Arthrin, a myofibrillar protein of insect flight muscle, is an actin-ubiquitin conjugate

Flight muscles of some insects contain a myofibrillar protein termed arthrin, which is closely related to actin (mw 43,000). Here we demonstrate that arthrin (mw 55,000) is ubiquitinated actin. We show that in Act88FM342, a flightless Drosophila mutant wherein the Act88F actin gene specifies a glu93----lys replacement, isoelectric points of both actin III and arthrin are shifted, revealing that both are encoded by the same gene. Arthrin reacts with an anti-ubiquitin antibody, which demonstrates that its extra mass results from ubiquitin ligation. Approximately one-seventh of myofibrillar actin is stably ubiquitinated, suggesting that there may be one arthrin molecule per actin-tropomyosin-troponin cooperative unit. Arthrin formation lags several hours behind that of actin III, implying that ubiquitination coincides with some aspect of myofibril assembly.     

AMP and IMP dissociate actomyosin into actin and myosin

We investigated to determine why heating of squid muscle at 60 degrees C induced the liberation of actin from myofibrils. When a mixture of a myofibrillar fraction and a low-molecular sarcoplasmic fraction prepared from squid muscle was heated at 60 degrees C, actin liberation occurred. When a myofibrillar fraction was heated with ATP, AMP, or IMP, actin liberation occurred. Hence, AMP is perhaps one of the factors causing actin liberation in postmortem squid muscle. It was found that AMP and IMP reversibly dissociated actomyosin of chicken, bovine, and porcine skeletal muscles into actin and myosin on incubation at 0 degrees C at pH 7.2 in 0.2 M KCl. These results led us to conclude that AMP and IMP were the most responsible factors causing actin liberation from myofibrils in the heated muscle and causing reversible dissociation of actomyosin on storage of skeletal muscle at a low temperature. Hence, AMP and IMP are possible factors causing the resolution of rigor mortis in muscles.     

Accumulation of myosin, actin, tropomyosin, and alpha-actinin in cultured muscles cells.

In an effort to understand the conditions that promote the assembly of myofibrillar proteins in muscle cells, the temporal sequence of accumulation of four myofibrillar proteins, actin, myosin, tropomyosin, and α-actinin, was monitored during the period of de novo assembly of myofibrils in differentiating muscle cells. Isotope dilution experiments indicated that all four proteins were accumulated simultaneously. Therefore, assembly of myofibrils may be occurring in the presence of a full complement of myofibrillar proteins.     

AMP and IMP Dissociate Actomyosin into Actin and Myosin

We investigated to determine why heating of squid muscle at 60 °C induced the liberation of actin from myofibrils. When a mixture of a myofibrillar fraction and a low-molecular sarcoplasmic fraction prepared from squid muscle was heated at 60 °C, actin liberation occurred. When a myofibrillar fraction was heated with ATP, AMP, or IMP, actin liberation occurred. Hence, AMP is perhaps one of the factors causing actin liberation in postmortem squid muscle. It was found that AMP and IMP reversibly dissociated actomyosin of chicken, bovine, and porcine skeletal muscles into actin and myosin on incubation at 0 °C at pH 7.2 in 0.2 M KCl. These results led us to conclude that AMP and IMP were the most responsible factors causing actin liberation from myofibrils in the heated muscle and causing reversible dissociation of actomyosin on storage of skeletal muscle at a low temperature. Hence, AMP and IMP are possible factors causing the resolution of rigor mortis in muscles.     

