ARTICLE |
Correspondence to: Richard L. Lieber, Dept. of Orthopaedics (9151), VA Medical Center and UC San Diego, 3350 La Jolla Village Drive, San Diego, CA 92161. E-mail: rlieber@ucsd.edu
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Summary |
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We describe a novel system that permits simultaneous confocal imaging of protein interactions and measurement of cell mechanical properties during passive loading. A mechanical apparatus was designed to replace the stage of a confocal microscope, enabling cell manipulation, force transduction, and imaging. In addition, image processing algorithms were developed to quantify the degree of connectivity between subcellular structures. Using this system, we examined the interactions among three cellular structures thought to be linked by the muscle's intermediate filament system: Z-disks, nuclei, and the costamere protein complexes located at the muscle cell surface. Fast Fourier transforms (FFTs) and autocorrelations (ACs) were implemented to quantify image periodicity and relative phase shifts among structures. We demonstrated in sample wild-type muscle cells that there was significant connectivity among Z-disks in the same fiber at various sarcomere lengths, as well as between Z-disks and the costamere complexes. This approach can be applied to any cell system in which structural periodicity and mechanical connectivity are of interest. (J Histochem Cytochem 51:1929, 2003)
Key Words:
costamere, talin, -actinin, sarcomere length, image analysis
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Introduction |
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UNDERSTANDING functional relationships among cellular structures is a fundamental problem facing biologists today. Elucidation of the mechanisms by which a protein or protein complex elicits a physiological response depends in part on the ability to accurately define a protein's physical connections to surrounding structures. Previous studies inferred functional roles of proteins on the basis of light or electron microscopy, snapshots of protein localization and/or co-localization (e.g.,
In striated muscle, passive mechanical properties have been measured in whole muscle (
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Materials and Methods |
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Experimental Model and Design
Experiments were performed on cells from the fifth toe muscle of the extensor digitorum longus muscle in wild-type (n=4, 1014 weeks; Taconic Farms, Germantown, NY) and desmin-null (n=1, aged 14 weeks; generously contributed by Yassemi Capetanaki, Baylor University) 129/Sv adult mice. The EDL fifth toe was chosen on the basis of its fiber length homogeneity and because it contains a single fiber type (
All procedures were performed in accordance with the NIH Guide for the Use and Care of Laboratory Animals and were approved by the University of California and Department of Veteran's Affairs Committees on the Use of Animal Subjects in Research. Each mouse was anesthetized with a cocktail composed of (mg/kg): ketamine 10, rompum 5, and acepromazine 1. Hindlimbs were transected proximal to the knee and immediately placed for further dissection into a mammalian Ringer's solution composed of (mM): NaCl 137, KCl 5, NaH2PO4 1, NaHCO3 24, CaCl2 2, MgSO4 1, and glucose 11 with 10 mg/liter curare. The mouse was then sacrificed by direct intracardiac injection of sodium pentobarbital.
Dissection of Single Fiber Segments and Attachment to Mechanical Apparatus
All dissection was performed under a dissecting microscope (Wild M-8; Heerbrugg, Switzerland). The EDL was quickly removed from each hindlimb and immersed for 60 min in a relaxing solution at pCa 8.0 and pH 7.1 consisting of (mM): imidazole 59.4, KCH4O3S 86.0, Ca(KCH4O3S)2 0.13, Mg(KCH4O3S)2 10.8, K3EGTA 5.5, KH2PO4 1.0, Na2ATP 5.1, and 50.0 µM of the protease inhibitor leupeptin (
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The mechanical apparatus was designed to replace the stage of an inverted laser scanning confocal microscope (Axiovert 100M; Zeiss, Thornwood, NY; Fig 1). One end of the fiber segment was tied to a titanium wire (Aldrich Chemical Corporation 26601-9; Milwaukee, WI) which was rigidly attached to a force transducer (Aurora Scientific 405A; Aurora, Ontario, Canada), and the other end to a titanium wire rigidly attached to a rotational bearing (Newport MT-RS; Irvine, CA) using 10-0 monofilament nylon suture. The positions of both the force transducer and rotational bearing were controlled in three dimensions using micrometer-controlled XYZ-translational stages (Newport, 460A), permitting alignment of the fiber segment with the microscope's optical axes. The rotational bearing was used to remove any fiber segment torsion that occurred during attachment. Fiber alignment was verified based on the maintenance of microscope focus at the same depth across the entire length of the fiber.
