Mechanical loading regulates expression of talin and its mRNA, which ...

Mechanical loading regulates expression of talin and its mRNA, which are concentrated at myotendinous junctions ´ RO ˆ ME FRENETTE AND JAMES G. TIDBALL JE Department of Physiological Science, University of California, Los Angeles, California 90095-1524 Frenette, Je´roˆme, and James G. Tidball. Mechanical loading regulates expression of talin and its mRNA, which are concentrated at myotendinous junctions. Am. J. Physiol. 275 (Cell Physiol. 44): C818–C825, 1998.—The hypothesis that mechanical loading regulates talin expression in developing and adult muscle was tested using in vitro and in vivo models. Talin was selected for study because it is a key structural link between the cytoskeleton and cell membrane. In the in vitro model, C2C12 myotubes were subjected to cyclic strains for 48 h. In the in vivo model, rat hindlimb muscles were unloaded for 10 days, then reloaded for 2 days. Cyclic loading of myotubes resulted in significant increases in the quantity of talin (68%) and its 190-kDa proteolytic fragment (70%), as well as talin mRNA (180%), relative to unloaded myotube cultures. Similarly, talin concentration and its mRNA increased by 68 and 136%, respectively, in soleus muscles reloaded for 2 days relative to ambulatory controls. Immunohistochemistry and in situ RT-PCR showed that talin and its mRNA are concentrated and colocalized at myotendinous junctions. Thus these findings indicate that increased mechanical loading promotes talin synthesis, which occurs principally at myotendinous junctions, according to talin mRNA distribution. rat; soleus muscle; cyclic loading; C2C12 myotubes

BIOLOGICAL STRUCTURES are typically constructed so that their mechanical characteristics do not greatly exceed the physical demands placed on the structure. This design reflects selective pressure to economize on materials needed to construct and maintain structures and reflects the ability of living systems to adapt structures if physical demands change over time (3). Further economy of design is apparent in cells in which proteins are synthesized near their site of function, so that there is no need for their active transport through the cell to their site of function after synthesis. This local synthesis of proteins is particularly important for large cells, such as skeletal muscle fibers, which can be several centimeters long and are constituted of different functional compartments. In the present investigation we test whether the design of skeletal muscle will reflect these simple principles of economic design in the synthesis of proteins involved in force transmission. Myotendinous junctions (MTJs) and their in vitro analogs, focal adhesions, are highly specialized sites present at the cell surface, where extensive cytoskeletal interactions with the cell membrane occur (15, 28). The complex of proteins found at these sites is capable of force transmission across the cell membrane (6),

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although it is unknown whether the synthesis of these force-transmitting proteins is regulated by mechanical loading. However, previous investigations have shown that increased mechanical loading of skeletal muscle in vivo or in vitro increases the synthesis of other cytoskeletal proteins in muscle, especially actin and myosin, and the expression of proteins regulating gene expression, such as c-fos and c-jun (22, 31). Although it is unknown whether the synthesis of proteins involved in force transmission across the muscle cell membrane occurs preferentially at MTJs, previous investigations have shown that individual muscle cells contain specific domains in which the synthesis of specialized proteins is restricted. For example, expression of ACh receptor (AChR) subunits is restricted to myonuclei near neuromuscular junctions (25), which contributes to the restricted distribution of AChR and its mRNA at these sites (1). In addition, the application of a chronic stretch to muscle in vivo can increase myosin heavy chain mRNA near MTJs (8). Because muscle lengthening results in sarcomere addition preferentially at the end of the myofibrils (33), the enrichment of myosin heavy chain mRNA at the MTJs of stretched muscle indicates that the site of protein synthesis after muscle stretch occurs primarily at the site where the protein will subsequently function. In the present investigation we test the hypothesis that mechanical loading is a positive regulator of talin expression and that talin expression occurs near the sites at which talin functions in muscle fibers. Talin was selected for examination because it is highly concentrated at MTJs (29) and focal adhesions (4) and its binding affinities for other members of a transmembrane complex of structural proteins indicate that it is an integral component of a force-transmitting assembly. Specifically, talin contains binding sites for b1integrin (13) and actin (12, 35) and several binding sites for vinculin (10, 18) and participates in the formation and maintenance of transmembrane structural assemblies in cultured myoblasts (24). We test whether talin is preferentially expressed at MTJs, where it is most concentrated in muscle fibers, by examining the distribution of talin mRNA in muscle cells that have experienced increased loading. Together, these findings will enable us to determine whether the regulation of talin synthesis reflects the general expectations of economic design in muscle. MATERIALS AND METHODS

