Tài liệu Báo cáo khoa học: Regulation of the actin–myosin interaction by titin doc

Regulation of the actin–myosin interaction by titin Nicolas Niederla¨ nder 1 , Fabrice Raynaud 2 , Catherine Astier 2 and Patrick Chaussepied 1 1 CRBM-CNRS, Montpellier, France; 2 EPHE-UMR5539-CNRS, Montpellier, France Titin is known t o interact with a ctin thin filaments within the I-band region of striated muscle sarcomeres. In this study, w e have used a titin fragment of 800 kDa (T800) purified from striated skeletal muscle to measure t he effect of this interaction on the functional properties of the actin– myosin complex. MALDI-TOF MS revealed that T 800 contains the entire titin PEVK (Pro, Glu, Val, Lys-rich) 1 domain. In the presence of tropomyosin–troponin, T800 increased the sliding velocity (both average and maximum values) of actin filaments on heavy-meromyosin (HMM)- coated surfaces and dramatically decreased the number of stationary filaments. These results were correlated with a 30% reduction in actin-activated HMM ATPase activity and w ith an i nhibition of HMM binding to actin N -terminal residues as shown by chemical cross-linking. At the same time, T800 did not affect the efficiency of the Ca 2+ - controlled on/off switch, nor did it alter the overall binding energetics of HMM t o actin, a s revealed b y cosedimentation experiments. These data are consistent with a competitive effect of PEVK domain-containing T800 on the electrostatic contacts at the a ctin–HMM interface. They also suggest that titin may participate in the regulation of the active tension generated by the a ctin–myosin complex. Keywords: ATPase; chemical cross-linking; mass spectro- metry; motility assay; muscle contraction. Titin is the largest known protein, containing more than 38 000 residues in its longest human striated muscle isoform. It represents the third most abundant component of vertebrate striated muscle, after myosin and actin, and is also present i n smooth muscle and nonmuscle c ells (recently reviewed in [1,2]). The importance of intact titin for normal muscle function has been demon strated in vitro [3–5], as well as in vivo through its implication in muscular dystrophies such as dilated cardiomyopathies and Udd’s tibial muscular dystrophy (reviewed i n [6]). In striated muscle, titin is involved in several fundamental processes, including sarcomere assembly, possibly in thick filament length control [4,7–9], maintenance of the sarco- meric structure, muscle elasticity and passive tension development [10–12]. These functions are related to three main structu ral properties o f the protein: titin s pans half a sarcomere, from the Z disks to the M line (connecting the Z d isks to myosin thick filaments), it contains subdomains that confer unusual e lastic properties, and i t interacts with several protein partners such as myosin, a ctin, M protein, C protein, MURF-1, calpain 3, myomesin, a-actinin, nebulin, telethonin and obscu rin. The elastic domains ar e made o f t andemly arranged immunoglobulin ( Ig)-like domains and a unique PEVK domain (Pro, Glu, Val, Lys-rich) whose size depends on the muscle fibre isotype. Specific structural properties and mechanical force/extension m easurements made o n muscle fibres or at the single molecule level suggest that the tandem Ig- and PEVK-domains are two elements of differential stiffness t hat function a s a two-spring system [13–24]. This elastic system i s now believed to b e a major contributor to the passive tension developed in striated m uscle . Another important feature of the I-band region was first revealed by electron microscopy images, which showed that in this region titin and actin can come close enough to associate w ith each other [25,2 6]. This associa tion has now been confirmed by numerous in vitro experiments involving actin and the titin PEVK domain [27–33]. The dynamics of this association seem to act together with the elastic elements of titin to modulate m uscle p assive stiffn ess [34–36]. Indeed, recent data suggest that the PEVK domain f rom cardiac muscle titin interacts with actin much more efficiently than does that f rom s keletal m uscle titin [36,37], supporting the idea that this interaction may be correlated with passive stiffness i n each muscle type. It is important to note, however, that both the size o f the PEVK domain, and the difficulty involved in extracting large amounts of native titin from muscle, have restricted these studies to examining the interaction between actin and bacterially expressed recombinant P EVK titin sub- fragments. In the case of the single in vitro motility assay that has been achieved using tissue-extracted titin, the experiments were d esigned to favour titin binding to the coverslip, which stopped actin motion during the assay [30]. In this study, we have further investigated the interaction of titin with actin by using two new experimental tools. First, we have used a native titin fragment of 800 kDa (encompassing the e ntire PEVK domain) that was isolated from the muscle sarcomeric I-band region. Second, we Correspondence to P. Chaussepied, Centre de Recherche de Biochimie Macromole ´ culaire, CNRS, 1919 Route de Mende, 34293 M ontpellier Cedex 5, France. Tel.: +33 467613334, Fax: +33 467521559, E-mail: patrick.chaussepied@crbm.cnrs-mop.fr Abbreviations: DTE, dithioerythritol; EDC, 1-ethyl-3-(3-dimethyl- aminopropyl)carbodiimide; NHS, N-hydroxysuccinimide; F-actin, filamentous actin; HM M, heavy meromyosin; T800, titin fragment of 800 kDa; Tm–Tn, tropomyosin–troponin complex. (Received 11 August 2004, revised 4 October 2004, accepted 11 October 2004) Eur. J. Biochem. 271, 4572–4581 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04429.x have worked with reconstituted thin filaments containing both actin and the regulatory tropomyosin–troponin (Tm– Tn) complex. Data obtained using these tools have confirmed the interaction between the PEVK domain- containing titin fragment and reconstituted thin filament. They have also shown that t he titin fragment reduces the number of contacts between myosin and the N-terminal part of actin, producing significant e ffects on both in vitro motility and the ATPase activitiy of the actin–myosin complex. Materials and methods Reagents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were from Sigma. a-Chymotrypsin was from Worthington. All other chemi- cals were of the h ighest analytical grade. Preparation of proteins All proteins were extracted from rabbit skeletal muscle. Myosin an d m yosin fragments were prepared as described by Offer et al. [ 38]. Heav y meromyosin (HMM) was obtained after a-chymotrypsin digestion of myosin (enzyme/substrate mass ratio of 1 : 400) for 15 min at 25 °C in 10 m M NaH 2 PO 4 , 600 m M NaCl, 1 m M MgCl 2 ,1m M dithioerythritol (DTE) pH 7.0. After the reaction was stopped b y phenylmethanesulfonyl fluoride ( phenyl- methanesulfonyl fluoride/substrate mass r atio of 1 : 200), the solution was dialysed overnight against 20 m M Mops, 0.2 m M DTE, pH 7 .0, and centrifuged 20 m