Regulation of the actin–myosin interaction by titinNicolas Niederla¨nder1, Fabrice Raynaud2, Catherine Astier2and Patrick Chaussepied1 1 CRBM-CNRS, Montpellier, France; 2 EPHE-UMR5539-CN
Trang 1Regulation of the actin–myosin interaction by titin
Nicolas Niederla¨nder1, Fabrice Raynaud2, Catherine Astier2and Patrick Chaussepied1
1 CRBM-CNRS, Montpellier, France; 2 EPHE-UMR5539-CNRS, Montpellier, France
Titin is known to interact with actin thin filaments within
the I-band region of striated muscle sarcomeres In this
study, we have used a titin fragment of 800 kDa (T800)
purified from striated skeletal muscle to measure the effect
of this interaction on the functional properties of the actin–
myosin complex MALDI-TOF MS revealed that T800
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 with an inhibition 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 Ca2+ -controlled on/off switch, nor did it alter the overall binding energetics of HMM to actin, as revealed by cosedimentation experiments These data are consistent with a competitive effect of PEVK domain-containing T800 on the electrostatic contacts at the actin–HMM interface They also suggest that titin may participate in the regulation of the active tension generated by the actin–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 in smooth muscle and nonmuscle cells (recently
reviewed in [1,2]) The importance of intact titin for normal
muscle function has been demonstrated 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 in [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 structural properties of the protein: titin spans half a
sarcomere, from the Z disks to the M line (connecting the
Z disks to myosin thick filaments), it contains subdomains
that confer unusual elastic properties, and it interacts with
several protein partners such as myosin, actin, M protein,
C protein, MURF-1, calpain 3, myomesin, a-actinin,
nebulin, telethonin and obscurin
The elastic domains are made of tandemly 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 measurements made on muscle fibres or at the single molecule level suggest that the tandem Ig- and PEVK-domains are two elements of differential stiffness that function as a two-spring system [13–24] This elastic system is now believed to be a major contributor to the passive tension developed in striated muscle
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 with each other [25,26] This association 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 muscle passive stiffness [34–36] Indeed, recent data suggest that the PEVK domain from cardiac muscle titin interacts with actin much more efficiently than does that from skeletal muscle titin [36,37], supporting the idea that this interaction may
be correlated with passive stiffness in each muscle type It
is important to note, however, that both the size of 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 PEVK titin sub-fragments In the case of the single in vitro motility assay that has been achieved using tissue-extracted titin, the experiments were designed 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 entire 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 Montpellier
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; HMM, 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)
Trang 2have 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 the titin fragment reduces the
number of contacts between myosin and the N-terminal
part of actin, producing significant effects 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 highest analytical grade
Preparation of proteins
All proteins were extracted from rabbit skeletal muscle
Myosin and myosin fragments were prepared as described
by Offer et al [38] Heavy meromyosin (HMM)
was obtained after a-chymotrypsin digestion of myosin
(enzyme/substrate mass ratio of 1 : 400) for 15 min at 25C
in 10 mMNaH2PO4, 600 mMNaCl, 1 mMMgCl2, 1 mM
dithioerythritol (DTE) pH 7.0 After the reaction was
stopped by phenylmethanesulfonyl fluoride
(phenyl-methanesulfonyl fluoride/substrate mass ratio of 1 : 200),
the solution was dialysed overnight against 20 mMMops,
0.2 mMDTE, pH 7.0, and centrifuged 20 min at 100 000 g
HMM was purified by ion exchange chromatography on
SP-sephacryl (Pharmacia-Biotech) using a 0–200 mMNaCl
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 lM) at 4C in the presence of 120 mM
NaCl, 2.5 mMMgCl2 Polymerized actin was concentrated
by centrifugation at 190 000 g for 20 min and kept at 4C
(120 lM final concentration) in 100 mM NaCl, 2.5 mM
MgCl2, 50 mM Mops, pH 7.5 For the in vitro motility
assay, F-actin was not concentrated but rather used directly
at 40 lM for rhodamine–phalloidin labelling (see below)
Tropomyosin and troponin complex (troponin I, T and C)
were prepared from acetone-dried muscle powder according
to Smillie [40] and Potter [41], respectively They were stored
in the lyophilized form and used as a solution containing
equimolar amounts 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, 25C) and centrifugation at 5000 g for 5 min
[42,43] T800 was subsequently purified through gel
filtra-tion S300 HR (Pharmacia-Biotech) followed by Poros
HQ/H column (Boehringer) in 2 mM Tris, 1 mM DTE,
1 mM EDTA, pH 7.9 Pure T800 was eluted at 250 mM
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 A1%280 of 5.7 cm)1, 6.5 cm)1, 11.0 cm)1, 3.3 cm)1, 4.5 cm)1and 10.0 cm)1for 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 the ZipTip device (Millipore Inc) and analysed by MALDI-TOF MS (BiflexIII, Bruker) Database queries were performed using the Mascot search engine (Matrix Science at http://www.matrixscience.com/)