Cell culture. C2C12 muscle cells (American Type Culture Collection) were grown in wells of a mechanical cell stimulator (MCS; Cell Kinetics, Providence, RI). The MCS consists of a stainless steel plate containing wells in which the floor is a transparent Silastic membrane (Dow-Corning). The MCS

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REGULATION OF TALIN BY MECHANICAL LOADING

was autoclaved before use, and the membrane was coated with a 2-mm-thick layer of 2% gelatin and allowed to dry for 48 h at 37°C. C2C12 myoblasts were then added to each well in 10% fetal bovine serum (FBS) in DMEM containing penicillin and streptomycin at 37°C in an atmosphere of 5% CO2. Cultures were fed every 48 h for 6 days, at which time the membranes were covered with confluent myoblasts. The cultures were transferred to DMEM containing no FBS for 16 h to stimulate fusion and then returned to 10% FBS in DMEM. The cultures were maintained for 6–8 additional days to allow growth of myotubes. At the end of this period, the substratum was covered with 15- to 30-µm-diameter myotubes. Over 70% of all myonuclei present were located in myotubes. Cell loading. Myotubes were placed in fresh 10% FBS in DMEM and then loaded by cyclic deformation of the Silastic membrane. The center of the Silastic membrane was deflected by a piston, causing a 6.7% mean deformation of the membrane and adherent cells. Cells were loaded using five deformations during a 20-s period, then 10 s of no deformation. This cycle of loading was repeated two more times, followed by a 30-min period of no deformation. The mean strain rate during periods of loading was 3.4%/s. The entire sequence was repeated 95 times over the course of 48 h. An inverted microscope was used to visually inspect the cultures to confirm that the myotubes remained attached to the substratum at the end of the 48 h of cyclic loading. Culture wells in the MCS containing detached myotubes that did not cover and lie closely apposed to the underlying substratum were not used for further analysis. Approximately 15% of the cultures were eliminated according to this criterion. Control cultures were grown under identical conditions in the MCS, except they were not subjected to loading. Hindlimb unloading-reloading. Muscle unloading of adult Wistar rats (,250 g body wt) was achieved by hindlimb suspension using a modification (27) of the technique developed by Morey-Holton and Wronski (19). Hindlimbs were suspended for 10 days, then reloaded for 2 days. After they were reloaded, soleus muscles were used for immunohistochemistry, Western and Northern blots, and in situ RT-PCR. All animal treatments followed protocols approved by the University of California, Los Angeles, Animal Research Committee. Immunohistochemistry. After the experimental protocol, freshly dissected soleus muscles were frozen and sectioned as described previously (27). Longitudinal sections were then fixed in cold acetone for 10 min and air dried. After fixation, slides were washed in 50 mM sodium phosphate buffer containing 150 mM sodium chloride (PBS), pH 7.5, and placed in blocking buffer for 45 min. The samples were overlaid without antibody or incubated for 3 h with monoclonal anti-talin (1:20; Sigma Immunochemicals). Tissue sections were then rinsed in PBS and overlaid for 2 h with secondary antibody (Vector Laboratories; biotinylated anti-mouse IgG, 1:200). After secondary antibody incubation, the longitudinal sections were quenched with methanol and 0.3% hydrogen peroxide and rinsed with PBS. Finally, sections were reacted with Vectastain ABC reagent (Vector Laboratories) for 20 min, reacted with peroxidase, and mounted with coverslips. Western blots. Immediately after the last cycle of myotube loading, the supernatants were removed from all loaded and control cultures, and the cultures were rinsed briefly with DMEM. Myotubes in culture as well as rat muscles previously frozen in liquid nitrogen were then homogenized in reducing sample buffer (80 mM Tris · HCl, pH 6.8, 0.1 M dithiothreitol, 70 mM SDS, 1.0 mM bromphenol blue, glycerol). After homogenization, 30 µg of myotube extracts or 75