in at 100 000 g. HMM was purified by ion exchange chromatography on SP-sephacryl (Pharmacia-Biotech) using a 0–200 m M NaCl gradient, drop-frozen in liquid nitrogen, and stored at )80 °C. Filamentous actin (F-actin) was prepared from acetone powder and further purified by two cycles of polymerization-depolymerization [39]. The final polymer- ization step was performed by overnight incubation of monomeric actin ( 40 l M )at4°C in the presence of 120 m M NaCl, 2.5 m M MgCl 2 . Polymerized actin w as concentrated by centrifugation at 190 000 g for20minandkeptat4°C (120 l M final concentration) in 100 m M NaCl, 2.5 m M MgCl 2 ,50m M Mops, pH 7 .5. For the in vitro motility assay, F-actin was not concentrated but rather used directly at 40 l M for rhodamine–phalloidin labelling (see below). Tropomyosin and troponin complex (troponin I, T and C) were prepared from acetone-dried mu scle powder according to Smillie [40] and Potter [41], respectively. They were stored in the lyophilized form and u sed as a solution containing equimolar a mounts of tropomyosin and troponin (Tm–Tn). Titin fragment (T800) was obtained from rabbit back muscles (mainly trapezius and lattissimus dorsi muscles) after Staphylococcus aureus V8 protease treatment of myofibrils (enzyme/myofibril weight ratio of 1 : 200, 30 min, 25 °C) and centrifugation at 5000 g for 5 min [42,43]. T800 was subsequently purified through gel filtra- tion S300 HR (Pharmacia-Biotech) f ollowed by Poros HQ/H column (Boe hringer) in 2 m M Tris, 1 m M DTE, 1m M EDTA, p H 7 .9. P ure T800 was eluted at 250 m M NaCl. All proteins were used within 5–6 days and ultra- centrifuged (except F-actin) at 190 000 g for 20 min prior to each experiment. Protein concentrations were determined spectrophoto- metrically assuming extinction coefficients A 1% 280 of 5.7 c m )1 , 6.5 cm )1 ,11.0cm )1 ,3.3cm )1 ,4.5cm )1 and 10.0 cm )1 for myosin (500 kDa), HMM (360 kDa), actin (42 kDa), tropomyosin (66 kDa), troponin (70 kDa) and T800 (800 kDa), respectively. The extinction coefficient for T800 was estimated experimentally using the Bradford method [44] to measure the protein concentration of the T800-containing solution, using HMM for the standard curve. MS Proteins were in-gel digested by trypsin according to Rosenfeld et al. [45]. The resulting digests were cleaned using t he ZipTip device (Millipore Inc) and analysed by MALDI-TOF MS (BiflexIII, B ruker). Database queries were performed using the Mascot search engine (Matrix Science at http://www.matrixscience.com/). In vitro motility assay F-actin (0.6 l M ) was fi rst stabilized and labelled b y adding a twofold excess of tetramethyl-rhodamine phalloidin in motility buffer ( 50 m M KCl, 10 m M MgCl 2 ,40m M DTE, 60 m M Hepes pH 7 .8, 90 m M ionic strength). Labelled F-actin was then diluted (2 n M final concentration) in motility buffer containing 3.3 mg ÆmL )1 glucose, 0.37 mgÆmL )1 catalase, 0.11 mgÆmL )1 glucose oxidase, 0.5% (w/v) methylcellulose, and 0.1 m M CaCl 2 or 1 m M EGTA (only when t he Tm –Tn c omplex was present). The solution was supplemented b y Tm–Tn and T800 (both at 20 n M , conditions for a saturating effect), and ATP (2 m M ) was added to flow cells containing HMM-coated glass coverslips just prior to image recording. Coverslips were pretreated overnight at room temperature with 1 M HCl, rinsed with distilled w ater, 95% ethanol and air-dried. They were then treated with BSA/casein (10 m gÆmL )1 )for10minat20°C, air-dried, mounted on the flow cell, and coated with HMM (50 lgÆmL )1 solution containing 600 m M KCl, 10 m M Hepes, pH 7.0) for 10 min on ice prior to the a ddition of the a ctin solution. ÔDeadÕ HMM molecules were removed before the coating step by two consecutive ultracentrifugation steps at 190 000 g for 20 m in in the p resence of a threefold molar excess of F-actin-phalloidin and 2.5 m M ATP in 10 m M Hepes, 600 m M KCl pH 7.0. After each ultracentrifuga- tion step, the HMM concentration was evaluated by the Bradford method. The ÔdeadÕ HMM eliminated in this way corresponded to 5–10% of the total HMM in the preparation. Images of the m icrofilaments were obtained w ith a DMR B microscope (Leica, Bensheim, 2 Germany) using a PL APO 100 · objective (NA 1.40) with a 1.6 · tube factor and immersion oil Immersol 518 F (Zeiss, Go ¨ ttingen, 3 Germany). Preparations were illuminated with a 100 W HBO 103 W /2 light bulb (OSRAM, Regensburg, 4 Germany) through a N 2.1 filter cube (Leica) for the visualization of rhodamine fluorescence. The microscope was equipped with a homemade heating stage. The h eat regulation was Ó FEBS 2004 Effect of titin on actin–myosin activity (Eur. J. Biochem. 271) 4573 stabilized to prevent undesired minute up and down movements of the stage, which can upset the s tability of image focusing during time-lapse recording. The stability was further enhanced by the p resence o f a plexiglass box that p rotected the front part of the microscope (objective barrel, stage, etc. down to the bench) from surrounding air movements. The front part of the box consisted of a plastic curtain that a llowed e asy a ccess to the stage. Images w ere captured with an ORCA 100 (B/W) 10 bits cooled CCD camera (C mount 1x), C 4742-95 controller a nd HIPIC controller program (Hamamatsu, Shizuoka, 5 Japan) run b y a PC-compatible computer. Time-lapse recording of the images (time intervals ranging from 0.1 to 1 s) were carried out with the Camera Sequence option of the controller program, with a 2 · 2 binning of the d etector and camera gain set a t its ma ximum v alue. Sequences were saved as a suite of individual TIFF format images (up to 250 i n one sequence). Measurements were c arried out with META- MORPH 6.1 software (Universal Imaging Corporation, Downington, PA, USA) 6 by following the movement of the leading end of the actin filament. Statistical analyses were performed using PRISM 2.2 software (GraphPad Software, Inc., San Diego, CA, USA) 7 . Mann–Whitney test was used to compare sets of data and a P-value < 0.005 was used to determine s tatistical significance. Steady-state ATPase and actin binding assays Various mixtures containing F-actin (3 l M ) alone or with T800 (0.15 l M ), Tm–Tn (1.0 l M ) and HMM (0.25 l M in the ATPase a ctivity and 1.5 l M in the binding assay) were incubatedfor10minin50m M Hepes, 5 m M MgCl 2 , 50 m M KCl, 2 m M DTE ( 80 m M ionic strength), with o r without 0.1 m M CaCl 2 ,1 m M EGTA or 2 m M ATP, pH 7.8 (the binding assay was also conducted in the presence of 100 m M NaCl, that is in a final 180 m M ionic strength without ATP). The M g.ATPase activities were measured at 25 °C. The reaction was started by the addition of 2 m M ATP and stopped after 10 min by 5% trichloroacetic acid. The amount of P i liberated was evaluated c olorimetrically [46]. The actin binding assay w as carried out by ultracentri- fugation of t he reaction mixtures at 190 000 g for 20 m in. An aliquot of each supernatant was removed after centri- fugation and mixed with Laemmli solution [50 m M Hepes, 2% (w/v) N aDodSO 4 , 1% 