Overexpression of human fibroblast caldesmon fragment containing actin- , Ca++/calmodulin-, and tropomyosin-binding domains stabilizes endogenous tropomyosin and microfilaments

Fibroblast caldesmon is a protein postulated to participate in the modulation of the actin cytoskeleton and the regulation of actin-based motility. The cDNAs encoding the NH2-terminal (aa.1-243, CaD40) and COOH-terminal (aa.244-538, CaD39) fragments of human caldesmon were subcloned into expression vectors and we previously reported that bacterially produced CaD39 protein retains its actin-binding properties as well as its ability to enhance low M(r) tropomyosin (TM) binding to actin and to inhibit TM-actin-activated HMM ATPase activity in vitro (Novy, R. E., J. R. Sellers, L.-F. Liu, and J. J.-C. Lin. 1993. Cell Motil. Cytoskeleton. 26:248-261). Bacterially produced CaD40 does not bind actin. To study the in vivo effects of CaD39 expression on the stability of actin filaments in CHO cells, we isolated and characterized stable CHO transfectants which express varying amounts of CaD39. We found that expression of CaD39 in CHO cells stabilized microfilament bundles as well as endogenous TM. CaD39-expressing clones displayed an increased resistance to cytochalasin B and Triton X-100 treatments and yielded increased amounts of TM-containing actin filaments in microfilament isolation procedures. In addition, analysis of these clones with immunoblotting and indirect immunofluorescence microscopy with anti-TM antibody revealed that stabilized endogenous TM and enhanced TM-containing microfilament bundles parallel increased amounts of CaD39 expression. The increased TM observed corresponded to a decrease in TM turnover rate and did not appear to be due to increased synthesis of endogenous TM. Additionally, the phenomenon of stabilized TM did not occur in stable CHO clones expressing CaD40. Therefore, it is likely that CaD39 can enhance TM's binding to F-actin in vivo, thus reducing TM's rate of turnover and stabilizing actin microfilament bundles.     

Forced expression of a dominant-negative chimeric tropomyosin causes abnormal motile behavior during cell division

Forced expression of the chimeric human fibroblast tropomyosin 5/3 (hTM5/3) in CHO cell was previously shown to affect cytokinesis [Warren et al., 1995: J. Cell Biol. 129:697-708]. To further investigate the phenotypic consequences of misexpression, we have compared mitotic spindle organization and dynamic 2D and 3D shape changes during mitosis in normal cells and in a hTM5/3 misexpressing (mutant) cell line. Immunofluorescence microscopy of wild type and mutant cells stained with monoclonal anti-tubulin antibody revealed that the overall structures of mitotic spindles were not significantly different. However, the axis of the mitotic spindle in mutant cells was more frequently misaligned with the long axis of the cell than that of wild type cells. To assess behavioral differences during mitosis, wild type and mutant cells were reconstructed in 2D and 3D and motion analyzed with the computer-assisted 2D and 3D Dynamic Image Analysis Systems (2D-DIAS, 3D-DIAS). Mutant cells abnormally formed large numbers of blebs during the later stages of mitosis and took longer to proceed from the start of anaphase to the start of cytokinesis. Furthermore, each mutant cell undergoing mitosis exhibited greater shape complexity than wild type cells, and in every case lifted one of the two evolving daughter cells off the substratum and abnormally twisted. These results demonstrate that misexpression of hTM5/3 in CHO cells leads to morphological instability during mitosis. Misexpression of hTM5/3 interferes with normal tropomyosin function, suggesting in turn that tropomyosin plays a role through its interaction with actin microfilaments in the regulation of the contractile ring, in the localized suppression of blebbing, in the maintenance of polarity and spatial symmetry during cytokinesis, and in cell spreading after cytokinesis is complete.     