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µg of muscle extracts were separated by 8% SDS-PAGE (17). Proteins were then stained with Coomassie blue or electrophoretically transferred to a nitrocellulose membrane. The membranes were incubated with talin antibody (1:50), then with an alkaline phosphatase-conjugated second antibody, and chromogenic development was performed with 5-bromo-4chloro-3-indolyl phosphate and nitro blue tetrazolium. Northern blots. Total RNA was isolated from myotubes or whole muscles by the acid guanidinium method of Chomczynski and Sacchi (5) and fractionated on 1.2% agarose gels containing 2.2 M formaldehyde. RNA concentration was determined by measuring absorbance of samples at 260 nm. A 25-µg sample of RNA was loaded per lane, separated by electrophoresis, and transferred to uncharged nylon membranes. Transfer efficiency and uniformity of loading were checked by staining the membrane with methylene blue, then hybridization with a 32P-labeled mouse talin cDNA probe (generous gift from Dr. D. J. G. Rees, University of Oxford, Oxford, UK). The entire 1.2-kb insert was utilized as the probe (clone MT26C). After hybridization at 65°C, blots were washed with 0.05 M sodium phosphate, 0.75 M sodium chloride, 5 mM EDTA, and 0.1% SDS for 1 h and exposed to autoradiographic film for 24 h. Uniformity of loading and transfers was confirmed by methylene blue staining of the membranes before autoradiography. Densitometry. Relative protein and mRNA concentrations were determined by scanning densitometry (Alpha Innotec). Comparisons were made only between those experimental and control samples that were run in the same gel and probed in the same nitrocellulose blot for protein measurements or the same autoradiograph for mRNA measurements. Protein and mRNA concentrations were expressed in arbitrary units. The significance of differences between the concentration of any given protein or mRNA in loaded and control samples was tested by the Mann-Whitney test, with confidence limit set at P , 0.05. The sensitivity and linearity of measurements were determined by preparing an immunoblot of muscle extracts over a known range of concentrations spanning the sample loadings used in this investigation and then performing densitometry. The quantity of protein used for loading experimental gels was within the linear range of the loading-densitometry curve, determined empirically. We determined that differences in protein concentration .10% could be reliably measured by this technique. In situ RT-PCR. After 2 days of reloading, soleus muscles were dissected and fixed for 16 h at 4°C in sterile PBS containing 4% paraformaldehyde. After fixation, muscles were separated into bundles with MTJs preserved, dehydrated, and embedded in paraffin. Longitudinal sections were then cut and placed on silane-coated slides (26) and treated with 2 mg/ml of trypsin for 20 min before they were washed in sterile water and then in 100% ethanol. Sections were then incubated overnight with DNase. The RT reaction was performed using the SuperScript kit (GIBCO BRL). MgCl2 concentration and annealing temperature were optimized by standard PCR before in situ RT PCR was performed (Fig. 1). The cycling conditions were as follows: initial denaturation at 94°C for 2 min followed by 20 cycles of denaturation at 94°C for 30 s, annealing at 56°C, and extension at 72°C for 3 min each. The PCR mixture contained 10 mM Tris · HCl, pH 8.3, 4.5 µM MgCl2, 200 µM dNTPs, 0.1 U/ml Taq, 16 µM digoxigenin-11,28-deoxyuridine-58-triphosphate, 0.08% BSA, and 1.0 µM primers. The upstream (58-CAACATGCAGCCTCTGCCCCCAAGG-38) and downstream (58-GATGTCAGCGCAGCCACACCCCGCG-38) primers amplified nucleotides 2965–3563 of the talin coding sequence (23). After DNA

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amplification, sections were washed three times in solution A (100 mM Tris · HCl, pH 7.6, 150 mM NaCl) for 15 min, incubated in solution A containing 2% BSA for 30 min, and then incubated for 1 h at room temperature in alkaline phosphatase-conjugated anti-digoxigenin diluted 1:700 in solution A. After three washes in solution A, the sections were immersed in 100 mM Tris · HCl, pH 9.5, 150 mM NaCl, and 5 mM MgCl2 containing the 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium reagents to produce a dark-blue precipitate at the reaction sites. Each experiment included positive and negative controls. Positive controls consisted of omitting DNase treatment, so that all myonuclei containing the targeted nucleic acid sequence would stain after a successful PCR reaction. RT was omitted from negative controls. RESULTS