Labeling of -actinin, Talin, and Nuclei
Primary antibodies preconjugated to fluorescent secondary antibodies were used to reduce background signal from unconjugated secondary antibody and to minimize incubation time. Primary antibodies specific for the Z-disk protein -actinin (Sigma A7811; St Louis, MO) and the costamere protein talin (Santa Cruz Technology sc-7534; Santa Cruz, CA) were preconjugated to Alexa-Fluor 488 (Molecular Probes A-10235, Eugene, OR; emission
=488 nm) or Alexa-Fluor 594 (Molecular Probes A-10239; emission
=594 nm) secondary antibodies, respectively. Modifications were made per the product literature to compensate for various levels of antibody purity and protein concentration. The fiber segment was rinsed three times using relaxing solution with 1% bovine serum albumin (BSA), followed by incubation at 48C with gentle agitation in 15 µl/ml normal horse serum (Vector Laboratories S-2000; Burlingame, CA) in 0.1% BSA (15 min), 100 µl/ml normal mouse serum (Sigma M5905) in 0.1% BSA (15 min), and 0.1% BSA (three washes of 5 min each). After this series of blocking steps and washes, the fiber segment was incubated in either a 1:2000 dilution of DNA-binding (nuclear) dye (Molecular Probes, TOTO-3;
=660 nm) followed by 0.1% BSA (three washes of 15 min each) or in an antibodydye solution at 4C for various periods of time, depending on the desired labeling combination (Table 1). Preconjugated antibodies for talin and
-actinin were diluted in 0.1% BSA to concentrations of 50 µl/ml and 2 µl/ml of solution, respectively. After overnight antibody incubation, the fiber was washed three times for 15 min at room temperature to remove any unbound antibody.
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Control experiments were performed to determine the effect (if any) of antibody labeling on fiber passive mechanical properties. Muscle fiber segments were elongated from resting sarcomere length to 75% strain in 250 µm increments and permitted to stressrelax for 2 min between elongations. LaGrangian stress was calculated at each strain and elastic modulus computed for each fiber across the entire deformation range. No significant difference between labeled (82.64 ± 6.4 kPa) vs unlabeled fibers (80.02 ± 8.5 kPa) was observed (p>0.8).
Confocal Microscopy
Samples were excited using an ArKr laser with beam splitters and filter sets appropriate for excitation of the AF-488 and AF-594 dyes, and an HeNe laser for excitation of TOTO-3 dye. These dyes allowed simultaneous imaging of AF-488 and either AF-594 or TOTO-3 with no bleed-through across channels. A x32/0.4 Achroplan objective (#440851; Zeiss) was used with a correction cap (#44491; Zeiss), to compensate for the small gap between coverslip and fiber segment (necessary to prevent fiber contact with the coverslip).
Lateral resolution (xy plane, perpendicular to the z-axis of the laser), based on the radius of the Airy disk (rAIRY), was determined according to Rayleigh's criterion (
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(1) |
where =wavelength of emitted fluorescence and NA is the numerical aperture of the objective lens. A frame of 512 x 512 pixels was captured at an electronic zoom (decrease of imaging field size) of 4.0, for a pixel-to-pixel spacing of 140 nm. Each row of pixels was scanned twice and averaged for improved accuracy, and the pinhole size was set between 0.40.5 Airy units, resulting in increased axial (z-axis) resolution (2.53 µm). These imaging conditions do not allow the measurement of absolute structure dimensions beyond the lateral resolution (
, from Equation 1). However, they do allow very accurate measurement of the relative position of structures with respect to one another.
