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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.

221, 491497 (1996)

Elastic Properties of Single Titin Molecules Made Visible through Fluorescent F-Actin Binding
Mikls S. Z. Kellermayer and Henk L. Granzier1
Department of Veterinary Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, Washington 99164-6520 Received March 14, 1996 Titin (also known as connection) is a giant filamentous protein that spans the distance between the Z- and M-lines of the vertebrate muscle sarcomere [1-4]. Several indirect observations have implicated titin as playing a fundamental role in the generation of passive force of muscle [5,6], driven by titins elastic properties. A direct observation of the mechanical properties of titin, however, has not been demonstrated. Here we have used the recently shown strong actin-binding property of titin [7-9] to indirectly visualize and manipulate single molecules of titin. Titin molecules were immobilized on a microscope coverslip by attaching them to anti-titin antibody. The titin molecules were detected by attaching fluorescent actin filaments to them. The titin molecules were subsequently stretched by manipulating the free end of the attached actin filaments with a glass microneedle. Titin is shown here to possess a high degree of torsional and longitudinal flexibility. The molecule can be repetitively stretched at least fourfold, followed by recoil. Titins unloaded elastic recoil proceeded in two stages: an initial rapid process (15 ms time constant) was followed by a slower one (400 ms time constant). The force necessary to fully extend titinestimated by observing the breakage of the titin-bound actin filaments may reach above 001pN (longitudinal tensile strength of actin [10]). Attachment of fluorescent actin filaments to titin provides a useful tool to further probe titins molecular properties. 1996 Academic Press, Inc.
Passive force develops when a non-activated (passive) muscle is stretched, and this is the force that restores the muscle length following release. During muscle contraction, passive force limits sarcomere-length inhomogeneity along the muscle cell [11], and limits A-band asymmetry within the sarcomere [12]. Several observations have indicated that in the generation of passive force an important role is played by titin/connection. Titin is a -5.3million-dalton proteinthe largest protein known to datethat constitutes about 10% of the total muscle protein mass [1-4]. This giant molecule spans the half sarcomere, from the Z-line to the M-line. It is anchored to the Z-line and to the thick filaments of the A-band (via strong myosin-binding property). Upon stretch of the sarcomere, passive tension is generated by the extension of the I-band segment of titin [5,6], by virtue of the proteins elastic nature. The elastic property of titin has been indirectly shown by immunoelectron microscopic and mechanical experiments. Immunoelectron microscopic studies have shownby labelling several titin epitopes along the molecule, following varying degrees of muscle stretchthat the I-band domain of titin can indeed be extended [13,14]. Mechanical experiments have shownby measuring passive muscle force following selective removal of titinthat it is indeed titin that develops passive force in muscle [5,6]. A drawback of these studies is that they are indirect. Immunoelectron microscopic experiments provide a snapshop of titin epitope positions following varying degrees of muscle stretch; however, they fail to provide information about the concomitant mechanical events. Mechanical experiments, on the other hand, lack specificity; components other than titin might significantly affect the observations. Therefore,

1 To whom correspondence should be addressed at: Department of VCAPP, Wegner Hall, Room 205, Washington State University, Pullman, WA, 99164-6520. Fax: (509) 335-4650. E-mail: granzier@unicorn.it.wsu.edu. Abbreviations: BSA, bovine serum albumin; DTT, DL-dithiothreitol; EGTA, ethylene glycol-bis(-aminoethyl ether)N,N,N,N-tetraacetic acid; F-actin, filamentous actin.

