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• W2022323043 abstract "Down-regulation of several key actin-binding proteins, such as α-actinin, vinculin, gelsolin, and tropomyosins (TMs), is considered to contribute to the disorganized cytoskeleton present in many neoplastic cells. TMs stabilize actin filaments against the gel severing actions of proteins such as cofilin. Among multiple TMs expressed in non-muscle cells, tropomyosin-1 (TM1) isoform induces stress fibers and functions as a suppressor of malignant transformation. However, the molecular mechanisms of TM1-mediated cytoskeletal effects and tumor suppression remain poorly understood. We have hypothesized that the ability of TM1 to stabilize microfilaments is crucial for tumor suppression. In this study, by employing a variant TM1, which contains an N-terminal hemagglutinin epitope tag, we demonstrate that the N terminus is a key determinant of tropomyosin-1 function. Unlike the wild type TM1, the modified protein fails to restore stress fibers and inhibit anchorage-independent growth in transformed cells. Furthermore, the N-terminal modification of TM1 disorganizes the cytoskeleton and delays cytokinesis in normal cells, abolishes binding to F-actin, and disrupts the dimeric associations in vivo. The functionally defective TM1 allows the association of cofilin to stress fibers and disorganizes the microfilaments, whereas wild type TM1 appears to restrict the binding of cofilin to stress fibers. TM1-induced cytoskeletal reorganization appears to be mediated through preventing cofilin interaction with microfilaments. Our studies provide in vivo functional evidence that the N terminus is a critical determinant of TM1 functions, which in turn determines the organization of stress fibers. Down-regulation of several key actin-binding proteins, such as α-actinin, vinculin, gelsolin, and tropomyosins (TMs), is considered to contribute to the disorganized cytoskeleton present in many neoplastic cells. TMs stabilize actin filaments against the gel severing actions of proteins such as cofilin. Among multiple TMs expressed in non-muscle cells, tropomyosin-1 (TM1) isoform induces stress fibers and functions as a suppressor of malignant transformation. However, the molecular mechanisms of TM1-mediated cytoskeletal effects and tumor suppression remain poorly understood. We have hypothesized that the ability of TM1 to stabilize microfilaments is crucial for tumor suppression. In this study, by employing a variant TM1, which contains an N-terminal hemagglutinin epitope tag, we demonstrate that the N terminus is a key determinant of tropomyosin-1 function. Unlike the wild type TM1, the modified protein fails to restore stress fibers and inhibit anchorage-independent growth in transformed cells. Furthermore, the N-terminal modification of TM1 disorganizes the cytoskeleton and delays cytokinesis in normal cells, abolishes binding to F-actin, and disrupts the dimeric associations in vivo. The functionally defective TM1 allows the association of cofilin to stress fibers and disorganizes the microfilaments, whereas wild type TM1 appears to restrict the binding of cofilin to stress fibers. TM1-induced cytoskeletal reorganization appears to be mediated through preventing cofilin interaction with microfilaments. Our studies provide in vivo functional evidence that the N terminus is a critical determinant of TM1 functions, which in turn determines the organization of stress fibers. One of the most common and yet prominent features of neoplastic cells is the presence of disorganized actin microfilaments (1Lin J.J. Warren K.S. Wamboldt D.D. Wang T. Lin J.L. Int. Rev. Cytol. 