2-mercaptoethanol and 50% (v/v) glycerol, pH 8 .0]. Air-dried pellets were homo- genized in Laemmli solution and aliquots of both the supernatant and the resuspended pellets were analysed by PAGE after boiling the samples f or 3 m in. Two-step cross-linking experiments During the activating step, 80 l M F-actin was treated for 10 min at 20 °Cwith50m M NHS and 25 m M EDC in buffer C (50 m M NaCl, 5 m M MgCl 2 ,50m M Mops pH 7.0). The activating reaction was stopped with 100 m M b-mercaptoethanol. During the condensation step, an aliquot of activated F-actin (3 l M final c oncentration) was mixed with 0.15 l M T800 with or without 1.0 l M Tm–Tn and 1.5 l M HMM in buffer C in the presence of 0.1 m M CaCl 2 . Reactions were terminated 30 min after the addition of HMM by adding an aliquot of the reaction mixture to a boiling Laemmli solution. PAGE Gel electrophoresis was as described by Laemmli [47] using a 2–15% gradient acrylamide gel. Densitometric analysis of the scanned gels w as performed using METAMORPH 6.1 software. Results Localization of T800 within the I-band region of skeletal titin Some of us have previously demonstrated that mild treatment of m yofibrils with S. aureus V8 protease releases a soluble titin fragment of 800 kDa (T800) that can be purified to homoge neity [42]. In order to localiz e T800 within titin, we performed MALDI-TOF MS following in-gel digestion of T 800 by trypsin. The set of molecular weights corresponding to the resulting tryptic peptides was then examined by a search in the NCBI nonredundant protein database using the search engine ÔMascotÕ without any manual interpretation [ 48]. The results of this search are summarized in Fig. 1A in the form of a graph showing scores reflecting the probability that an observed match is a random event. A s core higher than 65 indicates identity or extensive homology with theoretical sequences in the database. Significant scores of 98 and 84 were obtained for a human skeletal titin f ragment ( correspond- ing to residues 4262–12 392) and full-length human skeletal titin (residues 1–26 926), respectively. Of 79 peptides analysed, 22 matched w ith the two proteins, with the difference between calculated and experimental molecular weights bein g lower than 0.1 Da. These 2 2 peptides were located between residues 4670 and 9070 of full-length human titin, within the I-band region of the skeletal muscle sarcomere and encompassing the entire PEVK domain (amino a cid segment 5618–7792; Fig. 1B) . Based on these experimentally determined boundaries, and considering that T800 contains approximately 7200 resi- dues, we estimate that the extreme borders of T800 could lie between residue 1870 (lower value) and residue 1–11 500 (higher value). These data demonstrated that T800 contains the P EVK domain a nd falls entirely within the I-band r egion of skeletal titin. T800 accelerates in vitro motility of the reconstituted thin filament Because the titin PEVK domain is known t o i nteract w ith actin, we studied the effects o f T800 on the m ovement of reconstituted actin filaments on HMM coated coverslips, using the in vitro motility assay. Figure 2A depicts a typical velocity–time pattern for one actin filament. Such a pattern was representative of the results obtained, regardless of the experimental condition s or of the presence of T800 and the regulatory proteins Tm–Tn. The filament motion displayed a ccele ration/decel- eration phases throughout the entire time course of the movement. T his periodicity, which has been reported earlier 4574 N. Niederla ¨ nder et al. (Eur. J. Biochem. 271) Ó FEBS 2004 [49,50], is p robably due to the heterogeneity of the HMM molecules coated on t he glass surface, alth ough other explanations such as intra-actin cooperativity have also been proposed. The recording time varied from 50 to 150 s and was generally limited by the loss of focus. Due to data scattering, we favoured a global analysis of the entire set of velocity values recorded for all the moving filaments (without stop events ), rather than an analysis of the average values for each filament. Depending on the experimental conditions, 850–1700 data points were collected. The data obtained for four different experi- mental conditions (actin alone, actin in the presence o f T800,actinwithTm–Tn,andactinwithTm–Tninthe presence of T800) are presented in Fig. 2B and C and in Table 1 . The average velocity obtained for actin alone (2.5 lmÆs )1 ) was lower than the values generally obtained with nitrocellulose pretreated coverslips, but it was very comparable to the value (about 3 lmÆs )1 ) obtained w ith untreated coverslips under very similar conditions, using HMM frozen in liquid nitrogen [51]. The most significant result is that the average velocity was increased by the addition of T800, from 2.5 to 3.4 lmÆs )1 and from 3.9 to 4.3 lmÆs )1 in the absence and the p resence of Tm–Tn, respectively (Table 1). This increase in the average velocity was accompanied by an increase in the maximum velocity (Fig. 2 B). Statistical analysis revealed that these differences were significant, with a P-value < 0.0001. More importantly, the number of stationary actin filaments was also found to be altered by th e addition of T800. While th is number was slightly increased in the absence o f T m–Tn (21.6% vs. 16.0%), it was d ramatically reduced in the presence of reconstituted thin filaments, containing Tm–Tn (5.6% vs. 21.0%; T able 1, Fig. 2C). Note that the mean filament length was not significantly affected by T800 in the absence of Tm–Tn (2.3 vs. 2.1 lm), an d was slightly decreased i n i ts presence (1.5 vs. 1.1 lm). N ote also that the presence of Tm–Tn on its own decreased t he me an filament l ength and increased sliding velocity, in good accordance with previously published data [52–54]. Finally, as expected for filaments that are normally regulated by Tm–Tn, we did not observe any movement in the absence of Ca 2+ , inde- pendent of the addition of T800. This result, together with the fact that T800 increased the average and maximum velocity v alues of moving filaments, both in the a bsence and in t he presence of Tm–Tn–Ca 2+ ,argues against a simple effect of T800 on the calcium sensitivity (pCa curve) of the movement and for an effect involving the actin–HMM interaction. Interestingly, the o rder of addition of the various actin- bound components turned out to be essential in these experiments, as mixing T800 with actin prior to the addition of Tm –Tn resulted in the immobilization of the thin filaments, even in the presence of Ca 2+ . This result demonstrated that T800 binds to actin filaments differently in the absence and in the presence of Tm–Tn, and can promote, when added prior to Tm–Tn, an unproductive Fig. 1. Identification of T800. (A) Mascot search result for T800 after its ru n in SD S gel (inset), in-gel digestion with trypsin, and ana- lysis with automated MALDI-TOF MS, fol- lowed by a search in the NCBR nonredundant protein database. (B) Schematic representa- tion of human skeletal muscle titin (gi|17066105; score 84) and a human skeletal muscle titin fragment (gi|7512404; score 98). The loc ation of matching peptides around the PEVK domain and the two predicted extreme boundaries (residues 1870–9070 and 4670– 11500) of T800 are also shown. Ó FEBS 2004 Effect of titin on actin–myosin activity (Eur. J. Biochem. 