Cyclic loading increases the concentration of protein and mRNA in myotube cultures. The pattern of cyclic loading used in the present investigation was chosen for technical and biological reasons; the strain applied to the cells is within the range normally experienced by muscle cells and below the value shown previously to have negative effects on muscle cell growth or protein synthesis in vitro (31). Cyclic strains were selected, rather than prolonged, static strains because the surface area available for cell attachment and growth remained constant, except during the brief cycles of loading. In static loading experiments, there is an experimentally induced increase in surface area of the culture available for cell growth that is not available to cells in control cultures, which may influence experimental findings. The loading protocol used in the present study significantly increased the quantity of cellular protein in each well in loaded cultures by 19% compared with controls: 2.1 6 0.6 and 1.76 6 0.2 mg/well for loaded and control cultures, respectively (n 5 6). Similarly, loading increased the quantity of total RNA in each well contain-

Fig. 1. PCR product from talin primer set on a standard 0.8% agarose gel, with rat cDNA as template. A: 1-kb ladder standards. B: negative control (no cDNA). C: PCR product generated by talin primers utilized in in situ RT-PCR experiments. PCR product is 0.6 kb (arrowhead), which is expected size on the basis of rat cDNA clone (23). No other PCR products were generated using these primers.

Fig. 2. Top: immunoblot of myotubes for talin after 48 h of cyclic loading in vitro. * Position of intact, 230-kDa talin; ** position of 190-kDa proteolytic fragment of talin. A and B: control myotubes not subjected to loading. C and D: myotubes subjected to cyclic deformation. Middle: Northern blot of myotubes for talin mRNA after 48 cycles of loading in vitro. Bottom: methylene blue-stained Northern blot used to probe for talin mRNA showing 28S and 18S rRNA to indicate relative loading of lanes.

ing cyclically loaded myotubes by 96% compared with controls: 64.6 6 14.8 and 32.9 6 0.9 µg/well for loaded and control cultures, respectively (n 5 3). Talin protein and mRNA concentrations increase in myotubes experiencing cyclic loading in vitro. Talin showed a strong positive response to mechanical loading by increasing the proportion of total myotube protein that it comprised by 41% compared with controls (Figs. 2 and 3). The 190-kDa fragment of talin showed a similar change in loaded cells compared with controls, increasing by 43%. In view of the 19% increase of total protein in loaded cultures, these findings show ,68% more intact talin in loaded cultures than in unloaded controls and ,70% more 190-kDa talin fragment. The similar increases in the concentration of intact talin and talin fragment indicate that the increase in talin concentration is not attributable to increased stability of the intact protein in loaded cells, since that would be reflected by a decrease in concentra-


Fig. 3. Concentration of talin and its 190-kDa proteolytic fragment in C2C12 myotubes experiencing cyclic loading or in unloaded controls. Densitometric measurements of alkaline phosphatase reaction products in Western blots were normalized to talin concentration in unloaded controls. * Significantly different from unloaded, P , 0.05.

tion of its 190-kDa fragment. A previous study has demonstrated that activated calcium-dependent proteases (calpains) can cleave talin into 190- and 47-kDa fragments (21). The quantity of talin mRNA in C2C12 myotubes was significantly increased by cyclic loading (Fig. 2). Normalization relative to total RNA shows a 42% increase in talin mRNA in loaded myotube cultures, where the normalized concentration of talin mRNA, expressed in arbitrary units, was 19.7 6 4.2 (n 5 4) in control myotube controls and 28.0 6 9.6 (n 5 4) in loaded myotube cultures. Thus, not only did cyclic loading increase the total RNA in myotubes by 96%, but the proportion of this total RNA that was comprised by talin mRNA increased by 42%. From this we can estimate an overall increase in the quantity of talin mRNA of ,180% in loaded myotubes compared with unloaded, control myotubes. Talin protein and mRNA concentration are increased in soleus muscles after modified mechanical loading in situ. Talin protein concentration relative to total protein increased significantly by 57% in reloaded soleus muscles compared with ambulatory controls (Figs. 4 and 5). Talin protein concentration was also expressed relative to a-actinin concentration to compensate for the possibility that the proportion of total muscle mass constituted by muscle fibers may vary during modified loading (Fig. 5). For example, there may be a relative increase in connective tissue concentration during muscle unloading (9). When normalized with a-actinin, a 68% increase in talin concentration relative to ambulatory controls was observed (Fig. 5). There was no significant change in talin concentration when normalized to total protein or a-actinin in animals experiencing only hindlimb unloading compared with ambulatory controls (Fig. 5). Similarly, talin mRNA