In vitro motility assay F-actin (0.6 lM) was first stabilized and labelled by adding
a twofold excess of tetramethyl-rhodamine phalloidin in motility buffer (50 mMKCl, 10 mMMgCl2, 40 mMDTE,
60 mM Hepes pH 7.8, 90 mM ionic strength) Labelled F-actin was then diluted (2 nM final concentration) in motility buffer containing 3.3 mgÆmL)1 glucose, 0.37 mgÆmL)1 catalase, 0.11 mgÆmL)1 glucose oxidase, 0.5% (w/v) methylcellulose, and 0.1 mM CaCl2 or 1 mM EGTA (only when the Tm–Tn complex was present) The solution was supplemented by Tm–Tn and T800 (both
at 20 nM, conditions for a saturating effect), and ATP (2 mM) was added to flow cells containing HMM-coated glass coverslips just prior to image recording Coverslips were pretreated overnight at room temperature with 1M HCl, rinsed with distilled water, 95% ethanol and air-dried They were then treated with BSA/casein (10 mgÆmL)1) for 10 min at 20C, air-dried, mounted
on the flow cell, and coated with HMM (50 lgÆmL)1 solution containing 600 mM KCl, 10 mM Hepes, pH 7.0) for 10 min on ice prior to the addition of the actin solution Dead HMM molecules were removed before the coating step by two consecutive ultracentrifugation steps at
190 000 g for 20 min in the presence of a threefold molar excess of F-actin-phalloidin and 2.5 mM ATP in 10 mM Hepes, 600 mM KCl pH 7.0 After each ultracentrifuga-tion step, the HMM concentraultracentrifuga-tion was evaluated by the Bradford method The dead HMM eliminated in this way corresponded to 5–10% of the total HMM in the preparation
Images of the microfilaments were obtained with a DMR B microscope (Leica, Bensheim,
PL APO 100· objective (NA 1.40) with a 1.6 · tube factor and immersion oil Immersol 518 F (Zeiss, Go¨ttingen,
3Germany) Preparations were illuminated with a 100 W HBO 103 W/2 light bulb (OSRAM, Regensburg,
through a N 2.1 filter cube (Leica) for the visualization
of rhodamine fluorescence The microscope was equipped with a homemade heating stage The heat regulation was
Trang 3stabilized to prevent undesired minute up and down
movements of the stage, which can upset the stability of
image focusing during time-lapse recording The stability
was further enhanced by the presence of a plexiglass box
that protected 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 allowed easy access to the stage Images were
captured with an ORCA 100 (B/W) 10 bits cooled CCD
camera (C mount 1x), C 4742-95 controller and HIPIC
controller program (Hamamatsu, Shizuoka,
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 detector and camera
gain set at its maximum value Sequences were saved as a
suite of individual TIFF format images (up to 250 in one
sequence) Measurements were carried out with
META-MORPH 6.1 software (Universal Imaging Corporation,
Downington, PA, USA)
the leading end of the actin filament Statistical analyses
were performed using PRISM 2.2 software (GraphPad
Software, Inc., San Diego, CA, USA)
was used to compare sets of data and a P-value < 0.005 was
used to determine statistical significance
Steady-state ATPase and actin binding assays
Various mixtures containing F-actin (3 lM) alone or with
T800 (0.15 lM), Tm–Tn (1.0 lM) and HMM (0.25 lMin
the ATPase activity and 1.5 lMin the binding assay) were
incubated for 10 min in 50 mM Hepes, 5 mM MgCl2,
50 mM KCl, 2 mM DTE (80 mM ionic strength), with or
without 0.1 mMCaCl2, 1 mMEGTA or 2 mMATP, pH 7.8
(the binding assay was also conducted in the presence of
100 mM NaCl, that is in a final 180 mM ionic strength
without ATP)
The Mg.ATPase activities were measured at 25C The
reaction was started by the addition of 2 mM ATP and
stopped after 10 min by 5% trichloroacetic acid The
amount of Piliberated was evaluated colorimetrically [46]
The actin binding assay was carried out by
ultracentri-fugation of the reaction mixtures at 190 000 g for 20 min
An aliquot of each supernatant was removed after
centri-fugation and mixed with Laemmli solution [50 mM
Hepes, 2% (w/v) NaDodSO4, 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 for 3 min
Two-step cross-linking experiments
During the activating step, 80 lMF-actin was treated for
10 min at 20C with 50 mM NHS and 25 mM EDC in
buffer C (50 mM NaCl, 5 mM MgCl2, 50 mM Mops
pH 7.0) The activating reaction was stopped with
100 mMb-mercaptoethanol During the condensation step,
an aliquot of activated F-actin (3 lMfinal concentration)
was mixed with 0.15 lM T800 with or without 1.0 lM
Tm–Tn and 1.5 lMHMM in buffer C in the presence of
0.1 mMCaCl 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 was performed usingMETAMORPH6.1 software
Results
Localization of T800 within the I-band region
of skeletal titin Some of us have previously demonstrated that mild treatment of myofibrils with S aureus V8 protease releases
a soluble titin fragment of 800 kDa (T800) that can be purified to homogeneity [42] In order to localize T800 within titin, we performed MALDI-TOF MS following in-gel digestion of T800 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 Mascot 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 score 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 fragment (correspond-ing to residues 4262–12 392) and full-length human skeletal titin (residues 1–26 926), respectively Of 79 peptides analysed, 22 matched with the two proteins, with the difference between calculated and experimental molecular weights being lower than 0.1 Da These 22 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 acid 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 PEVK domain and falls entirely within the I-band region of skeletal titin
