Effect of the Structure of the N Terminus of Tropomyosin on Tropomodulin Function*

Tropomodulins (Tmod) bind to the N terminus of tropomyosin and cap the pointed end of actin filaments. Tropomyosin alone also inhibits the rate of actin depolymerization at the pointed end of filaments. Here we have defined 1) the structural requirements of the N terminus of tropomyosin important for regulating the pointed end alone and with erythrocyte Tmod (Tmod1), and 2) the Tmod1 subdomains required for binding to tropomyosin and for regulating the pointed end. Changes in pyrene-actin fluorescence during polymerization and depolymerization were measured with actin filaments blocked at the barbed end with gelsolin. Three tropomyosin isoforms differently influence pointed end dynamics. Recombinant TM5a, a short non-muscle α-tropomyosin, inhibited depolymerization. Recombinant (unacetylated) TM2 and N-acetylated striated muscle TM (stTM), long α-tropomyosin isoforms with the same N-terminal sequence, different from TM5a, also inhibited depolymerization but were less effective than TM5a. All blocked the pointed end with Tmod1 in the order of effectiveness TM5a >stTM >TM2, showing the importance of the N-terminal sequence and modification. Tmod1-(1–344), lacking the C-terminal 15 residues, did not nucleate polymerization but blocked the pointed end with all three tropomyosin isoforms as does a shorter fragment, Tmod1-(1–92), lacking the C-terminal “capping” domain though higher concentrations were required. An even shorter fragment, Tmod1-(1–48), bound tropomyosin but did not influence actin filament elongation. Tropomyosin-Tmod may function to locally regulate cytoskeletal dynamics in cells by stabilizing actin filaments.

Actin filaments participate in many biological functions, including muscle contraction, cell migration, cell division, and organelle transport. A dynamic actin cytoskeleton allows the cell to respond rapidly to intracellular and extracellular signals by interacting with proteins that spatially and temporally influence the nucleation and kinetics of actin polymerization as well as the stability and state of assembly of the actin filament (1,2). Research in recent years has given insight into the mechanisms of assembly and disassembly of actin filaments and their regulation by cell signaling pathways. Now attention is turning to mechanisms that stabilize actin filaments within cells for participation in myosin-dependent contraction and structural functions.
Actin filaments are polar with "barbed" and "pointed" ends. The polarity, originally described by the arrowhead appearance of negatively stained filaments when myosin binds (3), is also evidenced by differences in critical concentration and affinity for specific binding proteins (4,5). In cells, rapid growth of actin filaments occurs by addition of actin monomer at free barbed ends, which in turn may be stabilized by the rapid binding of a capping protein. The fate of a filament then depends on the dynamics of the pointed end and the lability of the filament to severing proteins. The focus of the present work is to learn how two proteins, tropomyosin and tropomodulin, regulate the dynamics of the pointed ends of actin filaments.
Tropomyosins are a family of coiled coil proteins that bind head-to-tail along the sides of the helical actin filament (6). Best known for its function in cooperative regulation of actinmyosin interaction (7,8), tropomyosin is now recognized for its role in regulating actin filament stability (9). Tropomyosin stiffens actin filaments (10) and offers protection against severing and depolymerization by gelsolin (11,12), cofilin (13)(14)(15)(16), and DNase I (17). Tropomyosin also inhibits the rate of depolymerization from the pointed end (18,19) and inhibits Arp2/3 complex-nucleated branching (20). Tropomyosin isoforms differ in actin affinity and regulatory function and exhibit developmentally regulated and tissue-specific expression patterns (6) as well as different localizations within cells (21). The two major classes of tropomyosin, short (ϳ247 amino acids) and long (ϳ284 amino acids), differ in sequence and structure at the N terminus (22)(23)(24).
Tropomodulin, originally isolated as a tropomyosin binding protein from erythrocytes (25), is a family of widely expressed proteins that bind to tropomyosin and actin, capping the pointed end of actin filaments (26). Erythrocyte tropomodulin (Tmod1) 1 binds weakly to actin alone (K d ϳ0.3-0.4 mM) but much stronger in the presence of tropomyosin (K d ϳ50 pM) (27). Analysis of tropomodulin fragments and antibodies to specific domains has identified the actin and tropomyosin binding domains to be the C-and N-terminal parts of the molecule, respectively (28,29). The atomic structure of the C-terminal domain is a right-handed super-helix composed of alternating ␣-helices and ␤-strands (30). The N-terminal half has no definite tertiary structure but exhibits increases in ␣-helix and ␤-sheet stability when it binds to tropomyosin or tropomyosin model peptides (31,32).