1997; 170: 1-38Google Scholar, 2Pawlak G. Helfman D.M. Curr. Opin. Genet. Dev. 2001; 11: 41-47Google Scholar). A functionally defective cytoskeleton, arising from the disorganized microfilament architecture, has been shown to be responsible for the loss of normal cellular morphology and cell polarity; altered intracellular transport, cell motility, and cell adhesion; and defective cytokinesis. Suppression of several key actin-binding proteins, including tropomyosins (TMs), 1The abbreviations used are: TMs, tropomyosins; DT, doubly transformed; HA, hemagglutinin; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; PMSF, phenylmethylsulfonyl fluoride; DTNB, 5,5′-dithiobis-2-nitrobenzoic acid. occurs in many neoplastic cells, and this contributes to the assembly of disorganized cytoskeleton (reviewed in Refs. 3Button E. Shapland C. Lawson D. Cell Motil. Cytoskeleton. 1995; 30: 247-251Google Scholar and 4Janmey P.A. Chaponnier C. Curr. Opin. Cell Biol. 1995; 7: 111-117Google Scholar). Down-regulation of TMs in malignantly transformed cells has been known for about 2 decades and is widely reported (5Hendricks M. Weintraub H. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 5633-5637Google Scholar, 6Matsumura F. Lin J.J. Yamashiro-Matsumura S. Thomas G.P. Topp W.C. J. Biol. Chem. 1983; 258: 13954-13964Google Scholar, 7Cooper H.L. Feuerstein N. Noda M. Bassin R.H. Mol. Cell. Biol. 1985; 5: 972-983Google Scholar), and yet the role of TMs in neoplastic transformation of cells remains incompletely understood. TMs are a family of actin-binding proteins that stabilize microfilaments from the gel severing actions of proteins such as gelsolin and cofilin (1Lin J.J. Warren K.S. Wamboldt D.D. Wang T. Lin J.L. Int. Rev. Cytol. 1997; 170: 1-38Google Scholar, 8Pittenger M.F. Kazzaz J.A. Helfman D.M. Curr. Opin. Cell Biol. 1994; 6: 96-104Google Scholar). Multiple TMs are generated by alternative splicing with a high degree of tissue specificity. For example, fibroblasts express five different, closely related TMs that may be categorized into high and low Mr species containing 284 and 248 amino acids, respectively. TMs are key regulatory proteins of actin cytoskeleton in that they regulate almost all aspects of actin polymerization (9Wen K.K. Kuang B. Rubenstein P.A. J. Biol. Chem. 2000; 275: 40594-40600Google Scholar, 10Strand J. Nili M. Homsher E. Tobacman L.S. J. Biol. Chem. 2001; 276: 34832-34839Google Scholar, 11Blanchoin L. Pollard T.D. Hitchcock-DeGregori S.E. Curr. Biol. 2001; 11: 1300-1304Google Scholar, 12Ishikawa R. Yamashiro S. Matsumura F. J. Biol. Chem. 1989; 264: 16764-16770Google Scholar, 13Cooper J.A. Curr. Biol. 2002; 12: R523-R525Google Scholar). Although the function of TMs is better elucidated in skeletal and cardiac muscles, given their diverse and tissue-specific expression patterns, the importance of the existence of multiple TMs in nonmuscle cell physiology is poorly understood (8Pittenger M.F. Kazzaz J.A. Helfman D.M. Curr. Opin. Cell Biol. 1994; 6: 96-104Google Scholar). It has been suggested that TM isoforms perform distinct functions rather than being simply redundant (1Lin J.J. Warren K.S. Wamboldt D.D. Wang T. Lin J.L. Int. Rev. Cytol. 1997; 170: 1-38Google Scholar, 8Pittenger M.F. Kazzaz J.A. Helfman D.M. Curr. Opin. Cell Biol. 1994; 6: 96-104Google Scholar). Work from this and several other laboratories shows that the high Mr TMs are consistently down-regulated in many malignantly transformed cells (5Hendricks M. Weintraub H. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 5633-5637Google Scholar, 6Matsumura F. Lin J.J. Yamashiro-Matsumura S. Thomas G.P. Topp W.C. J. Biol. Chem. 1983; 258: 13954-13964Google Scholar, 7Cooper H.L. Feuerstein N. Noda M. Bassin R.H. Mol. Cell. Biol. 1985; 5: 972-983Google Scholar, 14Bhattacharya B. Prasad G.L. Valverius E.M. Salomon D.S. Cooper H.L. Cancer Res. 1990; 50: 2105-2112Google Scholar, 15Novy R.E. Lin J.L. Lin C.S. Lin J.J. Cell Motil. Cytoskeleton. 