T800 acceleratesin vitro motility of the reconstituted thin filament
Because the titin PEVK domain is known to interact with actin, we studied the effects of T800 on the movement 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 conditions
or of the presence of T800 and the regulatory proteins Tm–Tn The filament motion displayed acceleration/decel-eration phases throughout the entire time course of the movement This periodicity, which has been reported earlier
Trang 4[49,50], is probably due to the heterogeneity of the HMM
molecules coated on the glass surface, although 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 of
T800, actin with Tm–Tn, and actin with Tm–Tn in the
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 with
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)1and from 3.9 to
4.3 lmÆs)1 in the absence and the presence of Tm–Tn,
respectively (Table 1) This increase in the average
velocity was accompanied by an increase in the maximum
velocity (Fig 2B) 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 the addition
of T800 While this number was slightly increased in the absence of Tm–Tn (21.6% vs 16.0%), it was dramatically reduced in the presence of reconstituted thin filaments, containing Tm–Tn (5.6% vs 21.0%; Table 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), and was slightly decreased in its presence (1.5 vs 1.1 lm) Note also that the presence of Tm–Tn on its own decreased the mean filament length 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 Ca2+, inde-pendent of the addition of T800 This result, together with the fact that T800 increased the average and maximum velocity values of moving filaments, both in the absence and in the presence of Tm–Tn–Ca2+, 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 order 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 Ca2+ 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 run in SDS 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 location of matching peptides around the
PEVK domain and the two predicted extreme
boundaries (residues 1870–9070 and 4670–
11500) of T800 are also shown.
Trang 5interaction between HMM and actin It is likely that in adult native striated muscle, titin interacts with a preformed thin filament containing bound Tm–Tn, similar to the interactions described in 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 Mg2+-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 in the presence of T800 (varying from 0.17
to 0.19 s)1; Table 2) This result 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 Mg2+ -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 absence and presence of Tm–Tn (with Ca2+), 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 Ca2+-linked regulation of the actin–HMM ATPase is entirely consistent with the lack of effect on the
Ca2+-controlled on/off switch of thin filament 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 strengths (80 mM and
180 mM) 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 the 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 conditions: actin alone (Actin); actin + T800 (Actin +
T800); actin + Tm–Tn + CaCl 2 (Actin + Tm-Tn); actin + Tm–
Tn + T800 + CaCl 2 (Actin + Tm–Tn + T800) STOPS
corres-pond to the time filaments were stationary, expressed 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 the 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; Mann–Whitney test showed a P-value < 0.0001 com-paring either actin alone and actin + T800 or actin + Tm–Tn and actin + Tm–Tn + T800 STOPS correspond to the time that fila-ments were stationary, expressed as a percentage of total time of analysis for all moving filaments.
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.
Trang 6tion 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 of ATP, the amount of bound HMM was
very similar (average value of 52.5 ± 1.1%) in all
experi-ments except in the presence of Ca2+ and high ionic
strength (average value of 37.1 ± 4.0% for panels
Fs + ATP and Gs + ATP; see figure legend for the
detailed quantitative data) On the other hand, the
percent-age of T800 bound to actin was not affected by ATP and/or
by CaCl2but it was decreased by elevating ionic strength
from 80 to 180 mM with average values of 62.3 ± 2.2%
and 45.1 ± 3.5%, respectively (compare detailed values in
figure legend, panels B and D vs panels F and H)
Concerning the actin–HMM interface, we explored the
electrostatic contacts between the N-terminal part of actin
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 reducing 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 resi-dues within the actin–HMM interface [55,57] These cross-linked products were observed in the absence of the regulatory proteins, Tm–Tn (Fig 4, lane b), or 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 activities are the average values of three experiments performed as described in 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 of cosedimentation experiments performed as described in Materials 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 preincubated in the presence of CaCl 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), 39.9 (Fs + ATP), 34.2 (Gs + ATP), 52.6 (Hs + ATP) and 51.7 (Is + ATP) The per-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).