271) 4575 interaction between HMM a nd ac tin. It is likely that in adult native s triated muscle, titin interacts with a preformed thin filament containing bound Tm–Tn, similar to the interactions described i n the present study. T800 decreases actin-HMM ATPase activity In order to understand the effect of T800 on thin filament sliding velocity, we measured the Mg 2+ -ATPase activity of HMM and various actin–HMM complexes in the presence or absence of T800. The ATPase activity of HMM alone was not changed i n the presence of T800 (varying from 0.17 to 0.19 s )1 ; Table 2). This r esult was in accordance with the lack of interaction between the two proteins as revealed by the absence of cosedimentation of T800 with myosin during a low speed centrifugation experiment (data not shown), and by the absence of interaction between titin and HMM-coated coverslips during the in vitro motility assays. In contrast, T800 lowered the actin-activated HMM M g 2+ - ATPase activity at a saturating T800/actin molar ratio of 1 : 20, regardless of whether or not Tm–Tn was bound to actin. This inhibition was of 39% and 31% in the a bsence and p resence of T m–Tn (with Ca 2+ ), respectively. In addition, T800 did not significantly alter the EGTA-induced reduction of HMM ATPase activity, measured in the presence of Tm–Tn (62% vs. 69% reduction without and with T800, respectively). This small or nonexistent effect of T800 on the Ca 2+ -linked regulation of the actin–HMM ATPase is entirely consistent wit h the la ck o f effect on the Ca 2+ -controlled on/off switch of thin fi lament motion. T800 specifically reduces HMM binding to the N-terminal part of actin We studied in greater detail the simultaneous binding of T800 and HMM to reconstituted thin filaments containing the Tm–Tn complex at two ionic s trengths (80 m M and 180 m M ). As shown in Fig. 3, the presence of T800 did not have much effect on HMM binding to actin as judged by the constant amount of HMM in t he pellet of ultracentrifuga- Fig. 2. In vitro motility data. (A) Typical velocity vs. time trace obtained from the analysis of the movement of a single filament during the in vitro motility assay. (B and C) Box representation of the velo- cities (B) and the percentile of STOPS (C) obtained under four different experimental conditio ns: actin alone (Actin); actin + T800 (Actin + T800); actin + Tm–Tn + CaCl 2 (Actin + T m-Tn); actin + Tm– Tn + T800 + CaCl 2 (Actin + Tm–Tn + T800). STOPS corres- pond to the time filaments were stationary, ex pressed as a percentage of total time of analysis for each moving filament. Boxes extend from the 25th percentile to the 75th percentile of each data set with the horizontal line at the median. Whiskers show t h e range of the data. Detailed numbers and experimental conditions are reported in Table 1 and in Materials and methods. Table 1. In vitro motility assay analysis. Analyses were performed on three slides containing 81–91% moving filaments. Velocities were estimated on approximately 857–1709 points (without stops) for each experiment; M ann–Whitney test showed a P-value < 0.0001 com- paring either actin alone and actin + T800 or a ctin + Tm–Tn and actin + Tm–Tn + T800. STOPS correspond to the time that fila- ments were stationary, expresse d as a perc entage of to tal time of analysis for all moving fil aments. Actin alone Actin + T800 Actin + Tm–Tn Actin + Tm–Tn + T800 Velocity (lmÆs )1 ) 2.5 ± 1.3 3.4 ± 1.6 3.9 ± 2.0 4.3 ± 2.2 Stops (% time) 16.0 21.6 21.0 5.6 Filament length (lm) a 2.3 ± 2.3 2.4 ± 2.0 1.5 ± 1.7 1.1 ± 1.4 a Average length of more than 200 filaments for each experimental condition; values under 0.2 lm were excluded from all analysis. 4576 N. Niederla ¨ nder et al. (Eur. J. Biochem. 271) Ó FEBS 2004 tion experiments. Under rigor conditions, all HMM was bound to actin and remained in the pellet independently of the other components and the ionic strength of the mixture. In the presence o f A TP, the amount of bou nd H MM was very similar (average value of 52.5 ± 1.1%) in all experi- ments except in the presence of Ca 2+ and high ionic strength (average value of 37.1 ± 4.0% for panels Fs + ATP and Gs + ATP; see figure legend for t he detailed quantitative data). On the other hand, the percent- age of T800 bound to actin was not affected by ATP and/or by CaCl 2 but it was decreased by elevating ionic strength from 80 to 180 m M with average values of 62.3 ± 2.2% and 45.1 ± 3.5%, respectively (compare d etailed values in figure legend, panels B and D vs. panels F and H). Concerning the actin–HMM interface, w e explored the electrostatic contacts between the N -terminal p art o f a ctin and the positively charged segment (also called loop 2) of HMM using EDC-induced cross-linking experiments [55]. We used a two-step cross-linking reaction which has the property of only modifying reactive acidic residues on actin, thereby r educing the number of nonspecific cross-linking reactions. As previously described, the effect of EDC on the actin–HMM complex results in a covalent actin–HMM adduct that migrates as a double band (Fig. 4, [56]). This double band corresponds to two cross-linked products, which are each known to contain an equimolar actin– HMM complex, but involving different cross-linked r esi- dues within t he actin–HMM i nterface [55,57]. Thes e cross- linked products wer e ob served in the absence of the regulatory proteins, Tm–Tn (Fig. 4, lane b), o r when T800 was added to actin prior to Tm–Tn (Fig. 4 , lane c), but they were almost totally absent under physiological Table 2. Effect of T800 on HMM ATPase activity. ATPase acti vities are the average v alues of t hree e xperiments p erformed a s described i n Materials and methods. Proteins (in order of assembly) HMM alone +T800 +Actin +Actin +T800 +Actin +Tm–Tn (Ca 2+ ) +Actin +Tm–Tn +T800 (Ca 2+ ) +Actin +Tm–Tn (EGTA) +Actin +Tm–Tn +T800 (EGTA) ATPase (s )1 ) 0.17 ± 0.02 0.19 ± 0.02 2.8 ± 0.7 1.7 ± 0.3 2.0 ± 0.5 1.4 ± 0.1 0.6 ± 0.1 0.5 ± 0.1 Fig. 3. T800 and HMM binding to F-actin. Gel electrophoresis analysis o f cose dimentation experiments performed as described in Material s and methods. In all experiments, T800 was added to the preformed actin–Tm–Tn complex and HMM was added last. A mixture of all the proteins used is shown in (A). Proteins were preincubatedinthepresenceofCaCl 2 (B,C,F,G) or EGTA (D,E,H,I) with or without 2 m M ATP as indicated . After ultracentrifugation, supernatants (s) and pellets (p) were analysed. The percentages of HMM in the pellets were 52.8 (Bs + ATP), 50.9 (Cs + ATP), 52.4 ( Ds + ATP), 54.3 (Es + ATP), 3 9.9 (Fs + ATP), 34.2 (Gs + ATP), 52.6 (Hs + ATP) and 5 1.7 (Is + ATP). The pe r- centages of T800 in the pellets were 62.1 (Bs), 61.4 (Bs + ATP), 65.4 (Ds), 60.2 (Ds + ATP), 50.2 (Fs), 44.3 (Fs + ATP), 43.6 (Hs) and 42.3 (Hs + ATP). Ó FEBS 2004 Effect of titin on actin–myosin activity (Eur. J. Biochem. 