1993; 25: 267-281Google Scholar, 16Mahadev K. Raval G. Bharadwaj S. Willingham M.C. Lange E.M. Vonderhaar B.K.V. Salomon D. Prasad G.L. Exp. Cell Res. 2002; 279: 40-51Google Scholar). This suggests a role for TMs in the maintenance of normal growth and cytoskeletal organization, and that expression of TM1 may be incompatible with neoplastic growth. In support of this hypothesis, we have recently shown that TM isoform-1 (TM1) expression is widely and profoundly down-regulated in primary breast tumors (17Raval G.N. Bharadwaj S. Levine E.A. Willingham M.C. Geary R.L. Kute T. Prasad G.L. Oncogene. 2003; 22: 6194-6203Google Scholar). Furthermore, TM1 restores the microfilament organization in transformed cells, and suppresses malignant growth (16Mahadev K. Raval G. Bharadwaj S. Willingham M.C. Lange E.M. Vonderhaar B.K.V. Salomon D. Prasad G.L. Exp. Cell Res. 2002; 279: 40-51Google Scholar, 18Prasad G.L. Fuldner R.A. Cooper H.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7039-7043Google Scholar, 19Braverman R.H. Cooper H.L. Lee H.S. Prasad G.L. Oncogene. 1996; 13: 537-545Google Scholar, 20Prasad G.L. Masuelli L. Raj M.H. Harindranath N. Oncogene. 1999; 18: 2027-2031Google Scholar). Tumor suppression by TM1 is isoform-specific; for example, unlike TM1, closely related TMs such as TM2 failed to suppress transformed growth of highly malignant v-ki-ras-transformed NIH3T3 (DT) cells or MCF-7 human breast carcinoma cells (17Raval G.N. Bharadwaj S. Levine E.A. Willingham M.C. Geary R.L. Kute T. Prasad G.L. Oncogene. 2003; 22: 6194-6203Google Scholar, 19Braverman R.H. Cooper H.L. Lee H.S. Prasad G.L. Oncogene. 1996; 13: 537-545Google Scholar). Although these studies suggested TM1 is a class II tumor suppressor (17Raval G.N. Bharadwaj S. Levine E.A. Willingham M.C. Geary R.L. Kute T. Prasad G.L. Oncogene. 2003; 22: 6194-6203Google Scholar, 20Prasad G.L. Masuelli L. Raj M.H. Harindranath N. Oncogene. 1999; 18: 2027-2031Google Scholar, 21Sers C. Emmenegger U. Husmann K. Bucher K. Andres A.C. Schafer R. J. Cell Biol. 1997; 136: 935-944Google Scholar), the molecular basis of cytoskeletal organization and tumor suppression by TM1 remains unknown. TM1 lacks distinct catalytic activity or binding partners that readily explain the isoform-specific anti-oncogenic effects. Structurally, all TMs are predominantly α-helical proteins in which hydrophilic and hydrophobic amino acids occupy defined positions in a repeating heptapeptide (22Perry S.V. J. Muscle Res. Cell Motil. 2001; 22: 5-49Google Scholar). TM1, like other TMs, is a cytosolic structural protein that binds to actin stoichiometrically in micromolar range. For example, both TM1 and TM2 bind to actin at 1:7 ratio, although some differences in binding properties exist (23Pittenger M.F. Kistler A. Helfman D.M. J. Cell Sci. 1995; 108: 3253-3265Google Scholar). Nevertheless, it is intriguing that the biological effects of TM1 expression in tumor cells are remarkably different from those of other TM isoforms. We have considered that reorganization of microfilaments is a critical component of tumor suppression by TM1. The studies presented here demonstrate that the N terminus of TM1 is essential for TM1 functions and imply that TM1 is a key modulator of stress fibers. Cell Culture and Antibodies—Culture conditions and media for NIH3T3, NIH3T3/TM1, and DT/TM1 cells have been described previously 2(4Janmey P.A. Chaponnier C. Curr. Opin. Cell Biol. 1995; 7: 111-117Google Scholar). Doubly transformed (DT) cells are NIH3T3 cells transformed with two copies of the v-ki-ras oncogene (7Cooper H.L. Feuerstein N. Noda M. Bassin R.H. Mol. Cell. Biol. 1985; 5: 972-983Google Scholar). An epitope-tagged TM1 was constructed by cloning TM1 in-frame in pCGN vector to add hemagglutinin (HA) epitope at the N terminus. The variant TM1 thus produced would have an N-terminal extension, ASSYPYDVPDYASLGGPSR, but contains identical wild type TM1 sequence from the “R” onward. DT cells were cotransfected with the recombinant plasmid and pCMVneo to generate single cell-derived clones by standard transfection methods. N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate salts (Roche Applied Science) or LipofectAMINE (Invitrogen) were used for transfections. In experiments involving transient transfections, cells were routinely processed 48 h after transfection, unless otherwise indicated. A TM polyclonal antibody, generated in this laboratory, that detects multiple TMs was described previously (16Mahadev K. Raval G. Bharadwaj S. Willingham M.C. Lange E.M. Vonderhaar B.K.V. Salomon D. Prasad G.L. Exp. Cell Res. 2002; 279: 40-51Google Scholar). TM311 mouse monoclonal antibody that recognizes a common epitope found in all high Mr TMs (25Nicholson-Flynn K. Hitchcock-DeGregori S.E. Levitt P. J. Neurosci. 1996; 16: 6853-6863Google Scholar) was obtained from Sigma. HA (12CA5) mouse monoclonal antibody (Roche Applied Science) and antibodies against α-tubulin (Sigma), phosphocofilin (Upstate Biotechnology, Inc.), actin, and cofilin (Cytoskeleton, Inc., Denver, CO) were purchased. Monolayer Growth and Morphology—Ten thousand cells were plated and counted, and cell numbers were plotted against time of culture. Morphology of subconfluent monolayer cultures was recorded by fixing and staining with a HEMA 3 kit obtained from Fisher. The samples were photographed at ×40 magnification. Soft Agar Assays—Anchorage-independent experiments were performed in soft agar as described previously. One thousand cells were mixed in 0.36% agar, plated on a 0.8% agar base, and cultured for 10–14 days (18Prasad G.L. Fuldner R.A. Cooper H.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7039-7043Google Scholar). Cells were stained overnight with 0.05% nitro blue tetrazolium in PBS, and colonies were counted. Immunofluorescence—Cells were cultured in chamber slides (Nunc), fixed with 3.7% paraformaldehyde, and extracted with 0.5% Triton X-100 for 5 min (26Shah V. Bharadwaj S. Kaibuchi K. Prasad G.L. Oncogene. 2001; 20: 2112-2121Google Scholar). The samples were incubated with an appropriate primary antibody, followed by a second antibody conjugated to a fluorochrome, and finally with Texas Red-conjugated phalloidin. Slides were finally rinsed in water and mounted using Antifade kit (Molecular Probes). Images were recorded using a Zeiss LSM 510 confocal microscope and imported into Adobe Photoshop. Immunoblotting and Immunoprecipitations—Cells were lysed in a buffer containing Nonidet P-40, sodium deoxycholate, and protease inhibitors and clarified at 14,000 × g (26Shah V. Bharadwaj S. Kaibuchi K. Prasad G.L. Oncogene. 2001; 20: 2112-2121Google Scholar). Supernatants containing 50–100 μg of proteins were separated on 13% SDS-polyacrylamide gels and immunoblotted. For immunoprecipitations, 200 μg of protein lysate, precleared with protein G for 1 h, was incubated with the primary antibody (27Gimona M. Watakabe A. Helfman D.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9776-9780Google Scholar). The immune complexes were washed with immunoprecipitation buffer and subjected to SDS-PAGE. To quantify the protein expression, the exposed membranes were scanned, and the band intensities were calculated using the magic wand tool of the Adobe Photoshop (version 6.0). Metabolic Labeling of Cells—Subconfluent cultures were pre-incubated with labeling medium (Dulbecco's modified Eagle's medium containing 10% dialyzed fetal bovine serum) for 3 h. Metabolic labeling was carried out in the labeling medium containing [35S]methionine (50 μCi/ml) for 6 h (24Prasad G.L. Fuldner R.A. Braverman R. McDuffie E. Cooper H.L. Eur. J. Biochem. 