Caenorhabditis elegans kettin, a large immunoglobulin-like repeat protein, binds to filamentous actin and provides mechanical stability to the contractile apparatuses in body wall muscle

Kettin is a large actin-binding protein with immunoglobulin-like (Ig) repeats, which is associated with the thin filaments in arthropod muscles. Here, we report identification and functional characterization of kettin in the nematode Caenorhabditis elegans. We found that one of the monoclonal antibodies that were raised against C. elegans muscle proteins specifically reacts with kettin (Ce-kettin). We determined the entire cDNA sequence of Ce-kettin that encodes a protein of 472 kDa with 31 Ig repeats. Arthropod kettins are splice variants of much larger connectin/titin-related proteins. However, the gene for Ce-kettin is independent of other connectin/titin-related genes. Ce-kettin localizes to the thin filaments near the dense bodies in both striated and nonstriated muscles. The C-terminal four Ig repeats and the adjacent non-Ig region synergistically bind to actin filaments in vitro. RNA interference of Ce-kettin caused weak disorganization of the actin filaments in body wall muscle. This phenotype was suppressed by inhibiting muscle contraction by a myosin mutation, but it was enhanced by tetramisole-induced hypercontraction. Furthermore, Ce-kettin was involved in organizing the cytoplasmic portion of the dense bodies in cooperation with alpha-actinin. These results suggest that kettin is an important regulator of myofibrillar organization and provides mechanical stability to the myofibrils during contraction.     

Z-disc-associated,Alternatively Spliced,PDZ Motif-containing Protein (ZASP) Mutations in the Actin-binding Domain Cause Disruption of Skeletal Muscle Actin Filaments in Myofibrillar Myopathy

The core of skeletal muscle Z-discs consists of actin filaments from adjacent sarcomeres that are cross-linked by α-actinin homodimers. Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP)/Cypher interacts with α-actinin, myotilin, and other Z-disc proteins via the PDZ domain. However, these interactions are not sufficient to maintain the Z-disc structure. We show that ZASP directly interacts with skeletal actin filaments. The actin-binding domain is between the modular PDZ and LIM domains. This ZASP region is alternatively spliced so that each isoform has unique actin-binding domains. All ZASP isoforms contain the exon 6-encoded ZASP-like motif that is mutated in zaspopathy, a myofibrillar myopathy (MFM), whereas the exon 8–11 junction-encoded peptide is exclusive to the postnatal long ZASP isoform (ZASP-LΔex10). MFM is characterized by disruption of skeletal muscle Z-discs and accumulation of myofibrillar degradation products. Wild-type and mutant ZASP interact with α-actin, α-actinin, and myotilin. Expression of mutant, but not wild-type, ZASP leads to Z-disc disruption and F-actin accumulation in mouse skeletal muscle, as in MFM. Mutations in the actin-binding domain of ZASP-LΔex10, but not other isoforms, cause disruption of the actin cytoskeleton in muscle cells. These isoform-specific mutation effects highlight the essential role of the ZASP-LΔex10 isoform in F-actin organization. Our results show that MFM-associated ZASP mutations in the actin-binding domain have deleterious effects on the core structure of the Z-discs in skeletal muscle.     

Primary desminopathies

Mutations of the human desmin gene on chromosome 2q35 cause a familial or sporadic form of skeletal myopathy frequently associated with cardiac abnormalities. Skeletal and cardiac muscle from patients with primary desminopathies characteristically display cytoplasmic accumulation of desmin-immunoreactive material and myofibrillar changes. However, desmin-positive protein aggregates in conjunction with myofibrillar abnormalities are also the morphological hallmark of the large group of secondary desminopathies (synonyms: myofibrillar myopathies, desmin-related myopathies), which comprise sporadic and familial neuromuscular conditions of considerable clinical and genetic heterogeneity. Here, we will give an overview on the functional role of desmin in striated muscle as well as the main clinical, myopathological, genetic and patho-physiological aspects of primary desminopathies. Furthermore, we will discuss recent genetic and biochemical advances in distinguishing primary from secondary desminopathies.     

