Tropomyosin isoform 5b is expressed in human erythrocytes: implications of tropomodulin-TM5 or tropomodulin-TM5b complexes in the protofilament and hexagonal organization of membrane skeletons.

The human erythrocyte membrane skeleton consists of hexagonal lattices with junctional complexes containing F-actin protofilaments of approximately 33-37 nm in length. We hypothesize that complexes formed by tropomodulin, a globular capping protein at the pointed end of actin filaments, and tropomyosin (TM), a rod-like molecule of approximately 33-35 nm, may contribute to the formation of protofilaments. We have previously cloned the human tropomodulin complementary DNA and identified human TM isoform 5 (hTM5), a product of the gamma-TM gene, as one of the major TM isoforms in erythrocytes. We now identify TM5b, a product of the alpha-TM gene, to be the second major TM isoform. TM5a, the alternatively spliced isoform of the alpha-TM gene, which differs by 1 exon and has a weaker actin-binding affinity, however, is not present. TM4, encoded by the delta-TM gene, is not present either. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, hTM5 comigrated with the slower TM major species in erythrocyte membranes, and hTM5b comigrated with the faster TM major species. TM5b, like TM5, binds strongly to tropomodulin, more so than other TM isoforms. The 2 major TM isoforms, therefore, share several common features: They have 248 residues, are approximately 33-35 nm long, and have high affinities toward F-actin and tropomodulin. These common features may be the key to the mechanism by which protofilaments are formed. Tropomodulin-TM5 or tropomodulin-TM5b complexes may stabilize F-actin in segments of approximately 33-37 nm during erythroid terminal differentiation and may, therefore, function as a molecular ruler. TM5 and TM5b further define the hexagonal geometry of the skeletal network and allow actin-regulatory functions of TMs to be modulated by tropomodulin. (Blood. 2000;95:1473-1480)

HGF is an autocrine growth factor for skeletal muscle satellite cells in vitro.

Muscle satellite cell activation following injury is essential for muscle repair, and hepatocyte growth factor/scatter factor (HGF) was the first growth factor shown to be able to stimulate activation and early division of adult satellite cells in culture and in muscle tissue. In addition, HGF was shown to be present in uninjured and injured skeletal muscle. Experiments in this report demonstrate that cultured satellite cells also synthesize and secrete HGF. Reverse transcription-polymerase chain reaction (RT-PCR) was used to demonstrate the presence of HGF mRNA in cultured adult satellite cells as early as 12 h from the time of plating. Message content was detectable at early times in culture and appeared to increase between 36 and 48 h. HGF protein expression was demonstrated during this time period by immunofluorescence localization; HGF was localized to mononucleated cells and multinucleated myotubes. HGF message was not detectable in muscle-derived fibroblast clones, and fibroblast-like cells in satellite cell cultures were negative for HGF by immunofluorescence analysis. Furthermore, Western blot analysis revealed the presence of HGF in satellite cell culture conditioned medium, associated with the cell surface and inside cells. Finally, the addition of neutralizing HGF antibodies during the proliferation phase in culture (42-90 h) significantly reduced cell proliferation. These experiments indicate that HGF is expressed by cultured satellite cells and that endogenous HGF from satellite cells can act in an autocrine fashion. Because HGF plays a central role in satellite cell activation, it is likely that direct administration of HGF into damaged muscle may represent a potentially useful approach for stimulating muscle repair. This approach may also be useful in enhancing the efficiency of myoblast transplantation in vivo.

Microinjection of calpastatin inhibits fusion in myoblasts.

Rat satellite cells (RSC) were microinjected with purified calpastatin or m-calpain, and myoblasts from a C2C12 mouse line were microinjected with purified calpastatin. Microinjection with calpastatin completely prevented fusion of myoblasts from both sources, whereas microinjection with m-calpain significantly increased the rate of fusion of cultured RSC; 44% of the nuclei of RSC cultures were in multinucleated myotubes within 48 h after microinjection with m-calpain plus labeled dextran, whereas only 15% of the nuclei were in multinucleated myotubes after microinjection with dextran alone. Western analyses indicated that neither RSC nor C2C12 myoblasts contained detectable amounts of mu-calpain before fusion. The levels of calpastatin in C2C12 myoblasts increased as cells passed from the proliferative stage to the onset of fusion, and these levels increased substantially in both the C2C12 and the RSC cells as they progressed to the late or postfusion stage. Both RSC and C2C12 myoblasts contained an 80-kDa polypeptide that was labeled with an anti-m-calpain antibody in Western blots. The results are consistent with a role of the calpain system (m-calpain in these myoblast lines) in remodeling of the cytoskeletal/plasma membrane interactions during cell fusion.