271) 4577 conditions, when T800 was added to reconstituted thin filaments (Fig. 4, lane d). An additional faint band was observed at a bout 70 kDa when Tm–Tn was present. Th is latter product c orresponded p resumably to a cross-linking reaction between a ctin and the Troponin I subunit ( Fig. 4, lanes c and d). Finally, the absence of bands above T800 in all experiments argue against a cross-linking reaction of T800 to actin N-terminal s egment. Discussion T800 represents the first n ative titin fragment containing the entire PEVK domain to b e directly extracted from s keletal muscle myofibrils. This titin fragment has the ability to interact with reconstituted thin filaments, and has unex- pected effects on HMM sliding velocity and on HMM binding to actin filaments. These results provide an e xperi- mental basis to investigate a possible role for titin in the regulation of energetics and force generation in the actomyosin system. Identification o f T 800 w as performed by MALDI-TOF spectrometry. In fact, the 800 kDa titin fragment represents the largest protein fragment so far identified using MS and in-gel tryptic digestion approaches. T800 contains the titin PEVK domain and is entirely located within the I-band region of the skeletal muscle s arcomere. Its b oundaries are estimated to be at the most around residues 1870 and 11 500 of skeletal muscle titin, two loci where titin is free of interaction with its protein partners and could easily be attacked by V8 protease [58]. Outside the I-band region, it is likely that the interactions of titin with myosin (in the A-band region) and actin or a-actinin (in Z-disk and its periphery) are strong enough to protect it against the formation (or prevent the release) of other proteolytic fragments. Such protection could a ctually explain w hy, besides T800, only one additional 150 kDa fragment, also belonging to the I-band region, was generated by the proteolytic treatment of skeletal myofibrils [42]. It is also noteworthy t hat we u sed in this study rabbit back m uscles which are heterogeneous in their fibre-type content [59]. Nevertheless, both the homogeneity of the T 800 preparation and the results of the mass peptide analysis suggest that the proteolysis and purification protocols selected preferentially the longest skeletal m uscle titin isoform. Our d ata clearly indicate that T800 bind s t o actin thin filaments, in good agreement with numerous w orks previ- ously published on PEVK domain-containing titin frag- ments (see Introduction for references). The PEVK domain remains the main actin-binding candidate identified in the I-band titin region and we propose, withou t totally exclu- ding other possibilities, that the interaction of T800 with actin is primarily mediated by the PEVK domain. Another actin-binding site candidate was proposed within stretch of residues 1791–2126 of cardiac titin [ 31], but we are still not certain whether this s tretch of residues belongs t o T 800 a s the corresponding residues 1870–2205 of skeletal titin are located close to the hypothetic extreme N-terminal end o f T800 (Fig. 1 ). The interaction between actin and T800 is characterized by an apparent saturating T800/actin m olar ratio of 1 : 2 0 as determined by centrifugation experiments with increasing amounts of T800. This ratio suggests t hat T800 covers a rather long segment of actin filament and that either it sterically protects part of actin filament region around the interaction site or it contains multiple actin binding sites. This last suggestion is compatible with the presence of repeated stretches o f charged/uncharged resi- dues along the PEVK domain [ 13,60] and with the i onic strength dependence o f T800 binding to actin. T800 increases th e velocity of moving actin filaments. This acceleration is observed both in the absence and in the presence of Tm–Tn (with Ca 2+ ). However, the molecular explanations seem different i n the two cases as the number of s tationary filamen ts decreases in t he absence of T m–Tn and increases in its presence, and also because th e reduction in actin–HMM cross-linking occurs only in the presence of Tm–Tn. Note that in both cases, t he acceleration observed in the motility assay excludes a direc t interaction between T800 and the myosin motor domain, as reported for the fibronectin-like domains of the A band part of titin [61]. No attempt w as made to further explain the c hanges observed in the absence of Tm–Tn, as this situation is highly unlikely to occur under physiological conditions. In the presence o f Tm–Tn, it is very tempting to correlate the functional changes with the structural modification of the actin–HMM interface, which results in the inhibition of HMM cross- linking to the N-terminal part of a ctin. This change of the actin–HMM ionic interface would be in contrast to the lack of effect on the HMM binding to actin observed i n cosedimentation experiments. Such a discrepancy has been previously related to t he fact that the electrostatic contacts taking place at the N-terminal part of actin represent only a very weak ) sometimes considered non-specific ) compo- nent of the actin–myosin interface [57,62–66]. Moreover, it should be mentioned that a reduction in these ionic contacts is compatible with the high efficiency of the Tm–Tn–Ca 2+ - linked regulation observed in the presence of T800, as a recent report demonstrated that removing the negative charges i n th is re gion o f actin does not affect the pC a curves of the m otion of thin filaments [ 67]. Fig. 4. EDC-induced cross-linking at the actin–HMM interface. Gel electrophoresis analysis of th e cross-linking experiments performed on mixtures composed of (in the order of addition): F -actin + T800 (a), F-actin + T800 + HMM (b), F-actin + T800 + Tm–Tn + HMM (c) and F-actin + Tm–Tn + T800 + HMM (d). 4578 N. Niederla ¨ nder et al. (Eur. J. Biochem. 