Α-actinin Isoforms Research Articles

An Unbiased Proteomics Method to Assess the Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes.

Human pluripotent stem cell (hPSC)-derived cardiomyocytes exhibit the properties of fetal cardiomyocytes, which limits their applications. Various methods have been used to promote maturation of hPSC-cardiomyocytes; however, there is a lack of an unbiased and comprehensive method for accurate assessment of the maturity of hPSC-cardiomyocytes. We aim to develop an unbiased proteomics strategy integrating high-throughput top-down targeted proteomics and bottom-up global proteomics for the accurate and comprehensive assessment of hPSC-cardiomyocyte maturation. Utilizing hPSC-cardiomyocytes from early- and late-stage 2-dimensional monolayer culture and 3-dimensional engineered cardiac tissue, we demonstrated the high reproducibility and reliability of a top-down proteomics method, which enabled simultaneous quantification of contractile protein isoform expression and associated post-translational modifications. This method allowed for the detection of known maturation-associated contractile protein alterations and, for the first time, identified contractile protein post-translational modifications as promising new markers of hPSC-cardiomyocytes maturation. Most notably, decreased phosphorylation of α-tropomyosin was found to be associated with hPSC-cardiomyocyte maturation. By employing a bottom-up global proteomics strategy, we identified candidate maturation-associated markers important for sarcomere organization, cardiac excitability, and Ca2+ homeostasis. In particular, upregulation of myomesin 1 and transmembrane 65 was associated with hPSC-cardiomyocyte maturation and validated in cardiac development, making these promising markers for assessing maturity of hPSC-cardiomyocytes. We have further validated α-actinin isoforms, phospholamban, dystrophin, αB-crystallin, and calsequestrin 2 as novel maturation-associated markers, in the developing mouse cardiac ventricles. We established an unbiased proteomics method that can provide accurate and specific assessment of the maturity of hPSC-cardiomyocytes and identified new markers of maturation. Furthermore, this integrated proteomics strategy laid a strong foundation for uncovering the molecular pathways involved in cardiac development and disease using hPSC-cardiomyocytes.

Huntingtin associates with the actin cytoskeleton and α-actinin isoforms to influence stimulus dependent morphology changes.

One response of cells to growth factor stimulus involves changes in morphology driven by the actin cytoskeleton and actin associated proteins which regulate functions such as cell adhesion, motility and in neurons, synaptic plasticity. Previous studies suggest that Huntingtin may be involved in regulating morphology however, there has been limited evidence linking endogenous Huntingtin localization or function with cytoplasmic actin in cells. We found that depletion of Huntingtin in human fibroblasts reduced adhesion and altered morphology and these phenotypes were made worse with growth factor stimulation, whereas the presence of the Huntington’s Disease mutation inhibited growth factor induced changes in morphology and increased numbers of vinculin-positive focal adhesions. Huntingtin immunoreactivity localized to actin stress fibers, vinculin-positive adhesion contacts and membrane ruffles in fibroblasts. Interactome data from others has shown that Huntingtin can associate with α-actinin isoforms which bind actin filaments. Mapping studies using a cDNA encoding α-actinin-2 showed that it interacts within Huntingtin aa 399–969. Double-label immunofluorescence showed Huntingtin and α-actinin-1 co-localized to stress fibers, membrane ruffles and lamellar protrusions in fibroblasts. Proximity ligation assays confirmed a close molecular interaction between Huntingtin and α-actinin-1 in human fibroblasts and neurons. Huntingtin silencing with siRNA in fibroblasts blocked the recruitment of α-actinin-1 to membrane foci. These studies support the idea that Huntingtin is involved in regulating adhesion and actin dependent functions including those involving α-actinin.

Sarcomeric and nonmuscle α-actinin isoforms exhibit differential dynamics at skeletal muscle Z-lines.

