Roles of mu-calpain in cultured L8 muscle cells: application of a skeletal muscle-specific gene expression system

The goal of this work was to characterize the roles of mu-calpain in skeletal muscle protein degradation. Three approaches were developed to alter mu-calpain activity in rat myotubes. These included over-expression of antisense mu-calpain (mu-AS), dominant negative mu-calpain (mu-DN) and the antisense 30-kDa calpain subunit (30-AS). Constructs were expressed in rat L8 myotubes, and their effects on protein degradation and on concentrations of intact and/or degraded fodrin, desmin and tropomyosin were examined. An ecdysone-inducible expression system, in which we replaced a constitutively active CMV promoter with a skeletal muscle-specific alpha-actin promoter, was used to drive expression. Cell lines were evaluated by expression of the gene-of-interest following addition of ponasterone A (PA; ecdysone analog) to culture medium. Changes in calpain activity were assessed by evaluating fodrin degradation. 30-AS, which should alter both mu- and m-calpain activities, increased intact fodrin concentration. mu-DN and mu-AS reduced fodrin degradation products. mu-DN reduced total protein degradation by 7.9% (P<0.01) at 24 h and by 10.6% (P<0.01) at 48 h. mu-AS reduced total protein degradation by 6.4% at 24 h (P<0.05). 30-AS reduced total protein degradation by 13.4% (P<0.05) and 7.3% (P<0.05) following 24 and 48 h of PA administration, respectively. We assessed effects of mu-DN, mu-AS and 30-AS on concentrations of desmin and tropomyosin. Inhibition of calpains stabilized desmin, but had no effect on tropomyosin. These data indicate that fodrin and desmin are mu-calpain substrates and that mu-calpain accounts for a small proportion of total protein degradation in muscle cells. Tropomyosin is not degraded by calpain in muscle cells.     

Agrin induces alpha-actinin, filamin, and vinculin to co-localize with AChR clusters on cultured chick myotubes.

Agrin induces discrete high-density patches of acetylcholine receptors (AChRs) and other synaptic components on cultured myotubes in a manner that resembles synaptic differentiation. Furthermore, agrin-like molecules are present at developing neuromuscular junctions in vivo. This provides us with a unique opportunity to manipulate AChR patching in order to examine the role of cytoskeletal components. Cultured chick myotubes were fixed and labeled to visualize the distributions of actin, alpha-actinin, filamin, tropomyosin, and vinculin. Overnight exposure to agrin caused a small amount of alpha-actinin, filamin, and vinculin to reorganize into discrete clusters. Double-labeling studies revealed that 78% of the AChR clusters were associated with detectable concentrations of filamin, 70% with alpha-actinin, and 58% with vinculin. Filamin even showed congruence to AChRs within clustered regions. By contrast, actin (visualized with fluorescein-phalloidin) and tropomyosin did not show specific associations with agrin-induced AChR clusters. The accumulation of cytoskeletal components at AChRs clusters raised the possibility that cytoskeletal rearrangements direct AChR clustering. However, a time course of agrin-induced clustering that focused on filamin revealed that most of the early AChR clusters (3-6 h) were not associated with detectable amounts of cytoskeletal material. The accumulation of cytoskeletal material at later times (12-18 h) may imply a role in maintenance and stabilization, but it appears unlikely that these cytoskeletal elements initiate AChR clustering on myotubes.     

Phosphoinositide binding to the substrate regulates susceptibility to proteolysis by calpain

Calpain-mediated proteolysis regulates cytoskeletal dynamics and is altered during aging and the progression of numerous diseases or pathological conditions. Although several cytoskeletal proteins have been identified as substrates, how localized calpain activity is regulated and the mechanisms controlling substrate recognition are not clear. In this study, we report that phosphoinositide binding regulates the susceptibility of the cytoskeletal adhesion protein alpha-actinin to proteolysis by calpains 1 and 2. At first, alpha-actinin did not appear to be a substrate for calpain 2; however, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) binding to alpha-actinin resulted in nearly complete proteolysis of the full-length protein, producing stable breakdown products. Calpain 1 was able to cleave alpha-actinin in the absence of phosphoinositide binding; however, PtdIns(3,4,5)P(3) binding increased the rate of proteolysis, and phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P(2)) binding significantly inhibited cleavage. Phosphoinositide binding appeared to regulate calpain proteolysis of alpha-actinin by modulating the exposure of a highly sensitive cleavage site within the calponin homology 2 domain. In U87MG glioblastoma cells, which contain elevated levels of PtdIns(3,4,5)P(3), alpha-actinin colocalized with calpain within dynamic actin cytoskeletal structures. Furthermore, proteolysis of alpha-actinin producing stable breakdown products was observed in U87MG cells treated with calcium ionophore to activate the calcium-dependent calpains. Additional evidence of PtdIns(3,4,5)P(3)-mediated calpain proteolysis of alpha-actinin was observed in rat embryonic fibroblasts. These results suggest that PtdIns(3,4,5)P(3) binding is a critical determinant for alpha-actinin proteolysis by calpain. In conclusion, phosphoinositide binding to the substrate is a potential mechanism for regulating susceptibility to proteolysis by calpain.     