271) Ó FEBS 2004 This reduction in actin–HMM contacts could be due to a direct or indirect competition between T800 and HMM for binding to the negatively c harged residues of the N-terminal part of actin. Our data suggest that T800 acts indirectly a s T800 alone or added b efore Tm–Tn on actin filaments cannot displace HMM. This indirect effect could, for example, be mediated through interactions with Tm–Tn, as proposed recen tly by s olid phase experiments [68], or following structural rearrangements within actin as sugges- ted by the decrease in filament length that i s observed in the presence of T800. Interestingly, both T800 and T m–Tn binding to actin induces a shortening of filament length and an increase in myosin sliding velocity, suggesting a strong synergy between these two actin binding components in their functional a nd molecular effects on actin. Can the diminution of HMM contacts with the N-terminal part of actin account f or the d ecrease in actin–HMM ATPase activity and for the increase in thin filament sliding velocity while the se t wo activities are supposed to be correlated? Reduction of these contacts was foun d to induce the loss of correlation between the ATPase and sliding activities in numerous examples [69–73] and it was reported to inhibit the actin-activated myosin ATPase activity in the same way as T800 [64,69]. Inhibition of the ATPase activity was then explained by a slowing down of the formation of active complex in solution. On the other hand, the acceleration of the sliding velocity (and the lower number of stops) of thin filaments could be related to a diminution o f the load in the actin–myosin interface. The properties of T800 described in this paper diverge significantly from previous reports that supported the idea that binding of the PEVK domain to actin slows down or totally inhibits actin motion over the myosin motor domain [29,30,35,36]. An important issue to consider here is that most of these s tudies were performed w ith bacterially expressed titin fragments, an d not with musc le-extracted native fragments. The muscle extracted titin f ragment, T800, did not tend to aggregate as it remained soluble (in the supernatant after high speed centrifugation) for several days after its purification, nor did it interact in a nonspecific way with myosin or with the glass support during the motility assay. Moreover, T800 interacted very efficiently with actin filaments during cosedimentation experiments and never induced the formation of actin bundles. Note also that T800 did not perturb t he Tm–Tn–Ca 2+ -linked r egulation of the reconstituted thin filaments, neither i n the motility assay nor in the ATPase experiments, further underscoring its very specific effect on thin filaments. On the other hand, the facts that the recombinant fragments may have interacted with myosin or the coverslips during the motility assay, and that in some cases they induced actin bundles, could easily explain the observed differences between their functional properties and those characterizing T 800. But this is not the only parameter that one should consider, as we f ound for example that T 800 had a different effect on the actin–HMM complex depending on whether or not Tm–Tn was present (see above). Two previous studies on actin binding to titin or recombinant t itin fragments also used tropomyosin [31] or tropomyosin–troponin [30]. However, their results were controversial as the fi rst one found an inhibition while the second one reported an increase o f actin binding in the presence of calcium. In this work, c alcium did not change T800 binding to actin nor the functional effect of titin on the actin–HMM complex. How should we interpret the effects of T800 observed in vitro with respect to the in vivo functional properties of t he actin–myosin complex? Reducing the ATPase activity of the actin–myosin complex could have important effects on the energetic balance during m uscle activity, and s peeding up the movement of actin filaments could h ave conse- quences for the generation of active tension. In resting and stretching conditions, there is no overlap between myosin cross-bridges and the titin PEVK domain. Therefore, the effects described in this work are unlikely to take place under t hese conditions, unless it is demonstrated that they propagate over long distances on actin filaments. In contrast, during muscle shortening, such an overlap, and its functional consequ ences for t he actin-myosin co mplex, may occur. The facts that T800 interacts w ith a ctin in the presence of Tm–Tn, HMM, ATP and up to 0.1 m M CaCl 2 and that at 180 m M ionic strength more than 45% of T800 remains bound to actin, support the in vivo ext rapolation o f titin binding to actin in the sarcomeric I-band region and its functional consequences in striated muscle. However, before extrapolating our results to any physiological environment, one should consider the properties of an additional natural component of the thin filament framework, nebulin. Nebulin spans the entire length of thin filaments and is capable of modulating the r ate of formation of the a ctin– myosin complex [74,75]. Interestingly, nebulin also interacts with the titin PEVK domain in a calcium/calmodulin and calcium/S100 dependent manner [76]. These data pose questions regarding t he precise functional properties of t he entirely reconstructed thin filament (actin–Tm–Tn–nebulin) as they relate to myosin binding and activation, and concerning how these properties are regulated by titin. These two missing but essential pieces of information will have to be addressed experimentally both in vitro and in vivo before conclusions can be drawn about the functional consequences of titin binding to thin filaments. Finally, it will be important to investigate t he effec ts of titin on the actin–myosin complex using titin fragments extracted from other striated muscles, such as cardiac muscle. Titin isoforms from cardiac muscle have been shown to interact more strongly with actin than does the skeletal isoform, and titin is thought to be the main contributor to passive tension d evelopment i n c ardiac muscle [36,37,77]. Stud ying titin from smooth muscle or nonmuscle tissues will also be of particular interest for at least two reasons: the PEVK content of titin in these isoforms is not well characterized and the structural constraints in these tissues could conceivably allow t he PEVK do main to control myosin b inding to actin, and t o play an even more crucial role in the energetics and the generation of active tension within smooth muscle or nonmuscle stress fibers. Acknowledgements We are grateful to Jean Derancourt for his help in the mass spectrometry an alysis of the T800 fragment (Montpellier Genopole Proteome facilities, http://genopole.igh.cnrs.fr/), Pierre Travo (CRBM imaging facilities, http://www.crbm.cnrs-mop.fr/Imagcell.html) for advice and help s etting u p the in vitr o motility assay, an d Juliette Ó FEBS 2004 Effect of titin on actin–myosin activity (Eur. J. Biochem. 271) 4579 VanDijk for her critical reading of the manuscript. This work was supported b y t he French Centre National de la Recherc he Sc ientifi- que. References 1. Tskhovrebova, L. & Trinick, J. (2003) Titin: properties and family relationships. Nat. Rev. Mol. Cell Biol. 4, 679–689. 2. Keller,T.C.S., III, Higginbotham, M., Kazmierski, S., Kim, K.T. & Velichkova, M. (2000) Role of titin in non muscle an d sm ooth muscle cells. In Elastic Filaments of t he Ce ll (Gra nzier, H.L. & Pollack, G.H., eds), pp.265–281. Kluwer Academic/Plenum Pub- lishers, Dordrecht, the Netherlands. 8 3. Peckham, M., Young, P . & Gau tel, M. (1997) Constitutive and variable regions of Z-disk titin/connectin in myofibril formation: a dominant-negative screen. Cell Struct. Funct. 22, 95–101. 4. Van der Ven, P.F., B artsch, J.W., Ga utel, M., Jockusch, H . & Furst, D.O. (2000) A functional knock-out of titin results in defective myofibrils assembly. J. Cell Sci. 113, 1405–1414. 