In vitro and in vivo characterization of four fibroblast tropomyosins produced in bacteria: TM-2, TM-3, TM-5a, and TM-5b are co-localized in interphase fibroblasts

Most cell types express several tropomyosin isoforms, the individual functions of which are poorly understood. In rat fibroblasts there are at least six isoforms; TM-1, TM-2, TM-3, TM-4, TM-5a, and TM-5b. TM-1 is the product of the beta gene. TM-4 is produced from the TM-4 gene, and TMs 2, 3, 5a, and 5b are the products of the alpha gene. To begin to study the localization and function of the isoforms in fibroblasts, cDNAs for TM isoforms 2, 3, 5a, and 5b were placed into bacterial expression vectors and used to produce TM isoforms. The bacterially produced TMs were determined to be full length by sequencing the amino- and carboxy termini. These TMs were found to bind to F-actin in vitro, with properties similar to that of skeletal muscle TM. In addition, competition experiments demonstrated that TM-5b was better than TM-5a in displacing other TM isoforms from F-actin in vitro. To investigate the intracellular localization of these fibroblast isoforms, each was derivatized with a fluorescent chromophore and microinjected into rat fibroblasts. TM-2, TM-3, TM-5a, and TM-5b were each found to associate along actin filaments. There was no preferred cellular location or subset of actin filaments for these isoforms. Furthermore, co-injection of two isoforms labeled with different fluorochromes showed identical staining. At the level of the light microscope, these isoforms from the alpha gene do not appear to achieve different functions by binding to particular subsets of actin filaments or locations in cells. Some alternative possibilities are discussed. The results show that bacterially produced TMs can be used to study in vitro and in vivo properties of the isoforms.     

Tropomodulin-binding site mapped to residues 7-14 at the N-terminal heptad repeats of tropomyosin isoform 5

Tropomodulin is a globular protein that caps the pointed end of actin filaments by complexing with the N-terminus of a tropomyosin (TM) molecule. TM consists of coiled coils except for the N-terminus, which may be globular. Here we report that human TM isoform 5 (hTM5) lacking the N-terminal 18 residues lost its binding activity toward tropomodulin. We further characterized the tropomodulin-binding site by creating a series of deletion and missense mutations within this region, followed by a solid-phase binding assay. I7, V10, and I14, hydrophobic residues located at the a and d positions of N-terminal heptad repeats involving intertwine, are essential for tropomodulin binding. R12, a positively charged residue at the f position, is also involved in recognition. In contrast, A2R and G3Y mutations, each creating a bulky N-terminus, did not alter the binding. In addition, rat TM5b, which differs from hTM5 in residues 4-6, exhibits a similar binding affinity. The tropomodulin-binding site, therefore, is mapped to residues 7-14 at the beginning of the long heptad repeats. Column chromatography revealed that hTM5 mutants remained capable of dimerization. Results also suggest tropomodulin has a groove-type, rather than a cavity-type, binding site for hTM5. We also mapped the epitope of monoclonal antibody LC1 to residues 4-10 of hTM5 and showed the competition between mAb LC1 and tropomodulin in hTM5 binding. Since the N-terminal residues need to overlap with the C-terminus of TM in their head-to-tail association, this investigation elucidates the mechanisms by which the tropomodulin-hTM5 complex is formed and functions in regulating the actin filaments.     

Sorting of tropomyosin isoforms in synchronised NIH 3T3 fibroblasts: evidence for distinct microfilament populations

The nonmuscle actin cytoskeleton consists of multiple networks of actin microfilaments. Many of these filament systems are bound by the actin-binding protein tropomyosin (Tm). We investigated whether Tm isoforms could be cell cycle regulated during G0 and G1 phases of the cell cycle in synchronised NIH 3T3 fibroblasts. Using Tm isoform-specific antibodies, we investigated protein expression levels of specific Tms in G0 and G1 phases and whether co-expressed isoforms could be sorted into different compartments. Protein levels of Tms 1, 2, 5a, 6, from the alpha Tm(fast) and beta-Tm genes increased approximately 2-fold during mid-late G1. Tm 3 levels did not change appreciably during G1 progression. In contrast, Tm 5NM gene isoform levels (Tm 5NM-1-11) increased 2-fold at 5 h into G1 and this increase was maintained for the following 3 h. However, Tm 5NM-1 and -2 levels decreased by a factor of three during this time. Comparison of the staining of the antibodies CG3 (detects all Tm 5NM gene products), WS5/9d (detects only two Tms from the Tm 5NM gene, Tm 5NM-1 and -2) and alpha(f)9d (detects specific Tms from the alpha Tm(fast) and beta-Tm genes) antibodies revealed 3 spatially distinct microfilament systems. Tm isoforms detected by alpha(f)9d were dramatically sorted from isoforms from the Tm 5NM gene detected by CG3. Tm 5NM-1 and Tm 5NM-2 were not incorporated into stress fibres, unlike other Tm 5NM isoforms, and marked a discrete, punctate, and highly polarised compartment in NIH 3T3 fibroblasts. All microfilament systems, excluding that detected by the WS5/9d antibody, were observed to coalign into parallel stress fibres at 8 h into G1. However, Tms detected by the CG3 and alpha(f)9d antibodies were incorporated into filaments at different times indicating distinct temporal control mechanisms. Microfilaments in NIH 3T3 cells containing Tm 5NM isoforms were more resistant to cytochalasin D-mediated actin depolymerisation than filaments containing isoforms from the alpha Tm(fast) and beta-Tm genes. This suggests that Tm 5NM isoforms may be in different microfilaments to alpha Tm(fast) and beta-Tm isoforms even when present in the same stress fibre. Staining of primary mouse fibroblasts showed identical Tm sorting patterns to those seen in cultured NIH 3T3 cells. Furthermore, we demonstrate that sorting of Tms is not restricted to cultured cells and can be observed in human columnar epithelial cells in vivo. We conclude that the expression and localisation of Tm isoforms are differentially regulated in G0 and G1 phase of the cell cycle. Tms mark multiple microfilament compartments with restricted tropomyosin composition. The creation of distinct microfilament compartments by differential sorting of Tm isoforms is observable in primary fibroblasts, cultured 3T3 cells and epithelial cells in vivo.     