Tropomodulin binds to the N terminus of tropomyosin (33,34), the end oriented toward the pointed end of the actin filament (35). Binding studies of tropomodulin to full-length tropomyosins and to model peptides with the N-terminal se-quences from short or long tropomyosins showed that the first 14 -18 residues of the tropomyosin coiled coil are sufficient to bind the N-terminal domain of tropomodulin (31,32). An intact N-terminal coiled coil is required because modifications to the peptide with the long tropomyosin sequence that locally destabilize the coiled coil, such as removal of the N-terminal acetyl group or introduction of the nemaline-myopathy-causing mutation M8R (36,37) destroy tropomodulin binding (32). Mutations in the homologous region of a short tropomyosin also resulted in the loss of tropomodulin binding (34).
Here we have investigated regulation of the dynamics of the pointed end of the actin filament by different tropomyosin isoforms alone and in concert with Tmod1 and its fragments.

Constructions of Expression Vectors of Tropomodulin N-terminal
Fragments-The expression plasmids for the N-terminal fragments of chicken E-tropomodulin (Tmod1), named pET(His)Tmod1-  and pET(His)Tmod1- , were constructed using a QuikChange sitedirected mutagenesis kit (Stratagene, La Jolla, CA). The plasmids were amplified by PCR using PfuTurbo DNA polymerase with the tropomodulin expression plasmid pET(His)Tmod(N39) (38) as the template using two complementary sets of oligonucleotides.
The oligonucleotides contained two stop codons (in bold) and an AatII site (underlined). The original plasmid was digested using DpnI, and the mixture was used to transform Escherichia coli (DH5␣). After plasmid purification, the presence of the mutations was confirmed by restriction enzyme mapping and sequencing of the full tropomodulin fragment. Synthesis of all oligonucleotides and sequence determination were done at the UMDNJ DNA Synthesis and Sequencing Facility (Robert Wood Johnson Medical School, Piscataway, NJ).
Sequence analysis of pET(His)Tmod1-  showed there was just one stop codon, not two as designed. Instead of the first stop codon TGA (as in the oligonucleotide), the sequence was CGA. This did not change the amino acid sequence from the original tropomodulin (Arg in both cases) but made the fragment length 92 (instead of 91) amino acids plus a His tag and Met at the N terminus.
Chicken pectoral muscle skeletal actin was purified from acetone powder as described (39). G-actin was purified on a Sephacryl S-300 column (40) and was stored in liquid nitrogen. Actin was labeled with pyrenyl-iodoacetamide, and the labeling ratios were calculated according to Refs. 41 and 42. The degree of the labeling was 80 -99%. Before experiments, G-actin (labeled or unlabeled) was defrosted in a 37°Cwater bath and then centrifuged at 100,000 rpm (TLA-100, Beckman) for 30 min at 4°C.
N-acetylated striated muscle ␣-tropomyosin, stTM was purified from chicken pectoral muscle according to Ref. 43, and rat recombinant ␣-tropomyosins TM2 and TM5a were expressed in E. coli and purified as previously described (37). AcTM1bZip is a designed chimeric protein that contains 19 residues of short rat ␣-tropomyosin encoded by exon 1b and the 18 C-terminal residues of the GCN4 leucine zipper domain (24). It was synthesized by SynPep (Dublin, CA).
Protein purity was evaluated using SDS-PAGE (44). Native gel electrophoresis was done in 9% polyacrylamide gels that were polymerized in the presence of 10% glycerol without SDS.
The actin concentrations were calculated from the UV spectrum using an extinction coefficient of 11.0 (1% at 280 nm). Concentrations of other proteins were determined by measuring their difference spectra in 6 M guanidine-HCl between pH 12.5 and 6.0 (45) using for calculations the extinction coefficients of 2357 for tyrosine and 830 for tryptophan (46). Recombinant human gelsolin was a generous gift from Dr. Philip G. Allen.