Purification and characterization of UDP-GalNAc:polypeptide N-acetylgalactosaminyl transferase from swine trachea epithelium

It is well established that periods of increased contractile activity result in significant changes in muscle structure and function. Such morphological changes as sarcomeric Z-line disruption and sarcoplasmic reticulum vacuolization are characteristic of exercise-induced muscle injury. While the precise mechanism(s) underlying the perturbations to muscle following exercise remains to be elucidated, it is clear that disturbances in Ca²⁺ homeostasis and changes in the rate of protein degradation occur. The resulting elevation in intracellular [Ca²⁺] activates the non-lysosomal cysteine protease, calpain. Because calpain cleaves a variety of protein substrates including cytoskeletal and myofibrillar proteins, calpain-mediated degradation is thought to contribute to the changes in muscle structure and function that occur immediately following exercise. In addition, calpain activation may trigger the adaptation response to muscle injury. The purpose of this paper is to: (i) review the chemistry of the calpain-calpastatin system; (ii) provide evidence for the involvement of the non-lysosomal, calcium-activated neutral protease (calpain) in the response of skeletal muscle protein breakdown to exercise (calpain hypothesis); and (iii) describe the possible involvement of calpain in the inflammatory and regeneration response to exercise.     

Interaction of alpha-actinin, filamin and tropomyosin with F-actin

The abilities of alpha-actinin, filamin and tropomyosin to bind F-actin were examined by cosedimentation experiments. Results indicated that smooth muscle alpha-actinin and filamin can bind to actin filaments simultaneously with little evidence of competition. In contrast, tropomyosin exhibits marked competition with either filamin or alpha-actinin for sites on actin filaments.     

Calpain 1-gamma filamin interaction in muscle cells: a possible in situ regulation by PKC-alpha

Calpains are a family of calcium-dependent cysteine-proteases involved in cytoskeleton remodelling and muscle differentiation. In a recent study, we observed the presence of calpain 1 in the muscle contractile apparatus and specifically in the N1- and N2-lines. This calpain isoform was found to be involved in the degradation of muscle fibres via proteolysis of key proteins in Z-disk and costameric junctions. The goal of this study was to determine whether gamma-filamin--a specific muscle isoform of the filamin family--is a calpain 1 substrate and to characterise this interaction. Gamma-filamin is a major muscle architectural protein located in the Z-line and under the sarcolemmal membrane. This protein is a component of the chain binding the sarcolemma to the sarcomeric structure. In this study, we found that gamma-filamin formed a stable complex in vitro and in cells with calpain 1 in the absence of calcium stimulation. We also located the binding domains in the C-terminus of gamma-filamin with a cleavage site between serine 2626 and serine 2627 in the hinge 2 region. The catalytic (80 kDa) and regulatory (28 kDa) subunits of calpain 1 are both involved in high affinity binding at gamma-filamin. Moreover, we showed that phosphorylation of the filamin C-terminus domain by PKC alpha protected gamma-filamin against proteolysis by calpain 1 in COS cells. Stimulation of PKC activity in myotubes, prevented gamma-filamin proteolysis by calpain and resulted in an increase in myotube adhesion.     

Role of the calcium-calpain pathway in cytoskeletal damage after eccentric contractions.