Trang 7conditions, when T800 was added to reconstituted thin
filaments (Fig 4, lane d) An additional faint band was
observed at about 70 kDa when Tm–Tn was present This
latter product corresponded presumably to a cross-linking
reaction between actin 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 segment
Discussion
T800 represents the first native titin fragment containing the
entire PEVK domain to be directly extracted from skeletal
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
experi-mental basis to investigate a possible role for titin in the
regulation of energetics and force generation in the
actomyosin system
Identification of T800 was 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 sarcomere Its boundaries 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 actually explain why,
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 that we used in this study rabbit back muscles which are heterogeneous in their fibre-type content [59] Nevertheless, both the homogeneity of the T800 preparation and the results of the mass peptide analysis suggest that the proteolysis and purification protocols selected preferentially the longest skeletal muscle titin isoform
Our data clearly indicate that T800 binds to actin thin filaments, in good agreement with numerous works 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, without 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 stretch of residues belongs to T800 as the corresponding residues 1870–2205 of skeletal titin are located close to the hypothetic extreme N-terminal end of T800 (Fig 1) The interaction between actin and T800 is characterized by an apparent saturating T800/actin molar ratio of 1 : 20 as determined by centrifugation experiments with increasing amounts of T800 This ratio suggests that 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 of charged/uncharged resi-dues along the PEVK domain [13,60] and with the ionic strength dependence of T800 binding to actin
T800 increases the velocity of moving actin filaments This acceleration is observed both in the absence and in the presence of Tm–Tn (with Ca2+) However, the molecular explanations seem different in the two cases as the number
of stationary filaments decreases in the absence of Tm–Tn and increases in its presence, and also because the reduction
in actin–HMM cross-linking occurs only in the presence of Tm–Tn Note that in both cases, the acceleration observed
in the motility assay excludes a direct 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 was made to further explain the changes observed
in the absence of Tm–Tn, as this situation is highly unlikely
to occur under physiological conditions In the presence of 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 actin This change of the actin–HMM ionic interface would be in contrast to the lack
of effect on the HMM binding to actin observed in cosedimentation experiments Such a discrepancy has been previously related to the 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–Ca2+ -linked regulation observed in the presence of T800, as a recent report demonstrated that removing the negative charges in this region of actin does not affect the pCa curves
of the motion of thin filaments [67]
Fig 4 EDC-induced cross-linking at the actin–HMM interface Gel
electrophoresis analysis of the 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).
Trang 8This reduction in actin–HMM contacts could be due to a
direct or indirect competition between T800 and HMM for
binding to the negatively charged residues of the N-terminal
part of actin Our data suggest that T800 acts indirectly as
T800 alone or added before Tm–Tn on actin filaments
cannot displace HMM This indirect effect could, for
example, be mediated through interactions with Tm–Tn, as
proposed recently by solid phase experiments [68], or
following structural rearrangements within actin as
sugges-ted by the decrease in filament length that is observed in the
presence of T800 Interestingly, both T800 and Tm–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 and molecular effects on actin
Can the diminution of HMM contacts with the
N-terminal part of actin account for the decrease in
actin–HMM ATPase activity and for the increase in thin
filament sliding velocity while these two activities are
supposed to be correlated? Reduction of these contacts
was found 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 of 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 studies were performed with bacterially
expressed titin fragments, and not with muscle-extracted
native fragments The muscle extracted titin fragment,
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 the Tm–Tn–Ca2+-linked regulation of the
reconstituted thin filaments, neither in 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 T800 But this is not the
only parameter that one should consider, as we found for
example that T800 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 titin fragments also used tropomyosin [31] or
tropomyosin–troponin [30] However, their results were
controversial as the first one found an inhibition while the
second one reported an increase of actin binding in the
presence of calcium In this work, calcium 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 vitrowith respect to the in vivo functional properties of the actin–myosin complex? Reducing the ATPase activity of the actin–myosin complex could have important effects on the energetic balance during muscle activity, and speeding
up the movement of actin filaments could have 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 these 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 consequences for the actin-myosin complex, may occur The facts that T800 interacts with actin in the presence of Tm–Tn, HMM, ATP and up to 0.1 mMCaCl2 and that at 180 mMionic strength more than 45% of T800 remains bound to actin, support the in vivo extrapolation of 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 rate of formation of the actin– 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 the precise functional properties of the 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 the effects 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 development in cardiac muscle [36,37,77] Studying 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 the PEVK domain to control myosin binding to actin, and to 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 analysis 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 setting up the in vitro motility assay, and Juliette
Trang 9VanDijk for her critical reading of the manuscript This work was
supported by the French Centre National de la Recherche
Scientifi-que.
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