratin 19, possibly contributing to anchoring the rootlets to the keratin network. This prompted us to investigate the importance of plastin 1 in brush border assembly. Although in vivo neither villin nor espin is required for brush border structure, plastin 1-deficient mice have conspicuous ultrastructural alterations: microvilli are shorter and constricted at their base, and, strikingly, their core actin bundles lack true rootlets. The composition of the microvilli themselves is apparently normal, whereas that of the terminal web is profoundly altered. Although the plastin 1 knockout mice do not show any overt gross phenotype and present a normal intestinal microanatomy, the alterations result in increased fragility of the epithelium. This is seen as an increased sensitivity of the brush border to biochemical manipulations, decreased transepithelial resistance, and increased sensitivity to dextran sodium sulfate-induced colitis. Plastin 1 thus emerges as an important regulator of brush border morphology and stability through a novel role in the organization of the terminal web, possibly by connecting actin filaments to the underlying intermediate filament network.     

Actin's propensity for dynamic filament patterning

Actin, through its various forms of assembly, provides the basic framework for cell motility, cell shape and intracellular organization in all eukaryotic cells. Many other cellular processes, for example endocytosis and cytokinesis, are also associated with dynamic changes of the actin cytoskeleton. Important prerequisites for actin's functional diversity are its intrinsic ability to rapidly assemble and disassemble filaments and its spatially and temporally well-controlled supramolecular organization. A large number of proteins that interact with actin, collectively referred to as actin-binding proteins (ABPs), carefully orchestrate different scenarios. Since its isolation in 1994 [Machesky, L.M. et al. (1994) J. Cell Biol. 127, 107-115], the Arp2/3 complex containing the actin-related proteins Arp2 and Arp3 has evolved to be one of the main players in the assembly and maintenance of many actin-based structures in the cell (for review see [Borths, E.L. and Welch, M.D. (2002) Structure 10, 131-135; May, R.C. (2001) Cell Mol. Life Sci. 58, 1607-1626; Pollard, T.D. et al. (2000) Rev. Biophys. Biomol. Struct. 29, 545-576; Welch, M.D. (1999) Trends Cell Biol. 11, 423-427]). In particular, when it comes to the assembly of the intricate branched actin network at the leading edge of lamellipodia, the Arp2/3 complex seems to have received all the attention in recent years. In parallel, but not so much in the spotlight, several reports showed that actin on its own can assume different conformations [Bubb, M.R. et al. (2002) J. Biol. Chem. 277, 20999-21006; Schoenenberger, C.-A. et al. (1999) Microsc. Res. Tech. 47, 38-50; Steinmetz, M.O. et al. (1998) J. Mol. Biol. 278, 793-811; Steinmetz, M.O. et al. (1997) J. Cell Biol. 138, 559-574; Millonig, R., Salvo, H. and Aebi, U. (1988) J. Cell Biol. 106, 785-796] through which it drives its supramolecular patterning, and which ultimately generate its functional diversity.