Alterations in flightin phosphorylation inDrosophila flight muscles are associated with myofibrillar defects engendered by actin and myosin heavy-chain mutant alleles

Flightin is a 20-kD myofibrillar protein found in the stretch-activated flight muscles ofDrosophila melanogaster. Nine of the eleven isoelectric variants of flightin are generatedin vivo by multiple phosphorylations. The accumulation of these isoelectric variants is affected differently by mutations that eliminate thick filaments or thin filaments. Mutations in the myosin heavy-chain gene that prevent thick filament assembly block accumulation of all flightin variants except N1, the unphosphorylated precursor, which is present at much reduced levels. Mutations in the flight muscle-specific actin gene that block actin synthesis and prevent thin filament assembly disrupt the temporal regulation of flightin phosphorylation, resulting in premature phosphorylation and premature accumulation of flightin phosphovariants. Cellular fractionation of fibers that are devoid of thin filaments show that flightin remains associated with the thick filamentrich cytomatrix. These results suggest that flightin is a structural component of the thick filaments whose regulated phosphorylation is dependent upon the presence of thin filaments.This work was supported by National Science Foundation Grant IBN-9253045.     

An antiactin antibody that distinguishes between cytoplasmic and skeletal muscle actins

We elicited antibodies in rabbits to actin purified from body wall muscle of the marine mollusc, Aplysia californica. We found that this antiactin has an unusual specificity: in addition to reacting with the immunogen, it recognizes cytoplasmic vertebrate actins but not myofibrillar actin. Radioimmunoassay showed little or no cross-reaction with actin purified from either chicken gizzard or rabbit skeletal muscle. Immunocytochemical studies with human fibroblasts and L6 myoblasts revealed intense staining of typical cytoplasmic cables. Myofibrils were not stained after treatment of human and frog skeletal muscle with the antibody, although the distribution of immunofluorescence suggested that cytoplasmic actin is associated with membrane systems in the muscle fiber. The antibody may therefore be especially suited for studying the localization of cytoplasmic actin in skeletal muscle cells even in the presence of a great excess of the myofibrillar form.     

Myosin and actin expression and oxidation in aging muscle.

While the age-related loss in muscle mass partially explains the decline in strength, other yet undefined mechanisms contribute. This study investigates whether changes in myosin-actin stoichiometry and oxidative modification could help explain the decrement in muscle strength with aging. Protein expression and oxidation were evaluated in myosin and actin isolated from the soleus and semimembranosus muscles from young adult, old, and very old Fischer 344 rats. In the soleus muscle, actin and myosin content did not change with aging. In the semimembranosus, actin content was stable, but myosin exhibited decreased content in muscles from very old rats, resulting in a decrease in the myosin-to-actin ratio. 3-Nitrotyrosine and 4-hydroxy-2-nonenal were used as markers of protein oxidative damage. Although myosin and actin are modified with 3-nitrotyrosine and 4-hydroxy-2-nonenal, the extent of chemical modification does not increase with age. The results suggest that the decline in force production with age is not due to the accumulation of these two specific markers of protein oxidation on the myofibrillar proteins. Additionally, age-dependent changes in myofibrillar stoichiometry do not contribute to the decline in force production in the soleus, but may play a role in the semimembranosus with advanced age.     

Attaching effacing Escherichia coli and paradigms of Tir-triggered actin polymerization: getting off the pedestal

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) colonize the gut mucosa via attaching and effacing (A/E) lesions. For years cultured cells were used as model systems to study A/E lesion formation, which showed actin accumulation under attached bacteria that can be raised above the plasma membrane in a pedestal-shaped structure. Studies of prototypical strains revealed that although both converge on N-WASP EPEC and EHEC O157:H7 use different actin polymerization pathways. While EPEC use the Tir-Nck pathway, Tir_EHECO157 cooperates with TccP/EspF_U to activate N-WASP. However, recent in vitro studies revealed a common EPEC and EHEC Tir-dependent and Nck-independent inefficient actin polymerization pathway. Unexpectedly, bacterial populations studies demonstrated that most non-O157 EHEC strains and EPEC lineage 2 strains can utilize both the Nck and TccP2 pathways in vitro . Importantly, in vivo and ex vivo mucosal infections have shown efficient A/E lesion formation independently of Nck and TccP. This review covers the progression in our understanding of EPEC and EHEC infection, through the different milestones obtained using cultured cells, to the realization that EPEC and EHEC have much more in common than previously appreciated and that mucosal attachment and microvillous effacement may be the key events, rather than pedestal formation.     