Fluorescence Measurements-The rates of actin polymerization and depolymerization were measured using the change in pyrene-actin fluorescence (41) using a PTI fluorimeter (Lawrenceville, NJ) (excitation, 366 nm, and emission, 387 nm, with a 2-nm slit). To measure polymerization of actin at the pointed end, short filaments capped at the barbed ends with gelsolin were prepared by polymerization of 6 M G-actin in the presence of 28 nM gelsolin. The G-actin cation used for polymerization was changed from Ca 2ϩ to Mg 2ϩ by incubation with 50 M MgCl 2 and 0.2 mM EGTA for 10 min before the experiment. Polymerization was monitored by the increase in fluorescence when the filaments were diluted 5-fold with G-actin (10% pyrenylactin) in F-buffer (100 mM KCl, 2 mM MgCl 2 , 1 mM EGTA, 0.5 mM dithiothreitol, 0.2 mM ATP, 0.2 mM CaCl 2 , 1 mM NaN 3 , 10 mM imidazole, pH 7.0) containing tropomyosin and/or tropomodulin fragments. The final concentrations of F-and G-actin were 1.2 and 1.5 M, respectively. For depolymerization experiments, gelsolin-capped filaments were prepared by polymerization of 3 M G-actin (10% pyrenylactin) in the presence of 11.2 nM gelsolin. Depolymerization was monitored by the decreasing fluorescence of filaments diluted 5-fold into F-buffer. Tropomyosin and/or tropomodulin were added 15 min before dilution.
Gelsolin-capped filaments were prepared in sets of four, and fluorescence measurements were carried out in parallel in a 4-cuvette holder with actin as a control in each set. Exponential curves were fit to the polymerization or depolymerization data using SigmaPlot, and initial rates were calculated as the first derivatives at time 0. The S.D. for the initial rates calculated for actin polymerization at the pointed end with seeds prepared separately was 15-20% of the value of average initial rate, whereas it was just 1-2% in experiments when the seeds were from the same set. S.D. of the final fluorescence in both cases were 6 and 4%, respectively. Therefore the large error in initial rate determination depends on errors in gelsolin concentration (concentration of pointed ends), not the actin concentration.
Sedimentation Experiments-Binding of tropomyosin to actin was measured by cosedimentation according to Refs. 47 and 48, with modifications. Actin filaments polymerized in the presence of tropomyosin and/or tropomodulin were pelleted at 60,000 rpm, 20 min at 20°C, and twice washed with F-buffer. The pellet and supernatant compositions were analyzed by SDS-PAGE, Coomassie blue (R250) stained gels were scanned using an Amersham Biosciences model 300A computing densitometer (Sunnyvale, CA), and the tropomyosin:actin ratio was calculated for each sample.
Circular Dichroism Measurements-CD measurements were done using an Aviv model 62 spectropolarimeter (Lakewood, NJ) as previously described (23,36). The protein concentrations were 10 M AcTM1bZip, 10 M Tmod1-(1-92), and 10 M Tmod1-  in 100 mM NaCl, 10 mM sodium phosphate, pH 6.5. Binding constants were calculated according to Ref. 32. The CD measurements also were performed for Tmod1-(1-92) at pH 7.2, and the calculated binding constant was within the experimental error of the value obtained at pH 6.5.

RESULTS AND DISCUSSION
Our aims were 1) to determine the structural requirements and isoform specificity of the regulation by tropomyosin of actin polymerization and depolymerization from the pointed end of the actin filament, 2) to understand whether the structural requirements for tropomodulin binding to tropomyosin model peptides are the same as for blocking the pointed end of filaments, and 3) to define the tropomodulin subdomains required for regulation of pointed end polymerization and depolymerization with tropomyosin. We studied tropomyosins with three different N-terminal structures: recombinant TM5a, a short ␣-tropomyosin where the N terminus is encoded by exon 1b, and recombinant TM2 and striated muscle stTM, long ␣-tropomyosins where the N terminus is encoded by exon 1a. TM2 is unacetylated because it is expressed in E. coli, whereas stTM, isolated from chicken muscle, is N-acetylated. The C termini of TM2 and TM5a are identical (encoded by exon 9d) but different from stTM (encoded by exon 9a).