The α-actinin proteins are a highly conserved family of actin crosslinkers that mediate interactions between several cytoskeletal and sarcomeric proteins. Nonsarcomeric α-actinin-1 and α-actinin-4 crosslink actin filaments in the cytoskeleton, while sarcomeric α-actinin-2 and α-actinin-3 serve a crucial role in anchoring actin filaments to the muscle Z-line. To assess the difference in turnover dynamics and structure/function properties between the α-actinin isoforms at the sarcomeric Z-line, we used Fluorescence Recovery After Photobleaching (FRAP) in primary myofiber cultures. We found that the recovery kinetics of these proteins followed three distinct patterns: α-actinin-2/α-actinin-3 had the slowest turn over, α-actinin-1 recovered to an intermediate degree, and α-actinin-4 had the fastest recovery. Interestingly, the isoforms' patterns of recovery were reversed at adhesion plaques in fibroblasts. This disparity suggests that the different α-actinin isoforms have unique association kinetics in myofibers and that nonmuscle isoform interactions are more dynamic at the sarcomeric Z-line. Protein domain-specific investigations using α-actinin-2/4 chimeric proteins showed that differential dynamics between sarcomeric and nonmuscle isoforms are regulated by cooperative interactions between the N-terminal actin-binding domain, the spectrin-like linker region and the C-terminal calmodulin-like EF hand domain. Together, these findings demonstrate that α-actinin isoforms are unique in binding dynamics at the Z-line and suggest differentially evolved interactive and Z-line association capabilities of each functional domain.

An analysis of splicing, actin-binding properties, heterodimerization and molecular interactions of the non-muscle α-actinins

The non-muscle α-actinin isoforms (actinin-1 and -4) are closely related dimeric actin filament cross-linking proteins. Despite high sequence similarity, unique properties have been ascribed to actinin-4in particular. For example, actinin-4, but not actinin-1, is essential for normal glomerular function in the kidney, is overexpressed in several cancers and can translocate to the nucleus to regulate transcription. To understand the molecular basis for such isoform-specific functions we have, for the first time, comprehensively compared these proteins in terms of alternative splicing, actin-binding properties, heterodimer formation and molecular interactions. We find that the Ca2+-insensitive variant of actinin-4 is expressed only in the nervous system and thus cannot be regarded as a smooth muscle isoform, as is the case for the Ca2+-insensitive variant of actinin-1. The actin-binding properties of actinin-1 and -4 are similar and are unlikely to explain isoform-specific functions. Surprisingly, we reveal that actinin-1/-4 heterodimers, rather than homodimers, are the most abundant form of actinin in many cell lines. Finally, we use a proteomics approach to identify potential isoform-specific interactions. The results of the present study indicate that actinin-1 and -4 can readily form heterodimers composed of monomers that may have different properties and interacting proteins. This significantly alters our view of non-muscle actinin function.

The Alström Syndrome Protein, ALMS1, Interacts with α-Actinin and Components of the Endosome Recycling Pathway

Alström syndrome (ALMS) is a progressive multi-systemic disorder characterized by cone-rod dystrophy, sensorineural hearing loss, childhood obesity, insulin resistance and cardiac, renal, and hepatic dysfunction. The gene responsible for Alström syndrome, ALMS1, is ubiquitously expressed and has multiple splice variants. The protein encoded by this gene has been implicated in ciliary function, cell cycle control, and intracellular transport. To gain better insight into the pathways through which ALMS1 functions, we carried out a yeast two hybrid (Y2H) screen in several mouse tissue libraries to identify ALMS1 interacting partners. The majority of proteins found to interact with the murine carboxy-terminal end (19/32) of ALMS1 were α-actinin isoforms. Interestingly, several of the identified ALMS1 interacting partners (α-actinin 1, α-actinin 4, myosin Vb, rad50 interacting 1 and huntingtin associated protein1A) have been previously associated with endosome recycling and/or centrosome function. We examined dermal fibroblasts from human subjects bearing a disruption in ALMS1 for defects in the endocytic pathway. Fibroblasts from these patients had a lower uptake of transferrin and reduced clearance of transferrin compared to controls. Antibodies directed against ALMS1 N- and C-terminal epitopes label centrosomes and endosomal structures at the cleavage furrow of dividing MDCK cells, respectively, suggesting isoform-specific cellular functions. Our results suggest a role for ALMS1 variants in the recycling endosome pathway and give us new insights into the pathogenesis of a subset of clinical phenotypes associated with ALMS.