Measurement of protein degradation by release of labelled 3-methylhistidine from skeletal muscle and non-muscle cells

The rates of [³H]N^τ-methylhistidine (3-MH) accumulation in the medium, following pulse labelling of cells for 48 h with [³H]methionine, were used to measure myofibrillar protein degradation. In fused C₂C₁₂ myotubes, incubation for 24 or 48 h after the labelling period gave rates of myofibrillar degradation of 38 and 42%/day. In a leucine free medium, these rates were similar; 40 and 47%/day, respectively. Using identical conditions ± leucine, but in the absence of [³H]-methionine, rates of protein accretion and synthesis over 24–48 h were measured. From these data, rates of total protein degradation were calculated by difference and were similar to myofibrillar degradation rates. We have used the same pulse labelling protocol to assess whether the method is applicable to non-muscle cell lines based on the knowledge that 3T3 fibroblasts contain actin in the cytoskeleton. 3-MH was detected both in protein and upon its release into the medium. Actin degradation measured over a 48 h period gave a value half that obtained for total degradation, but the results suggest that the release of 3-MH by fibroblasts in vivo could be appreciable. The development of this methodology should provide a useful tool to investigate signalling mechanisms regulating actin degradation in a variety of cell types. © 1996 Wiley-Liss, Inc.     

Sarcomere alignment is regulated by myocyte shape

Cardiac organogenesis and pathogenesis are both characterized by changes in myocyte shape, cytoskeletal architecture, and the extracellular matrix (ECM). However, the mechanisms by which the ECM influences myocyte shape and myofibrillar patterning are unknown. We hypothesized that geometric cues in the ECM align sarcomeres by directing the actin network orientation. To test our hypothesis, we cultured neonatal rat ventricular myocytes on islands of micro-patterned ECM to measure how they remodeled their cytoskeleton in response to extracellular cues. Myocytes spread and assumed the shape of circular and rectangular islands and reorganized their cytoskeletons and myofibrillar arrays with respect to the ECM boundary conditions. Circular myocytes did not assemble predictable actin networks nor organized sarcomere arrays. In contrast, myocytes cultured on rectangular ECM patterns with aspect ratios ranging from 1:1 to 7:1 aligned their sarcomeres in predictable and repeatable patterns based on highly localized focal adhesion complexes. Examination of averaged alpha-actinin images revealed invariant sarcomeric registration irrespective of myocyte aspect ratio. Since the sarcomere sub-units possess a fixed length, this observation indicates that cytoskeleton configuration is length-limited by the extracellular boundary conditions. These results indicate that modification of the extracellular microenvironment induces dynamic reconfiguring of the myocyte shape and intracellular architecture. Furthermore, geometric boundaries such as corners induce localized myofibrillar anisotropy that becomes global as the myocyte aspect ratio increases.     

Paratropomyosin, a new myofibrillar protein, weakens rigor linkages formed between actin and myosin

We have used an enzymatic technique to determine the weakening effect of paratropomyosin, a new myofibrillar protein, on rigor linkages formed between actin and myosin, and to clarify the distinct function of paratropomyosin, as to that of tropomyosin. Paratropomyosin inhibited the Mg-ATPase activity and enhanced the K-ATPase activity of reconstituted actomyosin stoichiometrically, and its maximal binding to actin was estimated to occur at a molar ratio of 1: 12.5. Paratropomyosin also inhibited the myofibrillar Mg-ATPase activity by 49% and enhanced the myofibrillar K-ATPase activity to 126%, while tropomyosin had no effect on these ATPases. These results indicate that paratropomyosin is able to bind to thin filaments of myofibrils, because the binding site for paratropomyosin on F-actin is different from that for tropomyosin, and that, due to its greater affinity for the myosin binding site on actin, paratropomyosin competes for the binding site and helps weaken rigor linkages.     