Actin Polymerization in the Presence of Different Tropomyosin Isoforms-Actin filament dynamics at the pointed end were investigated in short actin filaments capped at the barbed end with gelsolin. Tropomyosin binding to gelsolin-capped actin filaments has been reported to remove gelsolin from the barbed end and then anneal filaments (49,50). Care was taken to minimize these processes that are proportional to the actin and tropomyosin concentrations and inversely proportional to filament length (50,51). The gelsolin-actin seeds were prepared in the absence of tropomyosin at 1:210 and 1:270 gelsolin:actin molar ratios for polymerization and depolymerization, respectively, long enough for cooperative tropomyosin binding (the length of 15-22 tropomyosin molecules) and to reduce the rate of annealing. The actin and tropomyosin concentrations were lower than those in published reports to further minimize rapid annealing. Tropomyosin and tropomodulin were mixed with G-actin, 20ϫ F-buffer was added, and this mixture was immediately added to seeds polymerized in cuvettes. The rates of polymerization reported are calculated initial rates.
Because the rate of annealing of actin filaments increases with tropomyosin concentration (50), we determined the minimum concentration of each tropomyosin isoform sufficient for saturation of gelsolin-capped actin filaments. Following polymerization (after one hour) in the presence of different concentrations of tropomyosin, 0.2-3 M, the actin (2.7 M) was sedimented and the bound tropomyosin was analyzed using SDS-PAGE. The pellets contained gelsolin, suggesting that detectable amounts remained bound to the filaments and that the filaments did not anneal (data not shown). In further polymerization experiments the concentrations of TM5a, stTM, and TM2 were 0.5, 1, and 2 M, respectively, concentrations where the F-actin was saturated with tropomyosin. No gelsolin was added with the pyrene-G-actin so as to maintain a constant number of pointed ends and to reduce spontaneous nucleation of actin filaments in the course of the experiment. Fig. 1 compares the rates of pointed end elongation in the presence of three tropomyosin isoforms. The gelsolin-actin seeds contained unlabeled actin, and the polymerization rate was measured using the increase in fluorescence during polymerization of pyrene-actin. In the absence of tropomodulin, tropomyosins had little effect on the initial rate of polymerization. Addition of TM5a, stTM, and TM2 in excess of saturation (1.5, 2, and 3 M, respectively) to polymerized pyrenylactin did not change the final fluorescence, suggesting they had no large effect on the overall critical concentration of F-actin, consistent with previous results (52), and that the binding of tropomyosin did not enhance or quench the pyrene fluorescence. The small effect on the rate of pointed end elongation by the tropomyosins is inconsistent with annealing.
Tropomodulin Capping Activity Depends on the Tropomyosin N-terminal Sequence and Structure-Full-length Tmod1 has a small nucleating effect on actin polymerization (53), (Fig. 2). To simplify the interpretation of our assays, we used a truncated form of Tmod1 that lacks the C-terminal 15 amino acids, Tmod1-(1-344) (38) and does not nucleate actin polymerization (Fig. 2). Tmod1-(1-344) alone slightly inhibited the rate of elongation (Fig. 1D). The results suggest that the proposed second actin binding site (53) is located at the extreme C terminus of Tmod1. All Tmod1 forms in this study had a sixresidue His tag at the N terminus. The His tag does not influence the rate of polymerization of G-actin because addition of an equimolar concentration of a six-residue His tag cleaved from a recombinant protein had no effect on the rate or final level of polymerization of 1 M pyrene actin (results not shown).