The mechanism(s) underlying eccentric damage to skeletal muscle cytoskeleton remain unclear. We examined the role of Ca(2+) influx and subsequent calpain activation in eccentric damage to cytoskeletal proteins. Eccentric muscle damage was induced by stretching isolated mouse muscles by 20% of the optimal length in a series of 10 tetani. Muscle force and immunostaining of the cytoskeletal proteins desmin, dystrophin, and titin were measured at 5, 15, 30, and 60 min after eccentric contractions and compared with the control group that was subjected to 10 isometric contractions. A Ca(2+)-free solution and leupeptin (100 microM), a calpain inhibitor, were applied to explore the role of Ca(2+) and calpain, respectively, in eccentric muscle damage. After eccentric contractions, decreases in desmin and dystrophin immunostaining were apparent after 5 min that accelerated over the next 60 min. Increased titin immunostaining, thought to indicate damage to titin, was evident 10 min after stretch, and fibronectin entry, indicating membrane disruption, was evident 20 min after stretch. These markers of damage also increased in a time-dependent manner. Muscle force was reduced immediately after stretch and continued to fall, reaching 56 +/- 2% after 60 min. Reducing extracellular calcium to zero or applying leupeptin minimized the changes in immunostaining of cytoskeletal proteins, reduced membrane disruption, and improved the tetanic force. These results suggest that the cytoskeletal damage and membrane disruption were mediated primarily by increased Ca(2+) influx into muscle cells and subsequent activation of calpain.     

Inhibitors arrest myofibrillogenesis in skeletal muscle cells at early stages of assembly

A three-step model for myofibrillogenesis has been proposed for the formation of myofibrils [Rhee et al., 1994: Cell Motil. Cytoskeleton 28:1-24; Sanger et al., 2002: Adv. Exp. Med. 481:89-105]: premyofibril to nascent myofibril to mature myofibril. We have found two chemically related inhibitors that will arrest development at both the first and second step. Cultured quail embryonic skeletal myoblasts were treated with ethyl methane sulfonate (EMS) or 2-aminoethyl-methanesulfonate (MTSEA+). When the myoblasts fused in the presence of either of these compounds, myosheets rather than myotubes formed. Treated cells were fixed and immunostained against multiple proteins commonly found in muscle cells. Protein expression and localization throughout the myosheet were similar to that of developing myotube tips. Cells treated with high concentrations of EMS (10 mM) stained for non-muscle myosin II, sarcomeric alpha-actinin, and tropomyosin. No zeugmatin (Z-band region of titin) or muscle myosin II antibody staining was detected in fibers in this treatment group. These fibers are comparable to premyofibrils in control myotubes. At lower concentrations of EMS (7.5 to 5 mM), fibers that formed stained for muscle myosin II and titin as well as for non-muscle myosin IIB, sarcomeric alpha-actinin, and tropomyosin. Muscle myosin II was in an unbanded pattern. These fibers are comparable to nascent myofibrils observed during normal myofibrillogenesis. Similar effects to those obtained by treating cells with EMS were obtained when we treated cultured cells with MTSEA+ (5 mM) and stained them with sarcomeric alpha-actinin. MTSEA+ is chemically related to EMS, and is a well-known inhibitor of ryanodine receptors in skeletal muscle cells. Some abnormalities such as nemaline-like rods and other protein aggregates also appear within the myosheet during EMS and MTSEA+ treatment. Removal of these two inhibitors of myofibrillogenesis allows the premyofibrils and nascent myofibrils to form mature myofibrils.     

Immunocytochemistry of cytoskeletal proteins in adult Schistosoma mansoni

Antisera to vertebrate actin and actin-binding proteins were used to characterize the cytoskeleton of adult Schistosoma mansoni. Actin, alpha-actinin and tropomyosin immunoreactivities were detected in the cytoplasm of the apical tegument. Antiserum to alpha-actinin bound to the tegumental spines and this protein may be involved in cross-linking of spine actin filaments. Actin, alpha-actinin and tropomyosin antisera bound to the musculature. Strongest immunoreactivity was seen in the parenchyma. Antisera to actin, alpha-actinin, tropomyosin and spectrin bound to parenchyma cells including those of the tubercles, suggesting that these proteins are located in muscle cell bodies. The distribution of cytoskeletal proteins is discussed in relation to tegumental repair processes.     