Functional Nonequivalence of Drosophila Actin Isoforms

We show that different Drosophila actinisoforms are not interchangeable. We sequenced the sixgenes that encode conventional Drosophilaactins and found that they specify amino acidreplacements in 27 of 376 positions. To test the significance ofthese changes we used directed mutagenesis to introduce10 such conversions, independently, into the Act88Fflight muscle-specific actin gene. We challenged these variant actins to replace the nativeprotein by transforming germline chromosomes of aDrosophila strain lacking flight muscle actin.Only one of the 10 reproducibly perturbed myofibrillarfunction, demonstrating that most isoform-specific aminoacid replacements are of minor significance. In order toestablish the consequences of multiple amino acidreplacements, we substituted portions of theDrosophila Act88F actin gene with correspondingregions of genes encoding other isoforms. Only one offive constructs tested engendered normally functioningflight muscles, and the severity of myofibrillar defects correlated with the number of replacementswithin the chimeric genes. Finally, we completelyconverted the flight muscle actin-encoding gene to onespecifying a nonmuscle isoform, a change entailing atotal of 18 amino acid replacements. Transformationof flies with this construct resulted in disruption offlight muscle structure and function. We conclude thatactin isoform sequences are not equivalent and that effects of the amino acid replacements,while minor individually, collectively confer uniqueproperties.     

Inhibition of contraction of cultured muscle fibers results in increased turnover of myofibrillar proteins but not of intermediate- filament proteins

Muscle fibers are maintained in culture in a fully contractile state and are relaxed by the addition of 10(-7) M tetrodotoxin (TTX). This toxin binds to muscle membrane Na+- channels, abolishes spontaneous contractions and causes failure of the fiber to accumulate myosin heavy chains. These effects are reversible on removal of TTX. Synthesis and accumulation kinetics have been obtained for myofibrillar and for cytoplasmic filament proteins in normal, active muscle and in TTX- relaxed muscle fibers in culture. In relaxed fibers the synthesis of most proteins remained normal or slightly elevated. However, the accumulation of all myofibrillar proteins examined was markedly inhibited in TTX-treated cultures, whereas the accumulation of cytoplasmic filament proteins was normal or slightly elevated. Myofibrillar proteins examined were alpha-actin, troponin-C, myosin fast light chain 1, myosin fast light chain 2, alpha, beta-tropomyosins and the phosphorylated forms of tropomyosin and fast light chain 2. Cytoplasmic filament proteins studied were vimentin, alpha, beta-desmin and beta, alpha-actin. We also examined the synthesis and accumulation of six unidentified muscle-specific proteins and nine unidentified nonmuscle-specific proteins. Most of these proteins showed a normal accumulation pattern in TTX-relaxed fibers. We concluded that muscle fibers made inactive by TTX display an increased instability of all myofibrillar proteins while cytoplasmic filament proteins and cytoplasmic proteins in general are relatively unaffected. We suggest that TTX interferes, in a manner as yet unidentified, with assembly and normal stability of myofibrils. Decreased assembly and/or increased instability of myofibrils would lead to increased rates of myofibrillar protein degradation.     