1994; 224: 1-10Google Scholar). Cells were washed with PBS and extracted in Nonidet P-40/deoxycholate lysis buffer. For immunoprecipitations and cross-linking experiments, lysates equivalent to 4 × 106 cpm were used. F-actin Quantitation in Cells—F-actin content in cells was measured using a protocol described by Zigmond and co-workers (28Tardif M. Huang S. Redmond T. Safer D. Pring M. Zigmond S.H. J. Biol. Chem. 1995; 270: 28075-28083Google Scholar) with minor modifications. Cells (1.5 × 105) were plated 12–24 h prior to experimentation. Monolayer cultures were washed with PBS, fixed, and stained in PBS containing 3.7% paraformaldehyde, 0.5% Triton X-100, and 0.2 μm TRITC phalloidin for 3 h at room temperature with constant rocking in the dark. Unincorporated TRITC phalloidin was removed by four PBS washes. Cell monolayer was extracted by using 1 ml of methanol, and the cell suspension was transferred to microcentrifuge tubes and incubated for 48 h with constant rocking at 4 °C. Cell debris was removed by centrifugation and fluorescence (540ex/575em) was read in an Aminco Bowman luminescence fluorimeter using the methanol solvent as blank. To determine background fluorescence, 2 μm of unlabeled phalloidin was added along with TRITC-phalloidin. To determine total actin content, cell lysates were probed with anti-actin antibody in immunoblots and expressed as a ratio with endogenous tubulin. Cell Cycle Analysis—To estimate the fraction of cells in each phase of cell cycle, asynchronously growing subconfluent cultures were trypsinized and stained with 50 μg/ml propidium iodide in PBS containing 0.06% Nonidet P-40 and 30 μg/ml RNase A. Cell cycle analyses were carried out using a BD FACS Star Plus flow cytometer. Flow cytometry of serum-starved (24 h) or stimulated cells was performed. For serum stimulation, serum-starved cells were cultured in regular medium for 28 h. HA-TM1 Purification—HA-TM1 was subcloned in pET3a (Novagen) and expressed in BL21 (DE3) pLysS bacteria. Cultures were grown to A600 0.4–0.6 and induced with 0.4 mm isopropyl-1-thio-β-d-galactopyranoside for 3 h. Cells were harvested and resuspended for sonication in lysis buffer containing 20 mm Tris (pH 7.5), 100 mm NaCl, 2 mm EDTA, 1× protease inhibitor mixture (Roche Applied Science), 1 mg/liter DNase, 1 mg/liter RNase A, 1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 5 mm EDTA, 5 mm EGTA, 5 mm PMSF, 5 mm benzamidine, and 1 mg of potato carboxypeptidase inhibitor (Calbiochem) and immediately subjected to ammonium sulfate precipitation to achieve ∼50% saturation. The protein pellet was resuspended and extensively dialyzed against a buffer containing 20 mm Tris (pH 7.5), 1 mm PMSF, and 1mm benzamidine. The salt concentration of the dialysate was adjusted to 50 mm and subjected to DEAE-cellulose chromatography. HA-TM1 was eluted with 0.3 m NaCl containing buffer (20 mm Tris (pH 7.5), 5 mm EDTA and 2.5 mm PMSF), reprecipitated with ammonium sulfate (100% saturation to concentrate the eluate), and dialyzed. The protein sample was further purified by affinity chromatography on HA epitope affinity column (Roche Applied Science) as per the manufacturer's instructions. HA-TM1 was eluted with 1 mg/ml HA epitope peptide (Roche Applied Science) and dialyzed against the binding assay buffer (10 mm Tris (pH 7.5), 150 mm NaCl, 2 mm MgCl2, 0.2 mm EGTA). In Vitro Actin Binding Studies—Actin binding assays were performed as described previously (29Heald R.W. Hitchcock-DeGregori S.E. J. Biol. Chem. 1988; 263: 5254-5259Google Scholar) with modifications (30Hammell R.L. Hitchcock-DeGregori S.E. J. Biol. Chem. 1996; 271: 4236-4242Google Scholar). HA-TM1 and wild type TM2 were cosedimented at 20 °C with chicken pectoral muscle F-actin (5 