Substrate-selective and Calcium-independent Activation of CaMKII by α-Actinin

Protein-protein interactions are thought to modulate the efficiency and specificity of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) signaling in specific subcellular compartments. Here we show that the F-actin-binding protein α-actinin targets CaMKIIα to F-actin in cells by binding to the CaMKII regulatory domain, mimicking CaM. The interaction with α-actinin is blocked by CaMKII autophosphorylation at Thr-306, but not by autophosphorylation at Thr-305, whereas autophosphorylation at either site blocks Ca(2+)/CaM binding. The binding of α-actinin to CaMKII is Ca(2+)-independent and activates the phosphorylation of a subset of substrates in vitro. In intact cells, α-actinin selectively stabilizes CaMKII association with GluN2B-containing glutamate receptors and enhances phosphorylation of Ser-1303 in GluN2B, but inhibits CaMKII phosphorylation of Ser-831 in glutamate receptor GluA1 subunits by competing for activation by Ca(2+)/CaM. These data show that Ca(2+)-independent binding of α-actinin to CaMKII differentially modulates the phosphorylation of physiological targets that play key roles in long-term synaptic plasticity.

Fiber-type specific expression of α-actinin isoforms in rat skeletal muscle

α-Actinins are actin-binding proteins, and two isoforms (α-actinin-2 and -3) are major structural components of the sarcomeric Z line in mammalian skeletal muscle. Based on human and knockout mice studies, α-actinin-3 is thought to be associated with muscle force output and high contraction velocities. However, fiber-type specific expression of α-actinin isoforms is not well understood and may vary among species. In this study, we investigated the expression of α-actinin isoforms and the difference between fiber types in rat skeletal muscle and compared it with those of humans and mice from previous reports. Soleus and plantaris muscles were analyzed immunohistochemically to identify muscle fiber types and α-actinin protein expression. α-Actinin-2 was stained in all muscle fibers in both the soleus and plantaris muscles; i.e., all α-actinin-3 co-expressed with α-actinin-2 in rat skeletal muscles. The proportions of α-actinin-3 expression, regardless of fiber type, were significantly higher in the plantaris (75.8±0.6%) than the soleus (8.0±1.7%). No α-actinin-3 expression was observed in type I fibers, whereas all type IIx+b fibers expressed α-actinin-3. α-Actinin-3 was also expressed in type IIa fibers; however, approximately 75% of type IIa fibers were not stained by α-actinin-3, and the proportion varied between muscles. The proportion of α-actinin-3 expression in type IIa fibers was significantly higher in the soleus muscle than the plantaris muscle. Our results showed that fiber-type specific expression of α-actinin isoforms in rats is more similar to that in humans compared to that of the mouse, whereas the proportion of α-actinin-3 protein varied between muscles.

Adaptation of Alpha-Actinin Isoforms to Endurance Exercise Training in Adult and Old Rat Plantaris Muscle

We recently reported that that the increase in α-actinin-3 expression was associated with a type I to type II myosin heavy chain (MyHC) shift, and a chronic increase in muscular activity imposed by sprint training enhanced α-actinin-2 expression in rat skeletal muscles (Ogura et al. 2008, 2009). PURPOSE: To clarify the effects of 1) ageing-induced type II to type I MyHC shift and 2) endurance training on the expressions of α-actinin isoforms in rat plantaris muscle. METHODS: Adult (18 mo, n = 12) and old (28 mo, n = 12) male Fischer 344 rats were assigned to either a sedentary or an endurance training group (4 groups, n = 6/each). Animals in the training groups ran on a treadmill for 8 wk. The training intensity was adjusted to be relatively equal for adult and old training groups. After the training program was completed, the plantaris muscles were taken for analyses of α-actinin-2 and α-actinin-3 (western blot), MyHC composition (SDS-PAGE), and citrate synthase (CS) activity (spectrophotometory). Data were analyzed by two-way ANOVA (age × endurance training). RESULTS: Because no interaction was found, data were interpreted with main effect. The proportion of type IIb MyHC was lower (P < 0.05), and that of type I MyHC was higher (P < 0.05) in old animals than in adult animals, suggesting ageing-induced type II to type I MyHC shift. α-Actinin-3 was lower in old animals than in adult animals (adult: 0.97±0.02 vs. old: 0.84 ± 0.04 OD unit, P < 0.05). No significant difference was found in α-actinin-2 and CS activity between adult and old animals. CS activity was higher (+25%, P < 0.05) in trained animals than in sedentary animals. The proportion of type IIb and IIx MyHC were lower (P < 0.05), and that of type IIa MyHC was higher (P < 0.05) in trained animals than in sedentary animals, indicating a fast to slow shift within type II MyHC isoforms by endurance training. α-Actinin-2 was higher in trained animals than in sedentary animals (sedentary: 0.99±0.07 vs. training: 1.40±0.10 OD unit, P < 0.05). No significant difference was found in α-actinin-3 between trained and sedentary animals. CONCLUSIONS: These results support the α-actinin adaptation at the isoform level and show that the α-actinin-3 expression depends on the amount of type II MyHC, whereas α-actinin-2 expression is associated with improvement of muscular aerobic capacity.