491 0006-291X/96 $18.00

Copyright 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 221, No. 3, 1996

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
it would be necessary to establish the mechanical properties of titin by using purified preparations, ideally single titin molecules. In this study, we demonstrate the elastic nature of purified, single titin molecules by taking advantage of titins recently shown strong actin-binding property [7-9]. We have attached titin molecules to a microscope coverslip coated with an anti-titin antibody, followed by the addition of fluorescent actin filaments. By mechanically manipulating the titin-bound actin filament, we succeeded in visualizing the repetitive stretch and recoil of titin. Our results revealed that titin possesses a high degree of torsional and longitudinal flexibility. MATERIALS AND METHODS
Preparation of proteins. Actin was purified according to established methods [15]. F-actin was fluorescently labelled with molar excess of tetramethyl-rhodamine-isothiocyanate-phalloidin (Molecular Probes, Eugene, OR). Titin was prepared from rabbit back muscle (longissimus dorsi) essentially according to the method of Soteriou et al [16]. Purity of titin was determined by SDS-polyacrylamide gel electrophoresis using 2.35-12% gels [17]. Antibodies. The 9D10 monoclonal anti-titin antibody used in our experiments, developed by Dr. Marion L. Greaser, was obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, and the Department of Biological Sciences, University of Iowa, Iowa City, IA 52242, under contract NO1-HD-2-3144 from the NICHD. The antibody was used at 50-fold dilution in assay buffer (25 mM imidazole-HCl, pH 7.4, 200 mM KCl, 4 mM MgCl2, 1 mM EGTA, 1 mM DTT). Mounting and visualization of single titin molecules. A flow-through microchamber (identical to the one used for the in vitro motility assay, internal volume 01l [18]) was first filled with 9D10 antibody at 50-fold dilution. The antibody was allowed to bind to the nitrocellulose-coated surface of the microchamber for one minute. Unbound antibody was washed out by the infusion of 100 l blocking solution [5% (w/v) BSA, 1% (w/v) gelatin, 0.2% (v/v) Tween-20 in assay buffer]. Blocking was carried out for 10 minutes at room temperature. Titin was subsequently added to the microchamber at a concentration of 10 g/ml and allowed to bind to the 9D10 antibody for one minute. Unbound titin was washed out by the infusion of 100 l 0.5 mg/ml BSA in assay buffer. Fluorescent actin filaments were then added, at a concentration of 70 ng/ml (in low-ionic strength assay buffer, 25 mM imidazole-HCl, pH 7.4, 25 mM KCl, 4 mM MgCl2, 1 mM EGTA, 1 mM DTT, 0.5 mg/ml BSA) and allowed to bind to titin for one minute. Unbound actin filaments were washed out by the infusion of low-ionic strength assay buffer containing 100 mM -mercaptoethanol and an oxygen scavenger enzyme system to reduce photobleaching [10]. Fluorescent actin filaments were visualized by using a Nikon Diaphot 300 inverted epifluorescence microscope equipped with rhodamine interference filter set (Omega Optical, Brattleboro, VT) and a 64x, 1.4 NA oil-immersion objective (Nikon), and using a microchannel-plate intensified CCD camera (ICCD-100F, VideoScope International, Ltd., Sterling, VA). The detected images were recorded on a Hi8mm VCR (SONY EV-S7000), and subsequently digitized by an LG-3 frame grabber board (Scion Corporation, Frederick, MD) in an Apple Power Macintosh 6100/60 computer using image analysis software (Scion Image Version 1.57c, based on NIH Image, Wayne Rasband, NIH, Bethesda, MD). Mechanical manipulation of single titin molecules. Single molecules of titin were stretched by using a nitrocellulosecoated glass microneedle attached to the titin-bound fluorescent actin filament. Microneedles were pulled from borosilicate

FIG. 1. Binding of actin filaments to titin molecules immobilized on anti-titin antibody-coated surface. Bar graph comparing the number of actin filaments per field of view bound to either the titin-antibody complex, or in the absence of titin, or in the absence of antibody. 492
FIG. 2. (A) Time-lapse images of a fluorescent actin filament bound to a titin molecule attached to anti-titin antibody. The filament is pivoting around a single point of attachment. The time between the frames is 1s. Scale bar equals 5 m. (B) Superimposed picture of three images of the pivoting actin filament taken at different time points. The overlap of the images of the filament in different orientations marks the single point of attachment (arrowhead). Scale bar equals 5 m. microcapillaries (O.D. 1 mm, I.D. 0.5 mm, Sutter Instrument Co., Novato, CA) using a Model 730 needle/pipette puller (David Kopf Instruments, Tujunga, CA). The needles were coated with 1% nitrocellulose in iso-amyl acetate (Fullam, Latham, N.Y.). A microneedle was mounted in a MX630L hydraulic micromanipulator (Newport Corp., Irvine, CA), and inserted in the microchamber. Actin filaments and the microneedle were simultaneously visualized by using epifluorescence and brightfield illuminations, respectively, with a 16ND filter placed in the brightfield optical path to reduce exposure of the intensified CCD camera.
RESULTS AND DISCUSSION Visualization of single titin molecules. Single titin molecules were indirectly visualized by using the actin-binding property of titin. The nitrocellulose-covered surface of a sample chamber was coated with anti-titin antibody. The surface was then blocked with a mixture of 5% (w/v) BSA, 1%
FIG. 3. Angular distribution of the pivoting actin filament. The angle (in degrees) was measured between an arbitrary reference line and the long axis of the actin filament. 493
FIG. 4. Sequence of images showing the stretching of a single titin molecule by micromanipulation of the actin filament. A fluorescent actin filament, bound with its tip to a single titin molecule on the anti-titin antibody, was pulled by help of a glass microneedle (the needle is outside of the image area). First, the actin filament was pulled to the right; upon returning the microneedle, the tip of the actin filament returned to the starting position (indicated by arrowhead). Subsequently, the filament was pulled to the left; upon returning the microneedle, the tip of the actin filament repeatedly returned to the starting position, driven by titins elastic recoil. Scale bar equals 10 m.
(w/v) gelatin, and 0.2% (v/v) Tween-20 to prevent nonspecific binding of titin. Control experiments indicated that such an extensive blocking was necessary to prevent the nonspecific binding of titin to the nitrocellulose surface. Following the blocking procedure, titin was added, and allowed to bind to the antibody. Fluorescent actin filaments were then added, which bound to titin. When either the antibody or titin was omitted from the control experiments, no actin filaments were seen, indicating that actin recognized the antibody-bound titin specifically (Figure 1). Most of the actin filaments were focally attached, as evidenced by their pivoting around a single point (Figure 2). Occasionally, an actin filament was found to be tethered at more than one point of attachment. Such an actin filament did not exhibit pivoting motion. The pivoting movement of the actin filaments indicated that they were attached to single titin molecules. Although the pivoting motion of the actin filaments is a good indication for the presence of single titin molecules at the tether, it is conceivable that the tether is formed by not single molecules but by dimers or even trimers. At the high ionic strengths used, however, titin has been shown to be mostly monomeric in solution [19]. Thus, it is likely that the pivoting actin filaments are indeed tethered to the antibody by single titin molecules. The angular distribution of the pivoting actin filaments had a range of 620 degrees (Figure 3). The profile of the angular distribution was found to be multimodal. The maxima of the distribution are separated by 200 degrees. The multimodal nature of the distribution profile indicates that the pivoting actin filaments are found in preferred angular positions separated by 200 degrees. Provided that neither the actin-titin bond nor the titin-antibody bond allow for slippage (that is, they do not let go under the thermally driven torsional load), the presence of preferred angular positions of the actin filament represents preferred torsional configurations of the titin molecule. Mechanical manipulation of single titin molecules. Using the actin filament bound to titin, we carried out experiments to mechanically manipulate a single titin molecule. The freely moving end of the actin filament bound to a titin molecule was attached to a nitrocellulose-coated glass microneedle. The microneedle was then translated by a hydraulic micromanipulator. By pulling the actin filament with the microneedle, the titin molecule was stretched. Upon moving the microneedle back, the end of the actin filament bound to titin returned to its starting position (Figure 4). The procedure could be repeated many times and in different directions; the tip of the actin filament attached to titin was always pulled back (by the elastic titin molecule) to its starting position. Figure 5 shows the deviation of the tip of the actin filament from the starting position. The maximal deviation in this experiment was 4.2 m. Considering the position of 9D10 epitope along the titin molecule [14,20], and that the actin filament could in principle bind anywhere along titin,