The Role of Calpain and Proteasomes in the Degradation of Carbonylated Neuronal Cytoskeletal Proteins in Acute Experimental Autoimmune Encephalomyelitis

The present study was designed to investigate the role of calpain and the proteasome in the removal of oxidized neuronal cytoskeletal proteins in myelin basic protein-induced experimental autoimmune encephalomyelitis (EAE). To this end, EAE rats received a single intrathecal injection of calpeptin or epoxomicin at the first sign of clinical disease. Forty-eight hours later, animals were sacrificed and lumbar spinal cord segments were dissected and used for biochemical analyses. The results show that calpain and proteasome activity is specifically, but partially, inhibited with calpeptin and epoxomicin, respectively. Calpain inhibition causes an increase in total protein carbonylation and in the amount of neurofilament proteins (NFPs), β-tubulin and β-actin that were spared from degradation, but no changes are seen in the oxidation of any of three NFPs. By contrast, proteasome inhibition has no effect on total protein carbonylation or cytoskeletal protein degradation but increases the amount of oxidized NFH and NFM. These results suggest that while the proteasome may contribute to removal of oxidized NFPs, calpain is the main protease involved in degradation of neuronal cytoskeleton and does not preferentially targets oxidized NFPs species in acute EAE. Different results were obtained in a cell-free system, where calpain inhibition rises the amount of oxidized NFH, and proteasome inhibition fails to change the oxidation state of the NFPs. The later finding suggests that the preferential degradation of oxidized NFH and NFM in vivo by the proteasome occurs via the 26S and not the 20S particle.     

Detection of a specific isoform of alpha-actinin with antisera directed against dystrophin

We have characterized a protein immunologically related to dystrophin, the protein product of the Duchenne muscular dystrophy gene. We identify this related protein as a fast-twitch glycolytic isoform (mouse extensor digitorum longus-specific) of myofibrillar alpha-actinin. This specific isoform of alpha-actinin exhibits a more restricted pattern of expression in skeletal muscle than fast-twitch-specific isoforms of both myosin and Ca2+-ATPase. Our results provide evidence that dystrophin and myofibrillar alpha-actinin are related proteins, reinforcing the previous data concerning the sequence homologies noted between nonmuscle cytoskeletal alpha-actinin and dystrophin. In addition, we describe the first antisera directed against a specific myofibrillar skeletal muscle isoform of alpha-actinin.     

Calpains in muscle wasting

Calpains are intracellular nonlysosomal Ca(2+)-regulated cysteine proteases. They mediate regulatory cleavages of specific substrates in a large number of processes during the differentiation, life and death of the cell. The purpose of this review is to synthesize our current understanding of the participation of calpains in muscle atrophy. Muscle tissue expresses mainly three different calpains: the ubiquitous calpains and calpain 3. The participation of the ubiquitous calpains in the initial degradation of myofibrillar proteins occurring in muscle atrophy as well as in the necrosis process accompanying muscular dystrophies has been well characterized. Inactivating mutations in the calpain 3 gene are responsible for limb-girdle muscular dystrophy type 2A and calpain 3 has been found to be downregulated in different atrophic situations, suggesting that it has to be absent for the atrophy to occur. The fact that similar regulations of calpain activities occur during exercise as well as in atrophy led us to propose that the calpains control cytoskeletal modifications needed for muscle plasticity.     

Calponin and SM22 as differentiation markers of smooth muscle: spatiotemporal distribution during avian embryonic development

Abstract. Calponin and SM 22 are two proteins related in sequence that are particularly abundant in smooth muscle cells. Here, the distribution patterns of calponin and SM 22 were compared with that of other smooth muscle contractile and cytoskeletal components in the avian embryo using immunofluorescence microscopy and immunoblotting. Like myosin-light-chain kinase and heavy caldesmon, both calponin and SM 22 were more or less exclusively found in smooth muscle cells, during embryonic development and in the adult. Labelling of other cell types including striated muscle was not observed. In contrast, tropomyosin, smooth muscle α-actin, filamin and desmin could also be detected in many other cell types in addition to smooth muscles, at least during part of embryonic life. Calponin and SM 22 appeared almost synchronously during the differentiation of all smooth muscle cell populations, though with a slight time difference in the case of the aorta. The appearance of calponin, SM22 and heavy caldesmon was generally delayed in relation to desmin, tropomyosin, smooth muscle α-actin, myosin-light-chain kinase and filamin and a marked increase in abundance of these proteins was observed in the late embryo and in the adult. From these observations we can conclude that both calponin and SM 22 belong to a group of late differentiation determinants in smooth muscle and may constitute convenient and reliable markers to follow the differentiation of most, if not all, smooth muscle cell populations.     