Distinct localizations of tropomyosin isoforms in LLC-PK1 epithelial cells suggests specialized function at cell-cell adhesions.

At least eight nonmuscle, nonbrain tropomyosin isoforms have been described. We used antibodies, microinjection, and transfection to characterize their expression and localization in LLC-PK1 kidney epithelial cells and compared them with other cells. Similar to primary enterocytes, LLC-PK1 cells exhibited predominantly TM-1 and TM-3 of the high-molecular-weight (HMW) isoforms; TM-5 and TM-5b of the low-molecular-weight (LMW) isoforms. Neither TM-4 nor TM-5a was detectable in the LLC-PKI cells. Immunofluorescence studies revealed that HMW isoforms were localized only on stress fibers, not adhesion belts, whereas the adhesion belts were stained by LMW isoform antibodies. When exogenous proteins are introduced either by transfection or microinjection, the HMW isoforms do not incorporate into the adhesion belt, whereas the LMW isoforms can incorporate into the stress fibers, thus indicating there are different mechanisms at work for the selective localization. Temporal changes in the microfilament system of the LLC-PK1 cells were studied during differentiation in culture as defined by spectrin expression and F-actin architecture. Western blot analysis indicated that TM-5b is only expressed in the LLC-PK1 cells after a certain degree of maturation in culture, which suggests isoform switching after the cell-cell contacts are developed. Collectively these results demonstrate that epithelial cells express a complex pattern of TM isoforms, which exhibit differential localizations within the cells and different patterns of expression depending on their origin and stage of differentiation. The implication of differential localization of TM isoforms on their specific functions is discussed.

Low molecular weight rat fibroblast tropomyosin 5 (TM-5): cDNA cloning, actin-binding, localization, and coiled-coil interactions.

Previous studies have shown that three distinct genes encode six isoforms of tropomyosin (TM) in rat fibroblasts: the alpha gene encodes TM-2, TM-3, TM-5a, and TM-5b, the beta gene encodes TM-1, and the TM-4 gene encodes TM-4. Here we report the characterization of a cDNA clone encoding the most recent rat fibroblast TM to be identified, herein referred to as TM-5, that is the product of a fourth gene that is homologous to the human hTMnm gene, herein referred to as the rat slow-twitch alpha TM gene. The cDNA clone is approximately 1.7 kb and encodes a protein of 248 amino acids. Using two-dimensional gel electrophoresis, the TM-5 protein was found to co-migrate with fibroblast TM-5a and 5b. Comparison of the amino acid sequences of TM-5 to other fibroblast isoforms encoded by the alpha, beta, and TM-4 genes revealed a high degree of homology, although there were regions of divergence among the different isoforms. The gene encoding TM-5 is expressed in all tissues examined including skeletal muscle, stomach, heart, liver, kidney, uterus, spleen, brain, and diaphragm. However, Northern blot and RNase protection analyses revealed the presence of different mRNAs in fibroblasts, striated muscle (skeletal and diaphragm), and brain, which are expressed via alternative RNA splicing and the use of alternative promoters. The TM-5 protein was expressed in a bacterial system and tested for its ability to bind actin in vitro and in vivo. The apparent TM association constant (Ka) was taken as the free concentration at half saturation and was found to be 3 microM for TM-5 compared to 2 microM for TM-5b at an F-actin concentration of 42 microM. When fluorescently-labeled TM-5 was microinjected into living rat fibroblasts, it localized to the stress fibers and ruffles of the leading lamella. The coiled-coil interactions of TM-5 with other low and high molecular weight TM isoforms were studied. TM-5 and TM-4 were capable of dimerizing with each other as well as with other low molecular weight isoforms (TM-5a and TM-5b), but not with the HMW isoforms (TM-1, TM-2, and TM-3). In addition, TM-5a and TM-5b were unable to heterodimerize with each other. The implications of these results in understanding the role of TM diversity in cytoskeletal dynamics are discussed.

A focal adhesion factor directly linking intracellularly motile Listeria monocytogenes and Listeria ivanovii to the actin-based cytoskeleton of mammalian cells.

