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+

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-

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

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

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

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,

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

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

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

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

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

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.

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

(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,

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

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

-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

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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