Functional association between nicotinic acetylcholine receptor and sarcomeric proteins via actin and desmin filaments

By affinity chromatography utilizing alpha-cobrotoxin from digitonin-solubilized fractions of rabbit skeletal muscle, we found that many proteins are associated with the nicotinic acetylcholine receptor (AChR). In addition to the proteins we previously reported to bind to AChR (including dystrophin-dystrophin-associated protein (DAP) complex, utrophin, rapsyn, and actin; Mitsui et al. [1996] Biochem. Biophys. Res. Commun.224:802-807), alpha-actinin, desmin, myosin, tropomyosin, troponin T, and titin are also identified to be associated with AChR. Alkaline treatment or Triton X-100 solubilization released dystrophin-DAP complex, utrophin, and rapsyn from the AChR fraction, while actin and desmin remained associated. These findings demonstrate that AChR is supported primarily by a submembranous organization of actin and desmin filaments, and is linked to sarcomeric proteins via these filaments. To further investigate whether the association has any functional role, we studied the effect of acetylcoline on ATPase activity of the AChR fraction. Acetylcholine (0.5-4 microM) significantly activated Mg(2+)-ATPase activity of digitonin-solubilized AChR fraction (P < 0.05). Furthermore, we found that desmin as well as actin activated myosin Mg(2+)-ATPase activity. From these findings, it is suggested that desmin and actin form a submembranous organization in the postsynaptic region, and function as mediators of excitation of AChR to the sarcomeric contraction system.     

Tetanic contractions impair sarcomeric Z-disk of atrophic soleus muscle via calpain pathway

The aim of this study was to determine whether or not over-activation of calpains during running exercise or tetanic contractions was a major factor to induce sarcomere lesions in atrophic soleus muscle. Relationship between the degrees of desmin degradation and sarcomere lesions was also elucidated. We observed ultrastructural changes in soleus muscle fibers after 4-week unloading with or without running exercise. Calpain activity and desmin degradation were measured in atrophic soleus muscles before or after repeated tetani in vitro. Calpain-1 activity was progressively increased and desmin degradation was correspondingly elevated in 1-, 2-, and 4-week of unloaded soleus muscles. Calpain-1 activity and desmin degradation had an additional increase in unloaded soleus muscles after repeated tetani in vitro. PD150606, an inhibitor of calpains, reduced calpain activity and desmin degradation during tetanic contractions in unloaded soleus muscles. The 4-week unloading decreased the width of myofibrils and Z-disk in soleus fibers. After running exercise in unloaded group, Z-disks of adjacent myofibrils were not well in register but instead were longitudinally displaced. Calpain inhibition compromised exercise-induced misalignment of the Z-disks in atrophic soleus muscle. These results suggest that tetanic contractions induce an over-activation of calpains which lead to higher degrees of desmin degradation in unloaded soleus muscle. Desmin degradation may loose connections between adjacent myofibrils, whereas running exercise results in sarcomere injury in unloaded soleus muscle.     

Non-viral cellular substrates for human immunodeficiency virus type 1 protease

A computer search revealed 10 proteins with homology to the sequence we originally identified in vimentin as the site of cleavage by human immunodeficiency virus type 1 (HIV-1) protease. Of these 10 proteins (actin, alpha-actinin, spectrin, tropomyosins, vinculin, dystrophin, MAP-2, villin, TRK-1 and Ig mu-chain), we show that 4 of the first 5 were cleaved in vitro by this protease, as are MAP-1 and -2 [(1990) J. Gen. Virol. 71, 1985-1991]. In these proteins, cleavage is not restricted to a single motif, but occurs at many sites. However, cleavage is not random, since 9 other proteins including the cytoskeletal proteins filamin and band 4.1 are not cleaved in the in vitro assay. Thus, the ability of HIV-1 protease to cleave specific components of the cytoskeleton may be an important, although as yet unevaluated aspect of the life cycle of this retrovirus and/or may directly contribute to the pathogenesis observed during infection.     

Agrin induces α-actinin,filamin, and vinculin to co-localize with AChR clusters on cultured chick myotubes

Agrin induces discrete high-density patches of acetylcholine receptors (AChRs) and other synaptic components on cultured myotubes in a manner that resembles synaptic differentiation. Furthermore, agrin-like molecules are present at developing neuromuscular junctions in vivo. This provides us with a unique opportunity to manipulate AChR patching in order to examine the role of cytoskeletal components. Cultured chick myotubes were fixed and labeled to visualize the distributions of actin, α-actinin, filamin, tropomyosin, and vinculin. Overnight exposure to agrin caused a small amount of α-actinin, filamin, and vinculin to reorganize into discrete clusters. Double-labeling studies revealed that 78% of the AChR clusters were associated with detectable concentrations of filamin, 70% with α-actinin, and 58% with vinculin. Filamin even showed congruence to AChRs within clustered regions. By contrast, actin (visualized with fluorescein-phalloidin) and tropomyosin did not show specific associations with agrin-induced AChR clusters. The accumulation of cytoskeletal components at AChRs clusters raised the possibility that cytoskeletal rearrangements direct AChR clustering. However, a time course of agrin-induced clustering that focused on filamin revealed that most of the early AChR clusters (3–6 h) were not associated with detectable amounts of cytoskeletal material. The accumulation of cytoskeletal material at later times (12–18 h) may imply a role in maintenance and stabilization, but it appears unlikely that these cytoskeletal elements initiate AChR clustering on myotubes.     