μm) in a buffer containing 150 mm NaCl, 10 mm Tris-HCl (pH 7.5), 2 mm MgCl2, and 0.5 mm dithiothreitol. The amounts of bound and free tropomyosin in the supernatants and pellets were quantitated by densitometry of SDS-polyacrylamide gels stained in Coomassie Blue using a Molecular Dynamics model 300A computing densitometer (Amersham Biosciences). To separate HA-TM1 from actin, the gels also contained 6 m urea. The free tropomyosin in the supernatants was calculated from standard curves for wild type tropomyosin. The curve for TM2 was fit using the Hill equation using SigmaPlot (SPSS Science, Chicago) that reported a Kapp. Cross-linking Studies—Homodimers of TM1 were stabilized by cross-linking with 5,5′-dithiobis-2-nitrobenzoic acid (24Prasad G.L. Fuldner R.A. Braverman R. McDuffie E. Cooper H.L. Eur. J. Biochem. 1994; 224: 1-10Google Scholar, 31Lehrer S.S. Joseph D. Arch. Biochem. Biophys. 1987; 256: 1-9Google Scholar) (DTNB) (Sigma), a sulfhydryl cross-linker, as described previously (24Prasad G.L. Fuldner R.A. Braverman R. McDuffie E. Cooper H.L. Eur. J. Biochem. 1994; 224: 1-10Google Scholar). Cell lysates were incubated with DTNB and were either subjected to immunoblotting (for unlabeled samples) or immunoprecipitation followed by SDS-PAGE and fluorography (for 35S-labeled samples). Dimers are detectable when 2-mercaptoethanol is omitted in the gel sample buffer. Statistical Analyses—Data are presented as mean ± S.D. from at least three independent determinations. p values were calculated by Student's two-tailed t test (32Glantz S.A. Primer of Biostatistics. 4th Ed. McGraw-Hill Inc., New York1997Google Scholar) using the software provided in Microsoft Excel (2002 edition). In vitro studies have indicated that the N- and C-terminal ends of TMs are critical for binding to actin. Several studies have shown that some TMs, including TM1, require the acetylated N terminus for optimal binding to actin. N-terminal extensions, depending on the length and sequence, can alter TM functions (33Cho Y.J. Liu J. Hitchcock-DeGregori S.E. J. Biol. Chem. 1990; 265: 538-545Google Scholar, 34Monteiro P.B. Lataro R.C. Ferro J.A. Reinach F. J. Biol. Chem. 1994; 269: 10461-10466Google Scholar, 35Urbancikova M. Hitchcock-DeGregori S.E. J. Biol. Chem. 1994; 269: 24310-24315Google Scholar). To elucidate the mechanism of TM1-mediated tumor suppression, we have modified the N-terminal end of TM1 molecule by introducing a hemagglutinin (HA) epitope. This epitope tag contains three prolines and would not be predicted to be α-helical. This tagged protein, referred to as HA-TM1, contains a 19-residue N-terminal extension, which otherwise is identical to the wild type protein, and retains all the actin binding domains of TM1. We tested the ability of HA-TM1 to regulate cytoskeletal organization and growth phenotype. N-terminal Modification Abolishes TM1-mediated Cytoskeletal Reorganization and Tumor Suppression—We have used DT (NIH3T3 cells transformed by v-Ki-ras) cells as a model and transfected them with HA-TM1. Stable single cell clones, designated as DT/HA-TM1, were isolated. DT cells express TM1 at 50% levels compared with normal NIH3T3 fibroblasts, and TM2 and TM3 at essentially undetectable levels (18Prasad G.L. Fuldner R.A. Cooper H.