Susceptibility of the TOTO-3 label to photobleaching was a potentially confounding variable in the determination of nuclear dimensions. Positive controls confirmed that nuclear aspect ratio (defined as length:width) was unchanged despite slight variations in absolute dimensions. In the first control, a nucleus was exposed to continuous excitation for 60 sec at 75% of maximal intensity. In the second control, a nucleus was exposed to 20 alternating cycles of excitation at 15% and 70% of maximal power for 15 and 2 sec, respectively (simulating the experimental protocol). Aspect ratio fluctuated from an initial value of 3.11 to no more than 3.25 (4.5% increase) during the first control experiment and from an initial value of 3.72 to a minimum of 3.64 (decrease of 1.8%), and a maximum of 3.87 (increase of 4.1%) during the second control. For both controls, there was no significant relationship between aspect ratio and exposure time (m=0.017 and 0.019; r 2=0.73 and 0.63, respectively).
Passive Mechanical Protocols and Analysis
To minimize effects of the temperature sensitivity of the force transducer head, the confocal mechanical apparatus was equilibrated to room temperature before initiation of mechanical experiments. To reduce lingering effects of temperature-related drift, drift voltage (estimated based on a linear function of drift voltage vs time, as observed in control experiments) was subtracted from recorded voltage. This reduced the effect of drift on recorded forces to less than 3 mV (0.3 mg) over 1 hr. The temperature of the solution was verified to remain at 2224C throughout the course of an experiment by use of a thermocouple (Model 73ATA; Yellow Springs Instruments, Yellow Springs, OH).
The fiber segment was brought to its resting length, determined as the knot-to-knot fiber segment length at which passive tension was just discernable. To standardize starting conditions, each segment was preconditioned by three loading cycles of 25% stretch, resulting in a change in resting length of less than 1% from the pre-preconditioned resting length. The segment was then loaded in increments of 10% of the segment length at a rate of 0.10.3 segment lengths/sec. At each length, after 2 min, a stack of confocal images with 2.12.3 µm slice thickness was captured and steady-state force (within 5% of final force value) recorded. During the 2 min of relaxation, force was recorded via a laptop computer running the LABVIEW software (National Instruments; Austin, TX), and the confocal stage was manipulated to keep the selected region of the fiber in the imaging field. Stress was calculated by dividing force by either initial cross-sectional area (LaGrangian stress) or current cross-sectional area (Cauchy stress). Cross-sectional areas were calculated using an elliptical approximation based on major and minor diameters of the imaged segment. Strain was calculated by dividing sarcomere length at a given length by initial sarcomere length. For stressrelaxation experiments, the fiber segment was rapidly loaded (<l sec) and a grid of 256 x 10 µm was imaged continuously (delay between capture of
0.23 msec), to observe structural changes along the longitudinal axis of the fiber segment. This grid size was selected to balance a rapid capture time with minimal distortion of images due to artifacts of fiber motion and scanning artifacts.
Image Processing
Sarcomere length was determined using two signal processing techniques. The first technique applied a fast Fourier transform (FFT) to the intensity profile for each row across the fiber width:
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(2) |
where X is the intensity profile, n is pixel number along a row n=(0, 1, ..., N-1), N is the row width, and j is the square root of -1. Sarcomere length was then calculated as the reciprocal of the peak frequency of the FFT power spectrum. The second technique calculated the autocorrelation function AF, a special case of the cross-correlation function CFG, of the intensity profile for each row along the fiber width. The unbiased cross-relation function at a point i over a window of size (2W + 1) is given by:
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(3) |
for -wm
w pixel lags. G(u)=F(u) for the autocorrelation function. Because this function generated a peak at pixel lags in phase with the periodicity of the intensity profile, sarcomere length equaled the periodicity of the autocorrelation function, again calculated using an FFT.
"Connectivity" between -actinin and talin was defined as the phase shift between the peaks of the
-actinin and talin autocorrelated intensity profiles, as determined by cross-correlation. Connectivity between myofibrils within the sarcomere lattice was quantified by first calculating the phase shift between peaks of autocorrelated
-actinin intensity profiles of rows
0.6 µm apart (approximating the myofibrillar diameter). Then the variance of the mean phase shift for all myofibrils across the fiber diameter was defined as connectivity (a sarcomere lattice with high connectivity yields a low variance). Calculations and basic statistics were performed using MATLAB (MATLAB 6.0 Professional version; MathWorks, Natick, MA).