Effects of ageing and endurance exercise training on alpha‐actinin isoforms in rat plantaris muscle

We recently reported that α-actinin adaptation occurs at the isoform level. This study was undertaken to clarify the effects of: (1) ageing-induced shift of myosin heavy chain (MyHC) composition and (2) endurance exercise training on α-actinin isoforms in rat plantaris muscle. Adult (18 mo) and old (28 mo) male Fischer 344 rats were assigned to either sedentary control or endurance exercise training groups. Animals in the training groups ran on a treadmill for 8 week with training intensity adjusted to be equal for adult and old groups. After the training was completed, the plantaris muscles were taken for analyses of α-actinin-2, α-actinin-3, and MyHC composition and metabolic enzyme activities. The proportion of type IIb MyHC was lower, and that of type I MyHC was higher in old animals than in adult animals. α-actinin-3 was significantly lower in old animals than in adult animals. No significant difference was found in α-actinin-2 and citrate synthase (CS) activity between adult and old animals. Citrate synthase activity was higher in trained animals than in sedentary animals. Endurance training produced a fast-to-slow shift within type II MyHC isoforms in both adult and old animals. α-actinin-2 was significantly higher in trained animals than in sedentary animals. No significant difference was found in α-actinin-3 between trained and sedentary animals. These results support the α-actinin adaptation at the isoform level and show that the α-actinin-3 expression depends on the amount of type II MyHC, whereas α-actinin-2 expression is associated with improvement of muscular aerobic capacity.

Response of Alpha-actinin Isoforms to Mechanical Ventilation-induced Diaphragmatic Atrophy

Alpha-actinins are actin-binding proteins and important structural components of the Z-line. In mammalian skeletal muscle, the two isoforms are expressed, α-actinin-2 and α-actinin-3. α-actinin-2 is expressed in all skeletal muscles fibers, whereas α-actinin-3 expression is restricted to the fast fibers only, thus, α-actinin-3 is assumed to be associated with fast muscle fiber function. We previously examined α-actinin-3 level in the soleus muscle by using hindlimb unloading. This type of disuse induces the atrophy particularly in slow muscle fibers. In contrast, fast fibers are also atrophied by diaphragmatic inactivity, indicate that response of α-actinin-3 to atrophic stimuli with mechanical ventilation (MV) may differ from results of locomotor muscle disuse. PURPOSE: To examine the expression levels of α-actinin isoforms in rat diaphragm after MV. METHODS: Male Wistar rats were randomly divided into a control (n=5) or a 12h of MV (n=5) group. Mechanically ventilated animals were anesthetized, tracheostomized, and ventilated with 21% O2. Animals in the control group were acutely anesthetized but were not exposed to MV. After the treatment, diaphragms were removed, frozen with lipid nitrogen and stored at -80°C. Western blotting analysis was performed to Myosin heavy chain (MyHC) composition and expression of α-actinin isoforms. RESULTS: MV resulted in a decrease (14.0%, p < 0.01) in the cross-sectional area of diaphragm. MyHC composition did not differ between MV (I: 21.4, IIa: 19.3, IId/x: 49.2, IIb: 10.4%) and control (I: 22.2, IIa: 18.3, IId/x: 48.3, IIb: 11.2%) animals. Furthermore, there was no significant difference in expression levels of α-actinin isoforms between MV and control groups. CONCLUSION: The expression levels of α-actinin isoforms in rat diaphragm are not changed by the 12-h MV.