In the presence of tropomyosin, Tmod1-(1-344) had the same capping activity as full-length tropomodulin (results not shown). Tmod1-(1-344) inhibited the rate of pointed end elongation with all three tropomyosin isoforms (Fig. 1), but the effectiveness depended on the structure of the N terminus (Fig.  1D). TM5a and stTM are much more effective than TM2, nearly completely inhibiting polymerization at 20 nM Tmod1-(1-344). The difference in elongation rates with TM5a and stTM is consistent with the higher affinity of Tmod1 for short nonmuscle tropomyosin than for long isoforms in full-length tropomyosins, as well as in model peptides (32,54). However, whereas recombinant TM2, with an unacetylated N terminus, had no detectable affinity for either Tmod1 or Tmod4 (32), when bound to actin with Tmod1-(1-344) TM2 inhibited elongation though higher tropomodulin concentrations were required. N-acetylation of long ␣-tropomyosins increases the stability of the N-terminal domain (36) and is required for the extreme N terminus to assume a coiled coil structure in model peptides (23,55). Our present results suggest that the binding of recombinant TM2 to the actin filament with Tmod1 partially overcomes the lack of N-acetylation and may induce coiled coil formation of the N terminus, allowing it to bind to Tmod1. The relatively normal regulatory function of unacetylated stTM when it binds to F-actin in the presence of troponin is another example of the same phenomenon (48).
The sequence of the tropomyosin C terminus appears not to influence pointed end dynamics in the presence or absence of Tmod1. TM5a and TM2 have the same C terminus and are identical except for the first 43 amino acids, yet they differ in function.

Effect of Short N-terminal Tropomodulin Fragments Tmod1-(1-92) and Tmod1-(1-48) on Tropomyosin Binding and Actin
Polymerization-Analysis of Tmod1 fragments has identified two pointed end capping domains: a C-terminal capping domain (residues 160 -359) that is independent of tropomyosin and a N-terminal capping domain (residues 1-130) that depends on and binds to the N terminus of tropomyosin (32,53). The tropomyosin binding domain has been further localized to residues 1-92 (38) and in the present work to residues 1-48 ( Figs. 3 and 4). Tmod1-(1-92) also reduced the rate of pointed end elongation in the presence of all tropomyosins, though higher concentrations were required than with Tmod1-(1-344). Nearly full inhibition was obtained only with TM5a (Fig. 1). The results show that at least part of the N-terminal capping domain resides in Tmod1-(1-92).
Secondary structure analysis (56) of the amino acid sequence of Tmod1-(1-92) predicts the presence of two ␣-helices, the first with a high probability to form a coiled coil (31,32). A plasmid was constructed to express a fragment with the N-terminal ␣-helix, Tmod1-(1-48). Analysis of binding using circular dichroism spectroscopy showed that both Tmod1-(1-92) (Fig.  3A) and Tmod1-(1-48) (Fig. 3B) formed complexes with AcTM1bZip, a model peptide with the same N-terminal sequence as TM5a, used in previous studies (32). The ellipticity and stability of the mixtures were increased relative to that of fragments. The binding constants estimated from the changes in ellipticity were 0.22 Ϯ 0.10 M and 0.14 Ϯ 0.11 for the binding of Tmod1-(1-92) and Tmod1-(1-48) , respectively, also similar to that reported for Tmod1-(1-130) (0.23 Ϯ 0.15) in Ref. 32. Binding to AcTM1bZip was directly analyzed using native polyacrylamide gel electrophoresis (Fig. 4). Additional bands corresponding to complexes between each tropomodulin fragment and AcTM1bZip appeared when they were combined. The tropomyosin binding domain of Tmod1 is therefore localized to residues 1-48. The capping activity, however, is not present because Tmod1-(1-48) had no effect on filament elongation in the presence of TM5a even at a concentration four times higher than that used for Tmod1-(1-92) (results not shown).
Influence of Tropomyosin Isoforms and Tropomodulin Fragments on Actin Depolymerization-Tropomodulin blocks the pointed end of actin filaments by inhibiting depolymerization as well as polymerization. To measure depolymerization, py- rene-labeled actin filaments with gelsolin at the barbed end were diluted to the critical concentration of the pointed end (4), and the rate of loss of fluorescence was measured. In the absence of Tmod1-(1-344), saturating amounts of TM5a inhibited the rate of pointed end depolymerization by 50%. StTM and TM2 were much less effective (Fig. 5D), consistent with previous work (19). Interestingly, when gelsolin-blocked actin filaments were preincubated with saturating amounts of tropomyosin and then diluted to 0.6 M actin without added tropomyosin, resulting in subsaturating concentrations, the inhibition was similar (Fig. 5, A-C).