Tissue-specific tropomyosin isoform composition.

Four distinct genes encode tropomyosin (Tm) proteins, integral components of the actin microfilament system. In non-muscle cells, over 40 Tm isoforms are derived using alternative splicing. Distinct populations of actin filaments characterized by the composition of these Tm isoforms are found differentially sorted within cells (Gunning et al. 1998b). We hypothesized that these distinct intracellular compartments defined by the association of Tm isoforms may allow for independent regulation of microfilament function. Consequently, to understand the molecular mechanisms that give rise to these different microfilaments and their regulation, a cohort of fully characterized isoform-specific Tm antibodies was required. The characterization protocol initially involved testing the specificity of the antibodies on bacterially produced Tm proteins. We then confirmed that these Tm antibodies can be used to probe the expression and subcellular localization of different Tm isoforms by Western blot analysis, immunofluorescence staining of cells in culture, and immunohistochemistry of paraffin wax-embedded mouse tissues. These Tm antibodies, therefore, have the capacity to monitor specific actin filament populations in a range of experimental systems.     

Dominant negative effect of cytoplasmic actin isoproteins on cardiomyocyte cytoarchitecture and function

The intracompartmental sorting and functional consequences of ectopic expression of the six vertebrate actin isoforms was investigated in different types of cultured cells. In transfected fibroblasts all isoactin species associated with the endogenous microfilament cytoskeleton, even though cytoplasmic actins also showed partial localization to peripheral submembranous sites. Functional and structural studies were performed in neonatal and adult rat cardiomyocytes. All the muscle isoactin constructs sorted preferentially to sarcomeric sites and, to a lesser extent, also to stress-fiber-like structures. The expression of muscle actins did not interfere with cell contractility, and did not disturb the localization of endogenous sarcomeric proteins. In sharp contrast, ectopic expression of the two cytoplasmic actin isoforms resulted in rapid cessation of cellular contractions and induced severe morphological alterations characterized by an exceptional outgrowth of filopodia and cell flattening. Quantitative analysis in neonatal cardiomyocytes indicated that the levels of accumulation of the different isoactins are very similar and cannot be responsible for the observed isoproteins- specific effects. Structural analysis revealed a remodeling of the cytoarchitecture including a specific alteration of sarcomeric organization; proteins constituting the sarcomeric thin filaments relocated to nonmyofibrillar sites while thick filaments and titin remained unaffected. Experiments with chimeric proteins strongly suggest that isoform specific residues in the carboxy-terminal portion of the cytoplasmic actins are responsible for the dominant negative effects on function and morphology.     