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7039-7043Google Scholar). Expression of HA-TM1 was detected by Northern blot (not shown) and immunoblot methods (Fig. 1A). The variant protein, because of the N-terminal extension, migrates slower than wild type TM1 on SDS-PAGE. The expression of endogenous TM1 in parental DT and DT/HA-TM1 cells is determined by the ratio of TM1: α-tubulin. In DT cells, the relative expression of TM1 was 0.7 ± 0.12, and in DT/HA-TM1 cells, the endogenous TM1 was expressed at 0.75 ± 0.08, indicating that the transfected HA-TM1 did not alter the levels of the endogenous wild type protein (p < 1). Morphologically DT cells are spindle-shaped, lack stress fibers, and are not contact-inhibited. Because TM1 induces reorganization of the cytoskeleton to restore stress fibers with attendant cell spreading, we examined whether HA-TM1 alters the cell morphology. Morphologically, DT/HA-TM1 cells resembled parental DT and empty vector transfected cells. DT/HA-TM1 cells displayed spindle-shaped morphology, were not subject to contact inhibition and formed multiple foci (Fig. 1B). Confocal microscopy revealed that HA-TM1 expression, unlike that of the wild type TM1 protein (18Prasad G.L. Fuldner R.A. Cooper H.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7039-7043Google Scholar), did not induce the formation of stress fibers (Fig. 1C). Because TM1 induces cytoskeletal reorganization with the assembly of stress fibers, it is likely that enhanced TM1 levels may increase the levels of F-actin. Therefore, we examined whether TM1 up-regulates F-actin levels and whether the inability of the variant protein to reorganize cytoskeleton is reflected in lower F-actin content. Overnight cultures of NIH3T3 cells, DT cells, DT/TM1, and DT/HA-TM1 cells were fixed, and the F-actin content was determined using TRITC-conjugated phalloidin. The fluorescent intensities were normalized to 1.5 × 105 cells (Table I). Consistent with the degree of microfilament organization, NIH3T3 cells contained most F-actin. DT and DT/HA-TM1 cells contained comparable amounts of F-actin, but exhibited significantly lower F-actin than NIH3T3 cells, as measured by fluorescence intensity (p < 0.02). Enhanced expression of TM1 resulted in the reemergence of microfilaments, which reflected in increased F-actin content. F-actin content was significantly higher in DT/TM1 cells (1.97 ± 0.32) compared with DT cells (1.01 ± 0.24) (p < 0.03). However, the total actin content in DT-derived cells was unchanged, when quantitated by immunoblotting and expressed relative to tubulin (Table I).Table ITM1 enhances the cellular F-actin content F-actin content was determined by measuring the fluorescence of TRITC-labeled phalloidin, as described under “Experimental Procedures.” Means ± S.D. are derived from three independent experiments. Relative fluorescence was normalized to 1.5 × 105 cells. Total actin and tubulin content were measured in 50 μg of protein from the indicated cell lines by immunoblotting. The ratio of actin to tubulin is taken as a measure of total actin content. The p values reported in the text are calculated by two-tailed paired t test.Cell typeF-actin contentTotal actin contentfluorescence unitsactin/tubulinNIH3T33.63 ± 0.811.36 ± 0.11DT1.01 ± 0.241.15 ± 0.2DT/TM11.97 ± 0.321.18 ± 0.11DT/HA-TM10.72 ± 0.011.13 ± 0.12 Open table in a new tab Next we determined whether HA-TM1 altered the growth properties of DT cells. Variant TM1 did not affect either the monolayer growth or anchorage-independent growth rates of DT cells (Fig. 2, A and B). In contrast, wild type TM1 significantly decreased monolayer growth compared with HA-TM1 (p < 0.005), comparable with the NIH3T3 cells. Consistent with its inability to alter the cytoskeletal organization and morphology, HA-TM1 expression did not affect the anchorage-independent growth of DT cells. DT/HA-TM1 cells grew rapidly and formed colonies in soft agar as efficiently as the parental DT cells or vector control cells. In contrast, DT/TM1 cells failed to grow under anchorage-independent growth conditions (18Prasad G.L. Fuldner R.A. Cooper H.