α-Actinin 1 and α-actinin 4: Contrasting roles in the survival, motility, and RhoA signaling of astrocytoma cells

α-Actinin is a prominent actin filament associated protein for which different isoforms exist. Here, we have examined whether the two highly homologous non-muscle α-actinin isoforms 1 and 4 exhibit functional differences in astrocytoma cells. The protein levels of these isoforms were differentially regulated during the development and progression of astrocytomas, as α-actinin 1 was higher in astrocytomas compared to normal brains whereas α-actinin 4 was elevated in high-grade astrocytomas compared to normal brains and low grade astrocytomas. RNAi demonstrated contrasted contributions of α-actinin 1 and 4 to the malignant behavior of U-373, U-87 and A172 astrocytoma cells. While α-actinin 1 appeared to favor the expansion of U-373, U-87 and A172 astrocytoma cell populations, α-actinin 4 played this role only for U-373 cells. On the other hand, downregulation of α-actinin 4, but not 1, reduced cell motility, adhesion, cortical actin, and RhoA levels. Finally, in the three astrocytoma cell lines examined, α-actinin 1 and 4 had contrasted biochemical properties as α-actinin 4 was significantly more abundant in the actin cytoskeleton than α-actinin 1. Collectively, these findings suggest that α-actinin 1 and 4 are differentially regulated during the development and progression of astrocytomas because each of these isoforms uniquely contributes to distinct malignant properties of astrocytoma cells.

Histomonas meleagridis possesses three α-actinins immunogenic to its hosts

Histomonas meleagridis is a protozoan parasite known to cause histomonosis (syn. typhlohepatitis) in poultry. Due to the economic losses which the disease entails, there is an urgency to understanding its pathogenesis. In the present investigation, three partial cDNA sequences encoding heterogenic α-actinins previously identified for H. meleagridis were completed and characterized. These three H. meleagridis α-actinin isoforms were named α-actinin1, α-actinin2, and α-actinin3. Through mRNA analysis, it was possible to estimate their expected complete coding lengths to ca. 1.9kb, 3.5kb, and 3kb, respectively. Protein structure predictions of completed cDNA sequences revealed that the three H. meleagridis α-actinins contain two N-terminal calponin homology (CH) domains that bind actin, a central rod domain of varying lengths, and only one C-terminal EF-Hand. The well conserved N- and C-termini of the H. meleagridis α-actinins show amino acid identities between 54% and 82.5% to those of the related trichomonad Trichomonas vaginalis. Additionally, all three α-actinins were shown to immune-react with turkey antiserum, while only the immune reaction of α-actinin2 and α-actinin3 was visible using chicken serum. This demonstrates the antigenic potential of α-actinins in host animals. The identification of α-actinins with varying rod domain lengths within one organism is a relatively new concept, described as yet only for Entamoeba histolytica.

Strength, power, fiber types, and mRNA expression in trained men and women with differentACTN3R577X genotypes

Alpha-actinins are structural proteins of the Z-line. Human skeletal muscle expresses two alpha-actinin isoforms, alpha-actinin-2 and alpha-actinin-3, encoded by their respective genes ACTN2 and ACTN3. ACTN2 is expressed in all muscle fiber types, while only type II fibers, and particularly the type IIb fibers, express ACTN3. ACTN3 (R577X) polymorphism results in loss of alpha-actinin-3 and has been suggested to influence skeletal muscle function. The X allele is less common in elite sprint and power athletes than in the general population and has been suggested to be detrimental for performance requiring high power. The present study investigated the association of ACTN3 genotype with muscle power during 30-s Wingate cycling in 120 moderately to well-trained men and women and with knee extensor strength and fatigability in a subset of 21 men performing isokinetic exercise. Muscle biopsies were obtained from the vastus lateralis muscle to determine fiber-type composition and ACTN2 and ACTN3 mRNA levels. Peak and mean power and the torque-velocity relationship and fatigability output showed no difference across ACTN3 genotypes. Thus this study suggests that R577X polymorphism in ACTN3 is not associated with differences in power output, fatigability, or force-velocity characteristics in moderately trained individuals. However, repeated exercise bouts prompted an increase in peak torque in RR but not in XX genotypes, suggesting that ACTN3 genotype may modulate responsiveness to training. Our data further suggest that alpha-actinins do not play a significant role in determining muscle fiber-type composition. Finally, we show that ACTN2 expression is affected by the content of alpha-actinin-3, which implies that alpha-actinin-2 may compensate for the lack of alpha-actinin-3 and hence counteract the phenotypic consequences of the deficiency.