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Results |
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Single- and Double-labeled Images
At least 8 hr of incubation with gentle agitation was required for complete labeling of -actinin through the fiber lattice (Fig 2A).
-Actinin single labeling resulted in the expected striation pattern with striations overlapping the Z-bands when observed under phase microscopy (not shown) and extending across the entire fiber diameter (Fig 2B). In contrast to the
-actinin labeling, talin labeling reached steady state after only 3 hr (Table 1) and resulted in striations restricted to the cell membrane (Fig 2C). Autofluorescence was never observed in the absence of antibody incubation.
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The relative positions of -actinin and talin were tracked at various strains in double-labeled fibers (Fig 3A3D). At all strains, talin co-localized with
-actinin at the Z-band periphery (Table 3). Another talin striation, of lower intensity, was observed between
-actinin striations, presumably at the M-line. A decrease in cell diameter was apparent with increasing strain (compare diameters, Fig 3A3D). In addition, Z-bands spanning the fiber width maintained similar shear-induced curvature along the fiber axis at a given stretch, a feature exaggerated with increasing strain (compare Z-disk label, Fig 3A3D). Shear was probably a result of the boundary conditions imposed by fiber attachment with suture.
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Nuclear labeling occurred predominantly at the cell surface and displayed the expected ellipitical shape (Fig 4). Nuclear aspect ratio increased with strain, indicating a coupling of nuclear geometry to fiber geometry. Furthermore, the nucleus maintained a constant relative position with respect to Z-disks at various fiber strains.
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StressRelaxation
Five distinct mechanical phases were noted during the stress-relaxation experiment (Fig 5). During the initial phase of stretch (T1T2), as a result of fiber movement through the field of view, distorted images (presumably imaging artifacts) were observed as the fiber rapidly translated from its resting position. Between T2 and T3, although force was still increasing, dramatic changes in Z-band orientation were observed. Surprisingly, even after stress had peaked (T3), changes in Z-disk orientation were still observed during the rapid relaxation phase (T3T4). Finally, during slower relaxation (T4T5), minor structural differences were observed. A trivial explanation for the changes in Z-band orientation observed between T2 and T4 was simply that different regions/optical planes of the fiber were moving into and out of the imaging field during stretch or relaxation. However, regions of the fiber segment corresponding to the initial and final regions imaged during stressrelation were also imaged in three dimensions before and after the stretch. Regions of similar Z-disk orientation to those seen during stress-relaxation were not found, suggesting that the significant changes in Z-band shearing within the region imaged were a real phenomenon during stressrelaxation.
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Image Processing
For the images shown in Fig 3A3D, both functions returned similar periodicity values that were consistent with sarcomere lengths measured manually on 1015 Z-disks (Table 3). Both techniques were also sensitive to the relative fluorescence intensity of individual rows. Towards the middle of the fiber width, where a slight decrease in fluorescence was seen (marked by double lines to the left of Fig 3B), there was a corresponding decrease in power spectrum intensity. Each technique was nevertheless sensitive enough to detect a dominant periodicity. The power spectrum for an FFT applied to the AC function (Fig 6B) was eight orders of magnitude higher than the spectrum for an FFT applied to a raw intensity profile, resulting in a greater signal-to-noise ratio. We noted that, at higher sarcomere lengths, when the talin label became less distinct, the FFT approach still yielded a periodicity, whereas the autocorrelation approach failed. Both approaches yielded similar periodicities for multiple processed images (t-test for 60 images at various sarcomere lengths from eight fibers: -actinin, p>0.5; talin, p>0.75), confirming that the findings were not dependent on the analytical method. Mean periodicities for AC were 3.653 ± 0.097 and 1.854 ± 0.05 for
-actinin and talin, respectively, and for FFT, 3.563 ± 0.094 and 1.832 ± 0.047.