Specification of actin filament function and molecular composition by tropomyosin isoforms

The specific functions of greater than 40 vertebrate nonmuscle tropomyosins (Tms) are poorly understood. In this article we have tested the ability of two Tm isoforms, TmBr3 and the human homologue of Tm5 (hTM5(NM1)), to regulate actin filament function. We found that these Tms can differentially alter actin filament organization, cell size, and shape. hTm5(NM1) was able to recruit myosin II into stress fibers, which resulted in decreased lamellipodia and cellular migration. In contrast, TmBr3 transfection induced lamellipodial formation, increased cellular migration, and reduced stress fibers. Based on coimmunoprecipitation and colocalization studies, TmBr3 appeared to be associated with actin-depolymerizing factor/cofilin (ADF)-bound actin filaments. Additionally, the Tms can specifically regulate the incorporation of other Tms into actin filaments, suggesting that selective dimerization may also be involved in the control of actin filament organization. We conclude that Tm isoforms can be used to specify the functional properties and molecular composition of actin filaments and that spatial segregation of isoforms may lead to localized specialization of actin filament function.     

Immunolocalization of the gamma isoform of nonmuscle actin in cultured cells

In many vertebrate nonmuscle cells, the microfilament subunit protein, actin, exists as two isoforms, called beta and gamma, whose sequences differ only in their amino-terminal regions. We have prepared a peptide antibody specifically reactive with the amino-terminal sequence of gamma actin. This antibody reacted with nonmuscle actin as determined by Western blots of SDS gels, and reacted with the gamma, but not the beta, nonmuscle actin isoform as shown by Western blots of isoelectric focusing gels. In immunofluorescence experiments, the gamma peptide antibody stained microfilament bundles, ruffled edges, and the contractile ring of a variety of cultured cells, including mouse L cells, which have previously been reported to contain only the beta actin isoform (Sakiyama, S., S. Fujimura, and H. Sakiyama, 1981, J. Biol. Chem., 256:31-33). Double immunofluorescence experiments using the gamma peptide antibody and an antibody reactive with all actin isoforms revealed no differences in isoform localization. Thus, at the level of resolution of light microscopy, we have detected the gamma actin isoform in all microfilament-containing structures in cultured cells, and have observed no subcellular sorting of the nonmuscle actin isoforms.     

Polycystin-2 associates with tropomyosin-1, an actin microfilament component

Polycystin-2 (PC2) is the product of the second cloned gene (PKD2) responsible for autosomal dominant polycystic kidney disease and has recently been shown to be a calcium-permeable cation channel. PC2 has been shown to connect indirectly with the actin microfilament. Here, we report a direct association between PC2 and the actin microfilament. Using a yeast two-hybrid screen, we identified a specific interaction between the PC2 cytoplasmic C-terminal domain and tropomyosin-1 (TM-1), a component of the actin microfilament complex. Tropomyosins constitute a protein family of more than 20 isoforms arising mainly from alternative splicing and are present in muscle as well as non-muscle cells. We identified a new TM-1 splicing isoform in kidney and heart (TM-1a) that differs from TM-1 in the C terminus and interacted with PC2. In vitro biochemical methods, including GST pull-down, blot overlay and microtiter binding assays, confirmed the interaction between PC2 and the two TM-1 isoforms. Further experiments targeted the interacting domains to G821-R878 of PC2 and A152-E196, a common segment of TM-1 and TM-1a. Indirect double immunofluorescence experiments showed partial co-localization of PC2 and TM-1 in transfected mouse fibroblast NIH 3T3 cells. Co-immunoprecipitation (co-IP) studies using 3T3 cells and Xenopus oocytes co-expressing PC2 and TM-1 (or TM-1a) revealed in vivo association between the protein pairs. Furthermore, the in vivo interaction between the endogenous PC2 and TM-1 was demonstrated also by reciprocal co-IP using native human embryonic kidney cells and human adult kidney. Considering previous reports that TM-1 acts as a suppressor of neoplastic growth of transformed cells, it is possible that TM-1 contributes to cyst formation/growth when the anchorage of PC2 to the actin microfilament via TM-1 is altered.     