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Fig. 4. Immunoblot of talin from soleus muscles subjected to modified loading. A: ambulatory rats (controls). B: rats suspended for 10 days. C: rats suspended for 10 days, then subjected to 2 days of reloading.

concentration in soleus muscles of animals experiencing hindlimb reloading was 42% greater than in ambulatory controls and 51% greater than in soleus muscles of animals experiencing unloading only (Fig. 6). Talin protein and mRNA are concentrated at MTJs. Immunohistochemistry and in situ RT-PCR revealed that talin and its mRNA are more concentrated and colocalized at MTJs (Figs. 7 and 8). Negative controls for both experiments showed no labeling. The distribution of talin protein is similar to that previously reported (29). Talin mRNA was also detected in a periodic pattern along non-MTJ regions of the muscle fibers, at lower levels than at MTJs (Fig. 8).

Fig. 5. Concentration of talin in soleus muscles during modified loading. Densitometric measurements on Western blots were normalized with a-actinin or expressed relative to total protein. Values are means 6 SE (n 5 6 for each group); control values (ambulatory) were set at 100%. * Significantly different from suspended group, P , 0.05. # Significantly different from ambulatory group, P , 0.05.

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Fig. 6. Analysis of talin mRNA from soleus muscles subjected to modified loading. A: ambulatory rats (controls). B: rats suspended for 10 days. C: rats suspended for 10 days, then subjected to 2 days of reloading.

DISCUSSION

The results of the present investigation show that mechanical loading is a positive regulator of talin expression in myotubes experiencing cyclic loading in vitro and fully differentiated muscles experiencing reloading after periods of unloading in vivo. In addition, the elevated concentration of talin mRNA at MTJs of muscles experiencing increased loading indicates that there is more talin synthesis at the primary site of talin function in the muscle cell. Together, these findings support the hypothesis that regulation of talin expression will reflect an economy of design where its level and site of synthesis reflect the functional demands placed on the muscle. The present investigation showed an increase in talin concentration in response to mechanical stimulation in vitro and in vivo that can be attributed to changes in transcriptional or translational rates. The increase in talin mRNA in both loading systems suggests that increased talin concentration can be primarily associated with an increase in talin transcription or mRNA stability, rather than to mechanically induced changes in translation rate or talin turnover. However, previous investigations have shown that mechanical loading can also positively influence the rate of mRNA translation, so that the possibility that loading affects translation in the present system cannot be excluded. For example, cardiac myocytes expressed higher levels of contractile proteins during contraction than during quiescence, although the concentration of mRNA encoding the contractile proteins was unchanged (7). The increase in talin concentration during mechanical loading may also occur if there were decreases in talin proteolysis during muscle loading. Previous investigations have shown that changes in protease activation may be an important mechanism regulating protein concentration in myotubes experiencing mechanical loading in vitro. For example, 8–10 h of cyclic stretch of

Fig. 7. Immunoperoxidase labeling of talin in longitudinal sections of soleus muscles. A: negative control (no primary antibody) shows no staining. B: soleus from ambulatory control. C: soleus after 10 days of unloading. D: soleus after 10 days of unloading and 2 days of reloading. F, muscle fiber; T, tendon. Dark reaction product is at myotendinous junction. Scale bar, 25 µm.


Fig. 8. In situ RT-PCR of talin mRNA in muscle fibers from reloaded soleus muscle. A: negative control tissue (omission of RT enzyme from RT-PCR mixture). Muscle fiber is between 2 arrowheads. B: positive control (no DNase treatment). In situ RT-PCR shows strong labeling in myonuclei. C: experimental tissue. Talin mRNA is most concentrated at myotendinous junction (arrows). Muscle fibers terminate and connect to connective tissue to right of arrows. D: talin mRNA is distributed in transverse bands along muscle fibers. Scale bar, 25 µm.