In the experiments in Fig. 5, the actin filaments were incubated with saturating concentrations of tropomyosin for 15 min before dilution to ensure proper head-to-tail alignment on the filament. We were concerned that inhibition of the rate of depolymerization by TM5a may reflect annealing, resulting in a reduced concentration of ends and hence a slower rate. When gelsolin-actin filaments were incubated with TM5a for 15 or 60 min prior to dilution, the initial rate of depolymerization was about 52 Ϯ 14 and 57 Ϯ 12% of the control (no TM5a), compared with 70 Ϯ 9% of the control when added immediately prior to dilution (Fig. 6). The time of incubation with TM5a before dilution did not change the number of ends because the fluorescence after three hours was similar. The small difference in the initial rate could reflect a small amount of annealing or, more likely, the time it takes for tropomyosin to align properly with respect to the ends.
In the presence of Tmod1-(1-344), all three tropomyosin isoforms blocked depolymerization, though TM2 was least effective (Fig. 5D). Tmod1-(1-344) alone had almost no effect on the rate of depolymerization. Tmod1-(1-92) also inhibited the rate of depolymerization with tropomyosin, though the effect was much less than on polymerization. The part of Tmod1-(1-92) with capping activity (versus the tropomyosin binding site) may physically inhibit monomer addition at the pointed end even though it is too short to prevent depolymerization. Another possibility is that the C-terminal part of Tmod1-(1-92) binds actin, causing conformational changes in the actin molecule that prevent monomer addition without influencing depolymerization.
The complete blocking of depolymerization by subsaturating concentrations of tropomyosin in the presence of Tmod1 surprised us. Because troponin (48,57), caldesmon (58,59), and myosin (60) all greatly increase the affinity of tropomyosin for actin, we measured the effect of Tmod1 on stTM binding to actin (Fig. 7). Neither full-length Tmod1 nor Tmod1-(1-344) increased or decreased tropomyosin binding to actin. We suggest that a Tmod1-tropomyosin complex selectively binds with high affinity at the pointed end without influencing cooperative binding along the length of the filament. CONCLUSIONS We have defined the regions of Tmod1 and the N-terminal structural requirements of tropomyosin needed for capping of the pointed end of actin filaments blocked at the barbed end with gelsolin. In Tmod1, a 359-residue protein, only residues 1-48 are needed for high affinity tropomyosin binding. The first 92 residues are sufficient for blocking elongation, but complete capping of depolymerization requires additional regions of the molecule. Removal of the C-terminal 15 amino acids of Tmod1 destroys the nucleating activity of Tmod1 in the . Depolymerization curves were normalized to initial fluorescence value in each experiment and were fitted to exponential decay ones. Initial rates of depolymerization were 70 Ϯ 9, 52 Ϯ 14, and 57 Ϯ 12% of the control, respectively. absence of tropomyosin and gelsolin, but all tropomyosin-dependent activities are intact.
The ability of tropomyosin, with or without Tmod1, to influence polymerization or depolymerization at the pointed end is isoform-specific. The differential sorting of tropomyosin isoforms within cells (21) may locally influence actin dynamics along with other tropomyosin-dependent functions, including physical stabilization, inhibition of Arp2/3 complex-nucleated branching, cofilin severing, and cooperative regulation of myosin interaction. The influence of tropomyosins on the pointed end depends on the structure of the N terminus. TM5a, a short non-muscle isoform with a non-coiled coil N-terminal peptide, blocks depolymerization and is the most effective with Tmod1. Efficient capping ability with Tmod1 of long muscle and nonmuscle tropomyosins encoded by the ␣-TM gene, with a different N-terminal sequence, requires acetylation of the N-terminal Met. The results, in general, correlate with Tmod1-TM binding, though actin binding partially overcomes the need for N-acetylation.
The ability of Tmod1-tropomyosin to cap the pointed end of filaments at substoichiometric saturation with tropomyosin suggests a role for the proteins in regulating actin stability independent of imparting physical stability and resistance to severing proteins, which would require binding of tropomyosin along the length of the actin filament. We propose that binding of Tmod-TM to the pointed end of an actin filament may be an early step in the pathway to convert a nascent actin filament into a more stable structure.