The amino-terminal domain of the B subunit of vacuolar H+-ATPase contains a filamentous actin binding site

Vacuolar H(+)-ATPase (V-ATPase) binds actin filaments with high affinity (K(d) = 55 nm; Lee, B. S., Gluck, S. L., and Holliday, L. S. (1999) J. Biol. Chem. 274, 29164-29171). We have proposed that this interaction is an important mechanism controlling transport of V-ATPase from the cytoplasm to the plasma membrane of osteoclasts. Here we show that both the B1 (kidney) and B2 (brain) isoforms of the B subunit of V-ATPase contain a microfilament binding site in their amino-terminal domain. In pelleting assays containing actin filaments and partially disrupted V-ATPase, B subunits were found in greater abundance in actin pellets than were other V-ATPase subunits, suggesting that the B subunit contained an F-actin binding site. In overlay assays, biotinylated actin filaments also bound to the B subunit. A fusion protein containing the amino-terminal half of B1 subunit bound actin filaments tightly, but fusion proteins containing the carboxyl-terminal half of B1 subunit, or the full-length E subunit, did not bind F-actin. Fusion proteins containing the amino-terminal 106 amino acids of the B1 isoform or the amino-terminal 112 amino acids of the B2 isoform bound filamentous actin with K(d) values of 130 and 190 nm, respectively, and approached saturation at 1 mol of fusion protein/mol of filamentous actin. The B1 and B2 amino-terminal fusion proteins competed with V-ATPase for binding to filamentous actin. In summary, binding sites for F-actin are present in the amino-terminal domains of both isoforms of the B subunit, and likely are responsible for the interaction between V-ATPase and actin filaments in vivo.     

Microfilament bundles in cultured cells : Correlation with anchorage independence and tumorigenicity in nude mice

The distribution of actin microfilament bundles in cell lines 3T3B, CHO, HeLa and CLID extracted with 0.1% Triton X-100 was examined by indirect immunofluorescence using human actin antibodies and by electron microscopy of whole cells grown directly on support grids. Anchorage dependence as determined by growth in soft agar and tumorigenicity in nude mice was also investigated. Immunofluorescent staining showed that CHO and HeLa cells have normal numbers and distributions of actin microfilament bundles as compared with similarly spread control 3T3B cells. A significant fraction of the mouse CLID cells showed comparable numbers of microfilament bundles as 3T3B cells but their distribution was markedly different. In many cases the bundles radiated from a region close to the cell's centre or near its projections and usually penetrated the projections. The presence of diffuse staining in 4% of the cell population also indicated the existence in these cells of disorganized actin. Electron microscope studies of well spread regions of negatively-stained, Triton-extracted cells corroborated the observations made with the immunofluorescence technique. In 3T3B, CHO and CLID cells abundant microtubules were found, colinearly arranged with actin filaments in the thin cytoplasmic extensions. While CLID, CHO and HeLa cells showed the capacity to grow in soft agar, only CLID and HeLa cells produced tumours in athymic nude mice. The observations suggest that a reduction or disorganisation of the actin microfilament bundles may not in itself be essential at least for the non-virally transformed cells studied to show anchorage independence or to produce tumours in nude mice.     

Differential binding of tropomyosin isoforms to actin modified with m-maleimidobenzoyl-N-hydroxysuccinimide ester and fluorescein-5-isothiocyanate

Differential interactions of tropomyosin (TM) isoforms with actin can be important for determination of the thin filament functions. A mechanism of tropomyosin binding to actin was studied by comparing interactions of five αTM isoforms with actin modified with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and with fluorescein-5-isothiocyanate (FITC). MBS attachment sites were revealed with mass spectrometry methods. We found that the predominant actin fraction was cross-linked by MBS within subdomain 3. A smaller fraction of the modified actin was cross-linked within subdomain 2 and between subdomains 2 and 1. Moreover, investigated actins carried single labels in subdomains 1, 2, and 3. Such extensive modification caused a large decrease in actin affinity for skeletal and smooth muscle tropomyosins, nonmuscle TM2, and chimeric TM1b9a. In contrast, binding of nonmuscle isoform TM5a was less affected. Isoform’s affinity for actin modified in subdomain 2 by binding of FITC to Lys61 was intermediate between the affinity for native actin and MBS-modified actin except for TM5a, which bound to FITC–actin with similar affinity as to actin modified with MBS. The analysis of binding curves according to the McGhee–von Hippel model revealed that binding to an isolated site, as well as cooperativity of binding to a contiguous site, was affected by both actin modifications in a TM isoform-specific manner.     