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7039-7043Google Scholar). Thus, the N-terminal modification of TM1 abolishes the ability of TM1 to induce cytoskeletal reorganization and inhibits its anti-oncogenic properties. HA-TM1 Disrupts Microfilaments in Normal Fibroblasts— Next, we investigated whether the modification of the N terminus of TM1 would interfere with the ability of TM1 to associate with existing normal microfilament structures. NIH3T3 cells were transiently transfected with HA-TM1 (Fig. 3A). HA-TM1 incorporated into microfilaments and colocalized with F-actin. The HA-TM1 was distributed uniformly throughout the cytoskeleton, a finding consistent with a previous report (27Gimona M. Watakabe A. Helfman D.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9776-9780Google Scholar). However, examination of the microfilament architecture after extended periods (48–72 h) revealed severe disruptions in microfilaments (Fig. 3B). Cells expressing HA-TM1 generally lacked well defined linear microfilaments that traverse the cell, which are typical of NIH3T3 cells. Instead, the transfected cells displayed wavy and disorganized microfilaments containing the variant protein and F-actin filaments, and aberrant cellular morphology when compared with the untransfected cells. These findings suggest that HA-TM1 associates with microfilaments and subsequently perturbs the cytoarchitecture (Fig. 3B). Transient transfection typically produces an overabundance of the gene product, which could potentially result in artifacts. In this case, enhanced expression of a TM protein could perturb intracellular TM pools, leading to aberrant cytoskeleton, an effect independent of the N-terminal modification of TM1. To rule out the effects of overabundance of the transfected protein, we have examined the microfilament organization in NIH3T3 cell lines that were stably transduced with TM1 (NIH3T3/TM1 cells) (24Prasad G.L. Fuldner R.A. Braverman R. McDuffie E. Cooper H.L. Eur. J. Biochem. 1994; 224: 1-10Google Scholar). Enhanced expression of TM1 did not disorganize microfilaments, as did the variant protein (Fig. 3C). TM1 colocalized with linear, well defined stress fibers, and the normal cellular morphology was not compromised in NIH3T3/TM1 cells. These results indicate that enhanced TM1 expression per se does not induce disorganized cytoskeleton. Because TMs dimerize in “head to tail” fashion and associate with actin, modification of the N terminus of TM1 could have interfered with either dimerization or binding to actin, or both. To investigate further the effects of the N-terminal modification of TM1 molecule, we have stably transfected NIH3T3 cells with HA-TM1 (Fig. 3D). Immunofluorescence experiments show that microfilaments are often “pushed” to a side, and a substantial amount of variant TM1 occupies the perinuclear area. Variant TM1 is also colocalized with phalloidin-positive microfilaments, indicating that during dynamic reorganization of stress fibers, the HA-TM1 associates with filaments. The variant protein, because of its abundance, may compete with the endogenous TM1, although the F-actin binding and dimerization properties of TM1 and variant TM1 significantly differ (see below). However, it is possible that HA-TM1 and endogenous TM1 coexist in the stress fibers. Light microscopic observation of NIH3T3/HA-TM1 cells revealed the presence of high number of binucleated cells, indicating defects in cytokinesis in the transfected cells (Fig. 4). Asynchronously growing populations of NIH3T3/HA-TM1 cells con" @default.
• W2022323043 created "2016-06-24" @default.
• W2022323043 creator A5042949704 @default.
• W2022323043 creator A5048235189 @default.
• W2022323043 creator A5071830203 @default.
• W2022323043 creator A5076164186 @default.
• W2022323043 date "2004-04-01" @default.
• W2022323043 modified "2023-10-17" @default.
• W2022323043 title "N Terminus Is Essential for Tropomyosin Functions" @default.
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