5. Gotthardt, M., Hammer, R.E., Hubner, N ., Monti, J., Witt, C.C., McNabb, M., R ichardso n, J.A., Granzier, H., Labeit, S. & Herz, J. (2003) Conditional expression of mutant M-line titins results in cardiomyopathiy with altered sarcomere. J. Biol. Chem. 278, 6059–6065. 6. Nishino, I. & Ozawa, E. (2002) Muscular dystrophies. Curr. Opin. Neurol. 15, 539–544. 7. Whiting, A., Wardale, J. & Trinick, J. (1989) Does titin reg- ulate the length of muscle thick filament? J. Mol. Biol. 205, 263– 268. 8. Van der Ven, P.F., Ehler, E., Perriard, J.C. & Furst, D.O. (1999) Thick filament assembly occurs after the formation of a cytoske- letal scaffold. J. Muscle Res. Cell Motil. 20, 569–579. 9. Miller, G., Mus a, H., Gautel, M. & Peckham, M. (2003) A tar- geted d eletion o f the C-terminal end of t itin, including the t itin kinase domain, i mpairs myofibrillogenesis. J. Cell Sci. 116, 4811– 4819. 10. Horowits, R., Kempner, E .S., Bisher, M.E. & P odolsky, R.J. (1986) A p hysiological role for titin and nebulin in skeletal muscle. Nature 323, 160–164. 11. Magid, A. & Law, D.J. (1985) Myofibrils bear most of the resting tensioninfrogskeletalmuscle.Science 230, 1280–1282. 12. Wang, K., McCarter, R., Wright, J., Be ver ly, J. & R amirez - Mitchell, R. (1991) Regulation of skeletal muscle stiffness and elasticity by titin isoforms: a test of the segmental e xtension model of resting tension. Proc. Natl Acad. Sci. USA 88, 7101–7105. 13. Labeit, S. & Kolmerer, B. (1995) Titins: giant proteins in charge of muscle ultrastructure and elasticity. Science 270, 293–296. 14. Gautel, M. & Goulding, D. (1996) A molecular map of t itin/ connecting elasticity revea ls two different mechan isms a cting in series. FEBS Lett. 385, 11–14. 15. Tskhovrebova, L., Trinick, J.,Sleep,J.A.&Simmons,R.M. (1997) Elasticity and unfolding of sing le m olecules of t he giant muscle protein titin. Nature 387, 308–312. 16. Kellermayer, M.S.Z., Smith, S.B., Granzier, H.L. & Bustamante, C. (1997) Folding-unfolding transitions in s ingle titi n m olecules characterized with laser twee zers. Science 276, 1112–1116. 17. Linke, W.A., Ive meyer, M., Mundel, P., Stockmeier, M.R. & Kolmerer, B. (1998) Nature of PEVK-titin elasticity in skeletal muscle. Proc. Natl Acad. Sci. USA 95, 8052–8057. 18.Linke,W.A.,Stockmeier,M.R.,Ivemeyer,M.,Hosser,H.& Mundel, P. (1998) Characterizing titin’s I-band Ig domain region as an entropic spring. J. Cell Sci. 111, 1567–1574. 19.Linke,W.A.,Rudy,D.E.,Centner,T.,Gautel,M.,Witt,C., Labeit, S. & Gregorio, C.C. (1999) I-band titin in cardiac muscle is a three-elem ent mo lec ular s pring a nd is critical for maintaining thin filament structure. J. Cell Biol. 146, 631–644. 20. Trombita ´ s, K., Greaser, M., Labeit, S., Jin, J.P., Kellermayer, M ., Helmes, M. & Granzier, H.L. (1998) Titin extensibility in situ: entropic elasticity of permanently folded and permanently un- folded molecular segments. J. Cell Biol. 140, 853–859. 21. Trombita ´ s, K., R odkan, A., Freiburg, A., Centner, T., Wu , Y., Labeit, S. & Granzier, H.L. (2000) Extensibility of isoforms o f cardiac titin: variation in fractional extension provides a basis for diastolic stiffness diversity. Biophys. J. 79, 3226–3234. 22. Helmes, M., Trombita ´ s, K., Centner, T., Kellermayer, M., Labeit, S., L inke, W.A. & Granzier, H.L. (1999) Mechanic ally driven contour-length adjustment in rat cardiac titin’s uniqu e N2B sequen ce: t it in is a n ad jus ta ble spring. Circ. Res. 84, 1339–1352. 23. Li, H., Oberhauser, A.F., Redick, S .D., Carrion-Vazquez, M ., Herickson, H.P. & Fernandez, J.M. (2001 ) M ultiple c onforma- tions o f PEVK proteins detected by single-molecule te chniq ues. Proc. Natl. Acad. Sci. USA 98, 10682–10686. 24. Watanabe, K., Nair, P., Labeit, D., Kellermayer, M.S.Z., Greaser, M., Labeit, S. & Granzier, H.L. (2002) Molecular mechanics of cardiac titin’s PEVK and N2B spring elements. J. Biol. Chem. 277, 11549–11558. 25. Kimura, S., Maruyama, K. & Huang, Y.P. (1984) Interactions of muscle beta-connecting with myosin, actin, and actomyosin at low ionic strengths. J. Biochem. 96, 499–506. 26. Funatsu, T., Kono, E., Higuc hi, H., Kimura, S., Ish iwata, S., Yoshioka, T., Maruyama, K. & Tsukita, S. (1993) Elastic fila- ments in s itu in cardiac muscle: deep-etch replica analysis in combination with selective removal of actin and myosin filaments. J. Cell Biol. 120, 711–724. 27. Soteriou, A., G amage , M. & Trinick, J. (1993) A survey of th e interactions made by titin. J. Cell Sci. 104, 119–123. 28. Jin, J P. (1995) Cloned rat cardiac titin c lass I and II motifs. J. Biol. Chem. 270, 6908–6916. 29. Li, Q., Jin, J P. & Granzier, H.L. (1995) The effect of genetically expressed c ardiac titin fragmen ts on in vitro actin motility. Biophys. J. 69, 1508–1518. 30. Kellermayer, M.S. & G ranzier, H.L. (19 96) Ca lcium-dependent inhibition of in vitro thin-filament motility by native titin. FEBS Lett. 380, 281–286. 31. Linke, W.A., Ivemeyer, M., Labeit, S., Hinssen, H., Ruegg, J.C. & Gautel, M. (1997) Actin–titin interaction in cardiac myofibrils: probing a physiological role. Biophys. J. 73, 905–919. 32. Granzier, H.L., Kellermayer, M., Helmes, M. & Trombita ´ s, K. (1997) Titin elasticity a nd m echanism of passive force d evelop- ment in rat cardiac myocytes probed by thin-filament extraction. Biophys. J. 73, 2043–2053. 33. Trombita ´ s, K. & Granzier, H.L. (1997) Actin removal from car- diac myocytes shows that near Z. line titin attaches to actin while under tension. Am.J.Physiol.CellPhysiol.273, 662–670. 34. Jin, J.P. (2000) Titin thin filament interaction and potential role in muscle function. Adv. Exp. Med. Biol. 481, 319–333. 35. Kulke, M., Fujika-Becker, S., Rostokova, E., Neagoe, C., Labeit, D ., Manstein, D.J., Gautel,M.&Linke,W.A.(2001) Interaction between PEVK-titin and actin filaments. Circ. Res. 89, 874–881. 36. Yamasaki, R., Berri, M., Wu, Y., Trombita ´ s, K., McNabb, M., Kellermayer,M.S.Z.,Witt,C.,Labeit,D.,Labeit,S.,Greaser,M. & Granzier, H.L. (200 1) Titin– actin i nteraction in m o use m yo- cardium: passive tension modulation and its regu lation by cal- cium/S100A1. Biophys. J. 81, 2297–2313. 37. Linke, W.A., Kulke, M ., Li, H., Fujita-Becker, S., Neagoe, C., Manstein, D.J., Gautel, M. & Fernandez, J.M. (2002) PEVK domain of titin: an entropic spring with actin-binding properties. J. Struct. Biol. 137, 194–205. 38. Offer, G., Baker, H. & Baker, L. (1972) Interaction of monomeric and polymeric actin with myosin subfragment 1. J. Mol. Biol. 66, 435–444. 4580 N. Niederla ¨ nder et al. (Eur. J. Biochem. 271) Ó FEBS 2004 39. 9 Eisenberg, E. & Kielley, W.W. (1974) Troponin-tropomyosin complex: column chromatographic separation and activity of the three, active troponin components with and without tropomyosin present. J. Biol. Chem. 249, 4742–4748. 40. Smillie, L.B. (1982) Preparation and identification of alpha- and beta-tropomyosins. Methods Enzymol. 85, 234–241. 41. Potter, J.D. (1982) Preparation of troponin and its subunits. Methods Enzymol. 85, 241–263. 