抗人血栓调节蛋白单克隆抗体的制备与鉴定

为了制备特异性抗人血栓调节蛋白(human thrombomodulin,hTM)的单克隆抗体(monoclonal antibody,McAb),利用脂质体Lipofectamine 2000将包含hTM全长cDNA序列的重组表达质粒pThr402转染CHO细胞,经G418筛选及相关鉴定后获得高效稳定表达hTM的CHO-TM5细胞株。将CHO-TM5细胞直接免疫Balb/c小鼠,应用杂交瘤技术,通过细胞ELISA (cellular enzyme-linked immunoabsorbent assay,CELISA)筛选出阳性克隆后,将杂交瘤细胞株腹腔注射Balb/c小鼠诱生腹水。用CELISA、流式细胞术、免疫组织化学染色法及免疫印迹法对所获McAb的特异性进行鉴定。我们获得了1株可稳定分泌抗hTM的McAb的杂交瘤细胞株NH-1,其亚型为IgGl,McAb腹水效价为1×10~(-6),腹水抗体含量为20 mg/ml。NH-1对相应抗原具有较高的组织特异性,在体内与正常组织的交叉反应少,对人脐静脉内皮细胞、CHO-TM5有特异性结合反应,说明NH-1可特异性识别天然的hTM分子,为进一步应用此McAb进行hTM生物学功能及临床意义研究提供了基础。     

Actin-depolymerizing factor and cofilin-1 play overlapping roles in promoting rapid F-actin depolymerization in mammalian nonmuscle cells

Actin-depolymerizing factor (ADF)/cofilins are small actin-binding proteins found in all eukaryotes. In vitro, ADF/cofilins promote actin dynamics by depolymerizing and severing actin filaments. However, whether ADF/cofilins contribute to actin dynamics in cells by disassembling "old" actin filaments or by promoting actin filament assembly through their severing activity is a matter of controversy. Analysis of mammalian ADF/cofilins is further complicated by the presence of multiple isoforms, which may contribute to actin dynamics by different mechanisms. We show that two isoforms, ADF and cofilin-1, are expressed in mouse NIH 3T3, B16F1, and Neuro 2A cells. Depleting cofilin-1 and/or ADF by siRNA leads to an accumulation of F-actin and to an increase in cell size. Cofilin-1 and ADF seem to play overlapping roles in cells, because the knockdown phenotype of either protein could be rescued by overexpression of the other one. Cofilin-1 and ADF knockdown cells also had defects in cell motility and cytokinesis, and these defects were most pronounced when both ADF and cofilin-1 were depleted. Fluorescence recovery after photobleaching analysis and studies with an actin monomer-sequestering drug, latrunculin-A, demonstrated that these phenotypes arose from diminished actin filament depolymerization rates. These data suggest that mammalian ADF and cofilin-1 promote cytoskeletal dynamics by depolymerizing actin filaments and that this activity is critical for several processes such as cytokinesis and cell motility.     

Tropomyosin isoform 3 promotes the formation of filopodia by regulating the recruitment of actin-binding proteins to actin filaments

Tropomyosins are believed to function in part by stabilizing actin filaments. However, accumulating evidence suggests that fundamental differences in function exist between tropomyosin isoforms, which contributes to the formation of functionally distinct filament populations. We investigated the functions of the high-molecular-weight isoform Tm3 and examined the molecular properties of Tm3-containing actin filament populations. Overexpression of the Tm3 isoform specifically induced the formation of filopodia and changes in actin solubility. We observed alterations in actin-binding protein recruitment to filaments, co-incident with changes in expression levels, which can account for this functional outcome. Tm3-associated filaments recruit active actin depolymerizing factor and are bundled into filopodia by fascin, which is both up-regulated and preferentially associated with Tm3-containing filaments in the Tm3 overexpressing cells. This study provides further insight into the isoform-specific roles of different tropomyosin isoforms. We conclude that variation in the tropomyosin isoform composition of microfilaments provides a mechanism to generate functionally distinct filament populations.     

A chimeric toxin to study the role of the 21 kDa GTP binding protein rho in the control of actin microfilament assembly.