FIG. 5. Absolute distance (m) of the actin-filament tip from the starting point as a function of time (s). The fluorescent actin filament was repeatedly pulled away from and returned to the starting point. The maximal deviation of the actinfilament tip from the starting point was 4.2 m. 495
FIG. 6. Velocity of a short fragment of actin filament driven under unloaded conditions by titins elastic recoil. Following the stretching of a titin molecule, the actin filament broke and was observed to snap back, driven by titins recoil. Velocity is plotted as a function of time, and fitted with double exponential function (f abx + cdx).
the theoretically maximal unextended length of the titin molecule was 1m (distance from the 9D10 epitope to the M-line). Thus, at the extended length of 4.2 m, the titin molecule was stretched at least fourfold. The demonstrated fourfold extension of a single titin molecule supports previous predictions of titins extensibility [21]. At the minimally fourfold extension of titin, the protein domains (immunoglobulin, fibronectin type III, and PEVK [4]) from which titin is constructed are likely to be unfolded, as it has previously been proposed [21,22]. Figure 5 indicates that the mechanically induced unfolding-folding of titin is reversible. Upon stretching the actin-titin-antibody complex, the actin filament occasionally broke. In these instances, the broken piece of actin filament was observed to snap back toward the attachment point on the coverslip, driven by the elastic recoil of the titin molecule. The initial, resolvable velocity of the recoil was 150 m/s (Figure 6). This initial, very rapid recoil rate (15 ms time constant) was followed by a slower process (400 ms time constant, Figure 6). Such a two-stage process has been previously proposed [22], and is likely to be driven by the initial, rapid refolding of the domains into a molten globule, followed by a consolidation of the native structure. The actin filament in our system may be considered as a crude strain gauge. The tensile strength of a single actin filament has been measured to be 001pN [10]. The presence or absence of actin-filament breakage may reveal the tension in the antibody-titin-actin complex. Since pulling on the actin filament occasionally led to the breakage of the filament, the tension in the antibody-titin-actin complex exceeded 001pN, indicating that the passive force generated by stretching titin may exceed 001 pN per molecule. The forces involved in the high degree of titin unfolding are thus likely to be higher than the 5 pN proposed by Erickson [22], ranging up to at least 001pN (the tensile strength of actin). In summary, single molecules of titin were indirectly visualized and mechanically manipulated. Titin exhibited a considerable degree of torsional and longitudinal flexibility. The elastic nature of titin was directly and visually demonstrated at the molecular level. Such a single-molecule assay may lead to a better understanding of titins function and the molecular mechanisms of passive force generation in muscle. ACKNOWLEDGMENTS

We thank B. Stockman for assistance. This work was supported by grants from the American Heart Association, Washington Affiliate, the Whitaker Foundation, and by NIAMS (R29AR42652).

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