42. Astier, C., Labbe ´ , J P., Roustan, C. & Benyamin, Y. (1993) Effects of different enzymatic treatments on the release of titin fragments from rabbit skeletal myofibrils. Biochem. J. 290,731– 734. 43. Astier,C.,Raynaud,F.,Lebart,M.C.,Roustan,C.&Benyamin, Y. (1998) Binding of a native titin fragment to actin is regulated by PiP2. FEBS Lett. 429, 95–98. 44. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. 45. Rosenfeld, J., Capdevielle, J., Guillemot, J C. & Ferrara, P. (1992) In-gel digestion of p roteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal. Biochem . 203, 173–179. 46. Mornet, D., Bertrand, R., Pantel, P., Audemard, E. & Kassab, R. (1981)Proteolyticapproachtostructureandfunctionofactin recognition site in myosin heads, Biochemistry 20, 2110–2120. 47. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriolphage T4. Nature 227, 680– 685. 48. Perkins, D.N., Pappin, D.J.C., Creasy, D.M. & C ottrell, J.S. (1999) Probability-based protein identification by searching se- quence databases using mass spectrometry data. Electrophoresis 20, 3551–3567. 49. Marston, S.B., Fraser, E.D., Bing, W. & Roper, G. (1996) A simple method for automatic tracking o f actin fi lamen ts in t he motility assay. J. Muscle Res. Cell Motil. 17, 497–506. 50. DeBeer, E .L., Sontrop, A.M.A. , Kellermayer, M.S., Galambos, C. & Pollack, G.H. ( 1997) Actin-filamentmotionintheinvitro motility assay has a periodic component. Cell Motil. Cytoskeleton 38, 341–350. 51. Hamelik, W., Zegers, J.G., Treijtel, B.W. & Blange ´ , T. (1999) Path reconstruction as a tool for actin fi lament speed determination in the in vitro motility assay. Anal. Biochem. 273, 12–19. 52. Fraser, I.D.C. & Marston, S.B. (1995) In vitro motility analysis of actin-tropomyosin regulation b y troponin and c alcium. J. Bio l. Chem. 270, 7836–7841. 53. Gordon,A.M.,Chen,Y.,Liang,B.,LaMadrid,M.,Luo,Z.& Chase, P.B. (1998) Skeletal m uscle regulatory p roteins enh ance F-actin in vitro motility. Adv. Exp. Med. Biol. 453, 187–196. 54. Gordon, A.M., Homsher, E. & Regnier, M. (2000) Regulation of contraction in striated muscle. Physiol. Rev. 80, 853–924. 55. Van D ijk, J. , Fernandez, C. & Chaussepied, P. (1998) Effe ct of ATP a nalo gues on the a ctin–m yosin interface. Biochemistry 37 , 8385–8394. 56. Bonafe, N . & Chaussepied, P. (1995) A single myosin head can be cross-linked to t h e N termini of two adjacent actin mo nomers. Biophys. J. 68, 35S–43S. 57. Van Dijk, J., Celine, F., Barman , T. & Chaussepied, P. (2000 ) Interaction of myosin with F-actin: time-dependent changes at the interface are no t slow. Biophys. J. 78, 3093–3102. 58. Clark, K.A., McElhinny, A.S., Beckerle, M.C. & Gregorio, C.C. (2002) Striated muscle cytoarchitecture: an intricate web of form and func tion. Annu.Rev.CellDev.Biol.18, 637–706. 59.Barron,D.J.,Etherington,P.J.,Winlove,C.P.&Pepper,J.R. (2001) Scapular insertion of the rabbit latissimus dorsi muscle: gross anatomy and fibre-type c omposition . Cell Tiss ues O rgans 168, 312–318. 60. 10 Freiburg,A.,Trombitas,K.,Hell,W.,Cazorla,O.,Fougerousse, F., C entner, T., Kolmerer, B., Witt, C., Beckmann, J.S., Gegorio, C.C., Granzier, H. & Labeit, S. (2000) Serie s of exo n-skipping events in the elastic spring region of titin as the structural basis for myofibrillar elastic diversity. Circ. Res. 86, 1114–1121. 61. Muhle-Goll, C., Habeck, M., Cazorla, O., Nilges, M., Labeit, S. & Granzier, H.L. (2001) Structural and functional studies of titin’s fn3 modules reveal conserved surface patte rns a nd b inding to myosin S1 – a possible role in the Frank-Starling mechanism of the heart. J. Mol Biol. 313, 431–447. 62. Miller, C.J., Wo ng, W.W., Bobkova, E., Rubenstein, P.A. & Reisler, E. (1996) Mutational analysis of the role of the N-termi- nus o f actin in actomyosin interactions. Comparison with o ther mutant actins and implications for the cross-bridge cycle. Bio- chemistry 35, 16557–16565. 63. Wong, W.W., Doyle, T.C. & Reisler, E. (1999) Nonspecific weak actomyosin interactions: relocation of charged residues in sub- domain 1 of a ctin does not alter actomyosin function. Biochem- istry 38, 1365–1370. 64. Van Dijk, J., Furch, M., Lafont, C., Manstein, D.J. & Chausse- pied, P. (1999) Functional characterization of the secondary a ctin binding site of myosin II. Biochemistry 38, 15078–15085. 65. Van Dijk, J., Furch, M., Derancourt, J., Batra, R., Knescht, M.L., Manstein, D.J. & Chaussepied, P. (1999) Differences in the ionic interaction of actin w ith the motor domains of nonmuscle an d muscle myosin II. Eur. J. Biochem. 260, 672–683. 66. Chaussepied, P . & Van Dijk, J . (2002) Role of charges in a cto- myosin interactions. Results Probl Cell Differ. 36, 51–64. 67. Wong, W.W., Gerson, J.H., Rubenstein, P.A. & Reisler, E. (2002) Thin filament regulation and ionic interactions between the N-terminal region in actin a nd trop onin. Biophys. J . 83, 2726– 2732. 68. Raynaud, F., A stier, C. & B enyamin, Y. (2004) Evidence for a direct but seque ntial binding of titin to tropomyosin and actin filaments. Biochim. Biophys. Acta 1700, 171–178. 69. Uyeda, T.Q., Ruppel, K.M. & Spudich, J.A. (1994) Enzymatic activities correlate with chimaeric substitutions at the actin-bind- ing face of myosin. Nature 368, 567–569. 70. Umemoto, S. & Sellers, J .R. (1990) C haracterization of i n vitro motility assays using smooth muscle and cytoplasmic myosins. J. Biol. Chem. 265, 14864–14869. 71. Anson, M. (1992) Temperature d ependence and ar rhenius acti- vation energy of F-actin ve locit y generated in v itro by sk eletal myosin. J. Mol. Biol. 224, 1029–1038. 72. Bobkov, A.A., Bobkova, E.A., Lin, S.H. & Reisler, E. (1996) The role of surface loops (residues 204–216 a nd 627–646) in the motor function of the myosin head. Proc. Natl Acad. Sci. USA 93, 2285– 2289. 73. Bobkova, E.A., Bobkov, A.A., Levidsky, D.I. & Reisler, E. (1999) Effects of SH1 and SH2 modifi cations on myo sin: similarities an d differences. Biophys. J. 76, 1001–1009. 74. Kruger,M.,Wright,J.&Wang,K.(1991)Nebulinasalength regulator of thin filaments of vertebrate skeletal muscles: correla- tion of thin fil ament l ength, n ebulin size, a nd ep itope profi le. J. Cell Biol. 115, 97–105. 75. Root, D.D. & Wang, K. (2001) High-affinity actin binding nebulin fragments influence the ac toS1 complex. Bioc hemistry 40, 1171– 1186. 76. Gutierrez-Cruz, G., van Heerden, A.H. & Wang, K. (2001) Modular motif, structural folds and affinity profiles of the PEVK segment of human fetal skeletal muscle titin. J. Biol. Chem. 276, 7442–7449. 77. Leguennec, J.Y., Cazorla, O ., Lacampagne, O. & Vassort, G. (2000) Is titin the length sensor in cardiac m uscle: Physiological and pathophysiologic al perspectives. Adv. Exp. Mol. Biol. 481, 387–348. Ó FEBS 2004 Effect of titin on actin–myosin activity (Eur. J. Biochem. 271) 4581 . and remained in the pellet independently of the other components and the ionic strength of the mixture. In the presence o f A TP, the amount of bou nd H MM. on the calcium sensitivity (pCa curve) of the movement and for an effect involving the actin–HMM interaction. Interestingly, the o rder of addition of the