We have developed a new tool for studying the role of rho in actin stress fibre formation. Clostridium botulinum exoenzyme C3 which affects actin microfilament assembly by ADP-ribosylation of p21 rho was genetically fused in various ways to diphtheria toxin (DT). The resulting chimeric toxins were tested on Vero cells. Chimeras of C3 and both the A and B fragments of diphtheria toxin had reduced cell binding activities but were apparently able to penetrate into Vero cells by the same mechanism as DT. Upon exposure to low pH, DC3B, a fusion protein of C3 and DT B fragment, had a high affinity for the DT receptor, but was apparently not able to translocate to the cytosol upon acidification. In spite of this, addition of picomolar concentrations of DC3B to the growth medium caused disruption of the cell microfilament system associated with vinculin and blocked cell growth efficiently, indicating that the C3 part of DC3B reached the cytosol, albeit by a different mechanism than that of whole diphtheria toxin. The chimeric DC3B toxin was also applied to Vero cells infected by Listeria monocytogenes, a pathogenic bacterium that uses an unknown mechanism of actin polymerization to move rapidly in the cytosol. DC3B inhibited the bacterially induced microfilament assembly indicating that L. monocytogenes utilizes a cellular rho dependent mechanism in this process.     

Tropomyosin assembly intermediates in the control of microfilament system turnover

Tropomyosin is a coiled-coil alpha-helical protein, which self-associates in a head-to-tail fashion along polymers of actin to produce thin filaments. Mammalian non-muscle cells express a large number of tropomyosin isoforms, which are differentially regulated during embryogenesis and associated with specialized actin microfilament ensembles in cells. The function of tropomyosin in specifying form and localization of these subcellular structures, and the precise mechanism(s) by which they carry out their functions, is unclear. This paper reports that, while the major fraction of non-muscle cell tropomyosin resides in actin thin filaments of the cytomatrix, the soluble part of the cytoplasm contains tropomyosins in the form of actin-free multimers, which are isoform specific and of high molecular weight (MW(app) 180,000-250,000). Stimulation of motile cells with growth factors induces a rapid, actin polymerization-dependent outgrowth of lamellipodia and filopodia. Concomitantly, the levels of tropomyosin isoform-specific multimers decrease, suggesting their involvement in actin thin filament formation. Malignant tumor cells have drastically altered levels and composition of tropomyosin isoform-specific multimers as well as tropomyosin in the cytomatrix.     

Regulation of tropomyosin expression in transformed granulosa cell lines with steroidogenic ability

The expression of the different tropomyosin isoforms was analyzed in primary granulosa cell cultures and in established granulosa cell lines cotransfected with SV40 and Ha-ras DNA which retain a high steroidogenic response to cAMP stimulation. In contrast to normal cells which greatly reduce the expression of all tropomyosin isoforms during development of steroidogenic ability, in the doubly transformed cells only the synthesis of the high molecular weight isoforms nos 2 and 3 was decreased. The expression of isoforms 1 and 5 was elevated in the cotransfected lines and that of tropomyosin 1 was further enhanced by cAMP stimulation. The increased synthesis of tropomyosins 1 and 5 is unique to SV40 transformation, since it was observed also in cells transfected with SV40 DNA alone. These cells displayed a well organized microfilament system, but have lost the ability to differentiate. The reduced expression of tropomyosins 2 and 3 and a poorly organized microfilament system appear to be a dominant feature of both the highly differentiated normal- and transformed-granulosa cells. It is suggested that the switches in tropomyosin isoform expression during development of the steroidogenic phenotype and in cell transformation may account for necessary changes in microfilament organization which accompany these cellular processes.     

Vertebrate isoforms of actin capping protein beta have distinct functions In vivo

Actin capping protein (CP) binds barbed ends of actin filaments to regulate actin assembly. CP is an alpha/beta heterodimer. Vertebrates have conserved isoforms of each subunit. Muscle cells contain two beta isoforms. beta1 is at the Z-line; beta2 is at the intercalated disc and cell periphery in general. To investigate the functions of the isoforms, we replaced one isoform with another using expression in hearts of transgenic mice.Mice expressing beta2 had a severe phenotype with juvenile lethality. Myofibril architecture was severely disrupted. The beta2 did not localize to the Z-line. Therefore, beta1 has a distinct function that includes interactions at the Z-line. Mice expressing beta1 showed altered morphology of the intercalated disc, without the lethality or myofibril disruption of the beta2-expressing mice.The in vivo function of CP is presumed to involve binding barbed ends of actin filaments. To test this hypothesis, we expressed a beta1 mutant that poorly binds actin. These mice showed both myofibril disruption and intercalated disc remodeling, as predicted.Therefore, CPbeta1 and CPbeta2 each have a distinct function that cannot be provided by the other isoform. CPbeta1 attaches actin filaments to the Z-line, and CPbeta2 organizes the actin at the intercalated discs.