Drosophila α-actinin in ovarian follicle cells is regulated by EGFR and Dpp signalling and required for cytoskeletal remodelling

α-Actinin is an evolutionarily conserved actin filament crosslinking protein with functions in both muscle and non-muscle cells. In non-muscle cells, interactions between α-actinin and its many binding partners regulate cell adhesion and motility. In Drosophila, one non-muscle and two muscle-specific α-actinin isoforms are produced by alternative splicing of a single gene. In wild-type ovaries, α-actinin is ubiquitously expressed. The non-muscle α- actinin mutant Actn Δ233 , which is viable and fertile, lacks α-actinin expression in ovarian germline cells, while somatic follicle cells express α-actinin at late oogenesis. Here we show that this latter population of α-actinin, termed FC-α-actinin, is absent from the dorsoanterior follicle cells, and we present evidence that this is the result of a negative regulation by combined Epidermal growth factor receptor (EGFR) and Decapentaplegic signalling. Furthermore, EGFR signalling increased the F-actin bundling activity of ectopically expressed muscle-specific α-actinin. We also describe a novel morphogenetic event in the follicle cells that occurs during egg elongation. This event involves a transient repolarisation of the basal actin fibres and the assembly of a posterior β-integrin-dependent adhesion site accumulating α-actinin and Enabled. Clonal analysis using Actn null alleles demonstrated that although α-actinin was not necessary for actin fibre formation or maintenance, the cytoskeletal remodelling was perturbed, and Enabled did not localise in the posterior adhesion site. Nevertheless, epithelial morphogenesis proceeded normally. This work provides the first evidence that α-actinin is involved in the organisation of the cytoskeleton in a non-muscle tissue in Drosophila.

Multivalent Interactions of Calcium/Calmodulin-dependent Protein Kinase II with the Postsynaptic Density Proteins NR2B, Densin-180, and α-Actinin-2

Dendritic calcium/calmodulin-dependent protein kinase II (CaMKII) is dynamically targeted to the synapse. We show that CaMKIIalpha is associated with the CaMKII-binding proteins densin-180, the N-methyl-D-aspartate receptor NR2B subunit, and alpha-actinin in postsynaptic density-enriched rat brain fractions. Residues 819-894 within the C-terminal domain of alpha-actinin-2 constitute the minimal CaMKII-binding domain. Similar amounts of Thr286-autophosphorylated CaMKIIalpha holoenzyme [P-T286]CaMKII bind to alpha-actinin-2 as bind to NR2B (residues 1260-1339) or to densin-180 (residues 1247-1495) in glutathione-agarose cosedimentation assays, even though the CaMKII-binding domains share no amino acid sequence similarity. Like NR2B, alpha-actinin-2 binds to representative splice variants of each CaMKII gene (alpha, beta, gamma, and delta), whereas densin-180 binds selectively to CaMKIIalpha. In addition, C-terminal truncated CaMKIIalpha monomers can interact with NR2B and alpha-actinin-2, but not with densin-180. Soluble alpha-actinin-2 does not compete for [P-T286]CaMKII binding to immobilized densin-180 or NR2B. However, soluble densin-180, but not soluble NR2B, increases CaMKII binding to immobilized alpha-actinin-2 by approximately 10-fold in a PDZ domain-dependent manner. A His6-tagged NR2B fragment associates with GST-densin or GST-actinin but only in the presence of [P-T286]CaMKII. Similarly, His6-tagged densin-180 or alpha-actinin fragments associate with GST-NR2B in a [P-T286]CaMKII-dependent manner. In addition, GST-NR2B and His6-tagged alpha-actinin can bind simultaneously to monomeric CaMKII subunits. In combination, these data support a model in which [P-T286]CaMKIIalpha can simultaneously interact with multiple dendritic spine proteins, possibly stabilizing the synaptic localization of CaMKII and/or nucleating a multiprotein synaptic signaling complex.

Differential expression of α-actinin isoforms in dilated cardiomyopathy (DCM)

Differential expression of α-actinin isoforms in dilated cardiomyopathy (DCM)

Abnormalities in the expression of nebulin in chromosome-2 linked nemaline myopathy.