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myotubes in vitro causes an increase in creatine kinase efflux from the cells and a decrease in protein concentration (30), which indicate membrane damage that would permit influx of calcium into the cell and thereby activate calpain. Calpains are likely to be important in regulating talin turnover in muscle, in that they have been shown to cleave talin into two stable proteolytic fragments of 190 and 47 kDa (21). Our in vitro experiments do not support the possibility that the increase in talin concentration is attributable to a decrease in proteolysis, since mechanical stress increased intact talin by 68% and its 190-kDa proteolytic fragment by 70%. The most straightforward interpretation of this finding is that the cyclic loading imposed here causes an upregulation of talin synthesis and degradation rates. Recent findings indicate that calpain activity is also increased during muscle reloading after periods of hindlimb suspension, by demonstrating that reloaded plantaris muscles in vivo lead to a 90% increase in concentration of calpain II and a similar increase in the proportion of calpain II in an autolyzed state, indicating its activation (26). Thus neither the increase in talin concentration in loaded myotubes nor the increase in talin concentration in reloaded muscles in vivo is attributable to decreased activation of calpain. The observation that the concentration of talin in reloaded muscles significantly exceeded its concentration in ambulatory control muscles was an unexpected finding, but in hindsight it supports the relationship between mechanical loading and talin expression tested here. The stress placed on the soleus was proportional to the ratio of the body weight of the animal to muscle cross-sectional area. Thus the muscle stress associated with weight bearing by the soleus was greater at the onset of reloading than in ambulatory controls because the soleus mass decreased significantly during unloading for 10 days but the animal mass did not change significantly (27). Previous investigations have shown that soleus muscle mass does not return to control, ambulatory values until ,7 days after a 10-day period of unloading (27), so the MTJs of the soleus muscles would experience higher stress during this period. Our findings that talin mRNA was most highly concentrated at the site at which talin protein was most highly concentrated support our hypothesis concerning the general economy of cellular design, where talin synthesis is modified in response to modified loads, and our hypothesis that synthesis occurs at the sites where the protein will be incorporated into load-bearing assemblies. Although this enrichment of talin mRNA at the MTJ where talin is most concentrated is similar to the previously described codistribution of AChR and its mRNA at neuromuscular junctions, we also found talin mRNA distributed at lower concentrations throughout the muscle fiber, where it occurred in an apparently sarcomeric pattern. This resembles the distribution of talin protein, in that talin is found in periodic bands at the muscle cell surface in structures called costameres (29). Interestingly, a similar banding pattern for mRNA distribution in muscle has been shown for myosin

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heavy chain after electrical stimulation (2) and for vinculin during costamere development (20). Previous investigations have shown that polysomes have a binding site for cytoskeletal structures (14), which may partially account for the observed distribution of talin mRNA. However, targeting information that is more specific for select mRNAs may also be provided. For example, the 38-untranslated region (38-UTR) of the mRNAs for actin, c-myc, and myosin heavy chain have been shown previously to be involved in regulating the distribution of those mRNAs (16, 32, 34). Furthermore, mechanical activity has recently been demonstrated to influence the distribution of myosin heavy chain mRNA via a mechanism that is dependent on the presence of the 38-UTR (11). Whether the 38-UTR of talin is similarly responsible for regulating its distribution has not been tested. The results of the present investigation support the hypothesis that the expression of talin is positively regulated by mechanical loading of myotubes in vitro and fully differentiated muscle in vivo. Thus increased talin synthesis during increased mechanical loading reflects the economy of design apparent in many biological structures. This study also suggests that the increase in talin in muscle experiencing increased mechanical loading is explicable by an increase in transcription or an increase in mRNA stability and that changes in the rate of translation or proteolysis do not appear to contribute importantly to the modifications in talin concentration observed under the mechanical loading conditions employed here. Finally, these findings show that the distribution of talin and the distribution of its mRNA in muscle cells are very similar, which supports the view that the regulation of talin mRNA distribution plays a role in determining the distribution of talin within muscle. Brandon Lu provided valuable technical assistance. This investigation was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-40343 and the Fonds de la Recherche en Sante´ du Que´bec. Address for reprint requests: J. G. Tidball, Dept. of Physiological Science, 5833 Life Science Bldg., University of California, Los Angeles, CA 90095-1524.

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