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:
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 />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, Pierre Travo (CRBM
imaging facilities, 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.
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