The surface-bound ActA polypeptide of the intracellular bacterial pathogen Listeria monocytogenes is the sole listerial factor needed for recruitment of host actin filaments by intracellularly motile bacteria. Here we report that following Listeria infection the host vasodilator-stimulated phosphoprotein (VASP), a microfilament- and focal adhesion-associated substrate of both the cAMP- and cGMP-dependent protein kinases, accumulates on the surface of intracytoplasmic bacteria prior to the detection of F-actin 'clouds'. VASP remains associated with the surface of highly motile bacteria, where it is polarly located, juxtaposed between one extremity of the bacterial surface and the front of the actin comet tail. Since actin filament polymerization occurs only at the very front of the tail, VASP exhibits properties of a host protein required to promote actin polymerization. Purified VASP binds directly to the ActA polypeptide in vitro. A ligand-overlay blot using purified radiolabelled VASP enabled us to identify the ActA homologue of the related intracellular motile pathogen, Listeria ivanovii, as a protein with a molecular mass of approximately 150 kDa. VASP also associates with actin filaments recruited by another intracellularly motile bacterial pathogen, Shigella flexneri. Hence, by the simple expedient of expressing surface-bound attractor molecules, bacterial pathogens effectively harness cytoskeletal components to achieve intracellular movement.

Exploitation of microfilament proteins by Listeria monocytogenes: microvillus-like composition of the comet tails and vectorial spreading in polarized epithelial sheets.

Effective cell-to-cell spreading of the facultative intracellular pathogen Listeria monocytogenes requires the interaction between bacteria and the microfilament system of the host cell. By recruiting actin filaments into a 'comet tail' localized at one pole of the bacterial cell wall, Listeria become mobile and propel themselves through the cytoplasm. They create protrusions at the plasma membrane that can invaginate adjacent cells. In this work, we have analysed the structural composition of Listeria-recruited microfilaments in various epithelial cell lines by immunofluorescence microscopy. The microfilament-crosslinking proteins alpha-actinin, fimbrin and villin were localized around bacteria as soon as actin filaments could be detected on the bacterial surface. Surprisingly, the same was found for ezrin/radixin, proteins involved in linking microfilaments to the plasma membrane. We found that in a polarized cell line derived from brush border kidney epithelium (LLC-PK1), the actin filaments surrounding intracytoplasmic motile bacteria show the same immunoreactivity as the brush border-like microvilli, when analysed by a specific actin antibody. The successful invasion of polarized LLC-PK1 islets is vectorial, i.e. it progresses predominantly from the periphery of the islets towards the centre. Infection of the peripheral cells is sufficient for infiltration of the entire cellular islets, without any further contact with the extracellular milieu. This is in contrast to nonpolarized epithelial sheets, which can be invaded from the apical surface of any individual cell. The importance of active bacterial motility in this vectorial spreading is emphasized by our finding that an isogenic Listeria mutant that is unable to recruit actin filaments cannot colonize polarized epithelial layers but accumulates in the peripheral cells of the islets.

Chicken antibodies to rabbit muscle actin with a restricted repertoire of F-actin recognition.

This report describes a polyclonal antibody against actin with unexpected and unusual properties. The antibody was raised in chicken immunized with a complex of DNase I and rabbit skeletal muscle actin, and purified from egg yolk by affinity chromatography. In Western blots, it reacted with alpha, beta and gamma isoforms of actin. In immunofluorescence and dot blot assays, however, it recognized selectively actin filaments in myofibrils, microvilli of brush border-type epithelium and the "comet tails" of the intracellular parasite Listeria monocytogenes, while it did not react with stress fibers and peripheral belts of fibroblasts and epithelial cells, respectively. This reactivity pattern is reminiscent of that previously described for a monoclonal mouse antibody raised against smooth muscle actin (Sawtell et al., Cell Motil. Cytoskel. 11, 318, 1988). The data presented in this study are consistent with the hypothesis that the chicken antibody recognizes an actin epitope/actin epitopes either accessible in only a subpopulation of microfilaments, or expressed only in a particular conformation of F-actin.

Dynamic aspects of microfilament-membrane attachments.

Microfilament-membrane attachment sites are complex structures that are essential for tissue differentiation in animals. In this article, we focus on the assembly and dynamics of such contact sites as seen in two cell types differentiating in cultures of the embryonic chicken heart, cardiocytes and fibroblasts. Concentrating on the cytoplasmic domain, we refer to previous biochemical, light, and electron microscopic studies on the structure and dynamics of these regions and supplement them with our own recent data. Although many details are still to be elucidated, we would like to propose the following model. Actin, alpha-actinin and vinculin are the major structural components of all microfilament-membrane contacts. Various subtypes of junctions are characterised by additional structural components or by specific isoforms. Temporal regulation of contact sites is linked to assembly and disassembly of microfilaments and might be controlled by special regulatory proteins. Finally, the cytoplasmic domains of junctional complexes may serve as structural matrices for the positioning of proteins involved in signal transduction pathways.