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The intensity profile for talin was in phase with the profile for -actinin at all sarcomere lengths (Fig 7C; Table 3), confirming tight connectivity between the two proteins. To determine whether our image processing techniques could distinguish among muscles whose images varied dramatically, sample images from a wild-type fiber (Fig 7A) and a fiber lacking the intermediate filament desmin (Fig 7B) were analyzed at three different strains. In wild-type fibers, phase shifts measured along the fiber width successfully reflected the curvature of the Z-disks across the fiber width (Fig 7C and Fig 7D). Phase shifts increased with stretch, a result of the increasing shear shown in Fig 7A. However, the variance for each stretch remained low, suggesting that the fiber exhibited a high degree of myofibrillar connectivity even at high strains. On the other hand, images from the knockout fiber, although displaying clear periodicity along the length of the fiber, exhibited a low degree of connectivity, demonstrated by the higher variance (Fig 7B and Fig 7D; Table 2).
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An alternate indicator of connectivity was obtained by plotting the difference in the phase shift between consecutive myofibrils against position across the fiber width (Fig 7E and Fig 7F; Table 2). A mean close to zero and low variance implied that consecutive myofibrils across the fiber behaved in a similar fashion, whereas a mean differing from zero or a mean equal to zero with a high variance implied a lack of connectivity among myofibrils. This measure differed from the previous measure of connectivity in one critical aspect: whereas variance in the phase shift along the fiber measured the average connectivity across the entire fiber width, the phase shift difference measured connectivity locally, or between consecutive myofibrils. Although the wild-type images all displayed a mean close to zero, implying a well-connected network across the entire fiber width, the knockout images displayed a mean close to zero but with a high variance, implying low connectivity (Fig 7E and Fig 7F).
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Discussion |
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Here we report a novel experimental system that permits simultaneous confocal imaging of cell structure and measurement of cell segment mechanical properties. The system offers tremendous potential in muscle to characterize the functional connections among proteins within the cell. Specifically, we are now able to quantify the degree to which loads borne by the fiber affect fiber geometry and myofibrillar strain fields, and vice versa, information previously unavailable from either immunolocalization or biochemical studies. In addition, we have exploited the periodicity existing in skeletal muscle to develop analytical tools that quantify the relative position of structures within the cell.
We initially employed this system to examine the interactions among three structures believed to be linked by the intermediate filament system: Z-disks, nuclei, and the costamere protein complexes located at the cell surface (
In addition to imaging of steady-state passive mechanical properties of fiber segments, we also attempted to observe structural changes dynamically during stressrelaxation. We observed shifts in cytoskeletal orientation both during stretch and during the rapid stressrelaxation phase, and we speculate that, in addition to titin-mediated longitudinal reorganization of the sarcomere lattice, stress decreases immediately after stretch in part due to viscous energy lost during cytoskeletal reorganization.
Finally, we have attempted to introduce analytical tools to characterize images in a more systematic fashion, potentially aiding in the development of more accurate structural computational models for muscle mechanics or providing quantitative definitions of muscle pathology. The FFT has been used by muscle biologists to calculate sarcomere lengths (
Our system is particularly powerful when used in combination with appropriate combinations of antibody labels and animal models. The impact of specific variables on fiber performance, such as presence, absence, or modification of a protein, can be readily assayed. We are encouraged by the demonstrated differences in connectivity between the wild-type and desmin-null fiber tested using this system (Fig 7B, Fig 7D, and Fig 7F). A separate study using complete data sets, outside of the scope of the methodology reported here, will provide a more complete comparison between these genotypes.
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Acknowledgments |
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Supported by National Institutes of Health grants AR40050 and AR40539 and by the Department of Veterans Affairs Rehabilitation Research and Development Service.
We thank Dr Jan Fridén, Dr Gordon Lutz, Pat Reid, and Jennifer Davis for helpful discussions, and Dr Yassemi Capetanaki (Baylor University) for providing the desmin-null mouse used in this study.
Received for publication April 15, 2002; accepted August 10, 2002.
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