Huntington's Disease Induced Cardiac Amyloidosis Is Reversed by Modulating Protein Folding and Oxidative Stress Pathways in the Drosophila Heart

Amyloid-like inclusions have been associated with Huntington''s disease (HD), which is caused by expanded polyglutamine repeats in the Huntingtin protein. HD patients exhibit a high incidence of cardiovascular events, presumably as a result of accumulation of toxic amyloid-like inclusions. We have generated a Drosophila model of cardiac amyloidosis that exhibits accumulation of PolyQ aggregates and oxidative stress in myocardial cells, upon heart-specific expression of Huntingtin protein fragments (Htt-PolyQ) with disease-causing poly-glutamine repeats (PolyQ-46, PolyQ-72, and PolyQ-102). Cardiac expression of GFP-tagged Htt-PolyQs resulted in PolyQ length-dependent functional defects that included increased incidence of arrhythmias and extreme cardiac dilation, accompanied by a significant decrease in contractility. Structural and ultrastructural analysis of the myocardial cells revealed reduced myofibrillar content, myofibrillar disorganization, mitochondrial defects and the presence of PolyQ-GFP positive aggregates. Cardiac-specific expression of disease causing Poly-Q also shortens lifespan of flies dramatically. To further confirm the involvement of oxidative stress or protein unfolding and to understand the mechanism of PolyQ induced cardiomyopathy, we co-expressed expanded PolyQ-72 with the antioxidant superoxide dismutase (SOD) or the myosin chaperone UNC-45. Co-expression of SOD suppressed PolyQ-72 induced mitochondrial defects and partially suppressed aggregation as well as myofibrillar disorganization. However, co-expression of UNC-45 dramatically suppressed PolyQ-72 induced aggregation and partially suppressed myofibrillar disorganization. Moreover, co-expression of both UNC-45 and SOD more efficiently suppressed GFP-positive aggregates, myofibrillar disorganization and physiological cardiac defects induced by PolyQ-72 than did either treatment alone. Our results demonstrate that mutant-PolyQ induces aggregates, disrupts the sarcomeric organization of contractile proteins, leads to mitochondrial dysfunction and increases oxidative stress in cardiomyocytes leading to abnormal cardiac function. We conclude that modulation of both protein unfolding and oxidative stress pathways in the Drosophila heart model can ameliorate the detrimental PolyQ effects, thus providing unique insights into the genetic mechanisms underlying amyloid-induced cardiac failure in HD patients.     

Sequential disassembly of myofibrils induced by myristate acetate in cultured myotubes

The phorbol ester TPA induces the sequential disassembly of myofibrils. First the alpha-actin thin filaments are disrupted and then, hours later, the myosin heavy chain (MHC) thick filaments. TPA does not induce the disassembly of the beta- and gamma-actin thin filaments of stress fibers in presumptive myoblasts or fibroblasts, nor does it block the reemergence of stress fibers in 72-h myosacs that have been depleted of all myofibrillar molecules. There are differences in where, when, and how myofibrillar alpha-actin and MHC are degraded and eliminated from TPA-myosacs. Though the anisodiametric myotubes have begun to retract into isodiametric myosacs after 5 h in TPA, staining with anti-MHC reveals normal tandem A bands. In contrast, staining with mAb to muscle actin fails to reveal tandem I bands. Instead, both mAb to muscle actin and rhophalloidin brilliantly stain numerous disk-like bodies approximately 3.0 micron in diameter. These muscle actin bodies do not fuse with one another, nor do they costain with anti-MHC. All muscle actin bodies and/or molecules disappear in 36-h myosacs. The collapse of A bands is first initiated in 10-h myosacs. Their loss correlates with the appearance of immense, amorphous MHC patches. MHC patches range from a few micrometers to over 60 micron in size. They do not costain with antimuscle actin or rho-phalloidin. While diminishing in number and fluorescence intensity, MHC aggregates are present in 30% of the 72-h myosacs. Myosacs removed from TPA rapidly elongate, and after 48 h display normal newly assembled myofibrils. TPA reversibly blocks incorporation of [35S]methionine into myofibrillar alpha-actin, MHC, myosin light chains 1 and 2, the tropomyosins, and troponin C. It does not block the synthesis of beta- or gamma-actins, the nonmyofibrillar MHC or light chains, tubulin, vimentin, desmin, or most household molecules.