In vitro digestion of dystrophin by calcium-dependent proteases, calpains I and II.

Dystrophin is a cytoskeletal protein which is thought to play an important role in membrane physiology since its absence (due to gene deficiency) leads to the symptoms of Duchenne muscular dystrophy (DMD). Some disruption in the regulation of intracellular free Ca2+ levels could lead to DMD-like symptoms. In this study, calpains, which are very active calcium-dependent proteases, were examined for their capacity to hydrolyse dystrophin in vitro. The results show that calpains are able to split dystrophin and produce breakdown products of different sizes (the degree of cleavage being dependent on the incubation time with proteases). The time-course of protease degradation was examined by Western immunoblot using three polyclonal sera which were characterized as being specific to the central (residues 1173-1728) and two distal parts of the molecule ie specific to the N-terminal (residues 43-760) or the C-terminal (residues 3357-3660) extremities of the dystrophin molecule. The cleavage patterns of dystrophin showed an accumulation of some major protease-resistant fragments of high relative molecular mass (250-370 kDa). These observations demonstrate that calpains digest dystrophin very rapidly when the calcium concentration is compatible with their activation. For instance, it is clear that calpains first give rise to large dystrophin products in which the C-terminal region is lacking. These observations suggest that dystrophin antibodies specific to the central domain of the molecule should be used to detect dystrophin for diagnostic purposes and before any conclusion as to the presence or absence of dystrophin can be deduced from results obtained using immunoanalyses of muscle biopsies.     

Calpain 3 is activated through autolysis within the active site and lyses sarcomeric and sarcolemmal components

Calpain 3 (Capn3) is known as the skeletal muscle-specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. This enigmatic protease has many unique features among the calpain family and, importantly, mutations in Capn3 have been shown to be responsible for limb girdle muscular dystrophy type 2A. Here we demonstrate that the Capn3 activation mechanism is similar to the universal activation of caspases and corresponds to an autolysis within the active site of the protease. We undertook a search for substrates in immature muscle cells, as several lines of evidence suggest that Capn3 is mostly in an inactive state in muscle and needs a signal to be activated. In this model, Capn3 proteolytic activity leads to disruption of the actin cytoskeleton and disorganization of focal adhesions through cleavage of several endogenous proteins. In addition, we show that titin, a previously identified Capn3 partner, and filamin C are further substrates of Capn3. Finally, we report that Capn3 colocalizes in vivo with its substrates at various sites along cytoskeletal structures. We propose that Capn3-mediated cleavage produces an adaptive response of muscle cells to external and/or internal stimuli, establishing Capn3 as a muscle cytoskeleton regulator.     

Highly homologous filamin polypeptides have different distributions in avian slow and fast muscle fibers

The high molecular weight actin-binding protein filamin is located at the periphery of the Z disk in the fast adult chicken pectoral muscle (Gomer, R. H., and E. Lazarides, 1981, Cell, 23: 524-532). In contrast, we have found that in the slow anterior latissimus dorsi (ALD) muscle, filamin was additionally located throughout the l band as judged by immunofluorescence with affinity-purified antibodies on myofibrils and cryosections. The Z line proteins desmin and alpha-actinin, however, had the same distribution in ALD as they do in pectoral muscle. Quantitation of filamin and actin from the two muscle types showed that there was approximately 10 times as much filamin per actin in ALD myofibrils as in pectoral myofibrils. Filamin immunoprecipitated from ALD had an electrophoretic mobility in SDS polyacrylamide gels identical to that of pectoral myofibril filamin and slightly greater than that of chicken gizzard filamin. Two-dimensional peptide maps of filamin immunoprecipitated and labeled with 125I showed that ALD myofibril filamin was virtually identical to pectoral myofibril filamin and was distinct from chicken gizzard filamin.