Nemaline myopathy is clinically and genetically heterogeneous. The most common autosomal recessive form affecting infants (NEM2) links to chromosome 2q, and is caused by mutations in the gene for nebulin. We have examined the immunocytochemical expression of nebulin in skeletal muscle in 11 cases of nemaline myopathy, from ten families, with linkage compatible to chromosome 2q.22, the locus for nebulin. Mutations in the gene for nebulin have been found in eight of these cases. Immunolabelling with polyclonal antibodies to C-terminal regions of nebulin was compared with antibodies to fibre-type-specific myofibrillar proteins, including myosin heavy chain isoforms and alpha-actinin isoforms. No cases showed a complete absence of C-terminal nebulin, and no enhancement of labelling of the rods was seen with conventional fluorescence microscopy. In control muscle an antibody to the M176-181 repeat region of nebulin showed higher expression in fibres with slow myosin, while ones to the serine-rich domain and to the SH3 domain showed uniform expression. In some cases of nemaline myopathy differences in these patterns were observed. Two siblings with a homozygous mutation in exon 185, that produces a stop codon, showed an absence of labelling only with the SH3 antibody, and other cases showed uneven labelling with this antibody or some fibres devoid of label. Fibre type correlations also showed differences from controls, as some fibres had a fast isoform of one protein but a slow isoform of another. These results indicate that analysis of nebulin expression may detect abnormalities in some cases linked to the corresponding locus and may help to direct molecular analysis. In addition, they may also be relevant to studies of fibre type plasticity and diversity in nemaline myopathy.

Isoforms of alpha-actinin from cardiac, smooth, and skeletal muscle form polar arrays of actin filaments.

We have used a positively charged lipid monolayer to form two-dimensional bundles of F-actin cross-linked by alpha-actinin to investigate the relative orientation of the actin filaments within them. This method prevents growth of the bundles perpendicular to the monolayer plane, thereby facilitating interpretation of the electron micrographs. Using alpha-actinin isoforms isolated from the three types of vertebrate muscle, i.e., cardiac, skeletal, and smooth, we have observed almost exclusively cross-linking between polar arrays of filaments, i.e., actin filaments with their plus ends oriented in the same direction. One type of bundle can be classified as an Archimedian spiral consisting of a single actin filament that spirals inward as the filament grows and the bundle is formed. These spirals have a consistent hand and grow to a limiting internal diameter of 0.4-0.7 microm, where the filaments appear to break and spiral formation ceases. These results, using isoforms usually characterized as cross-linkers of bipolar actin filament bundles, suggest that alpha-actinin is capable of cross-linking actin filaments in any orientation. Formation of specifically bipolar or polar filament arrays cross-linked by alpha-actinin may require additional factors that either determine the filament orientation or restrict the cross-linking capabilities of alpha-actinin.

Human Skeletal Muscle-Specific α-Actinin-2 and -3 Isoforms Form Homodimers and Heterodimersin Vitroandin Vivo

α-actinins belong to a family of actin-binding and crosslinking proteins and are expressed in many different cell types. Multiple isoforms of α-actinin are found in humans and are encoded by at least four distinct genes. Human skeletal muscle contains two sarcomeric isoforms, α-actinin-2 and -3. Previous studies have shown that the α-actinins function as anti-parallel homodimers but the question of heterodimer formation between two different isoforms expressed in the same cell type has not been explored. To address this issue, we expressed both α-actinin-2 and -3in vitroand were able to detect their interaction by both blot overlay and co-immunoprecipitation methods. We were also able to demonstrate the presence of heterodimersin vivoin human skeletal muscle and in COS-1 cells transiently transfected with both isoforms. Our results clearly demonstrate the potential for α-actinin isoforms to form heterodimers which might have unique functional characteristics.

Projection Image of Smooth Muscle α-Actinin from Two-dimensional Crystals Formed on Positively Charged Lipid Layers

Two-dimensional crystalline arrays of chicken gizzard α-actinin have been formed on positively charged lipid layers. This is the first reported crystallization of α-actinin. The crystals have unit cell dimensions of a = 248 Å, b = 194 Å, γ = 106° and contain two α-actinin molecules. The two-sided group is P21. Projection images obtained from electron micrographs of negatively stained crystals have been calculated to a resolution of 25 Å. These images reveal a complex substructure. The molecule in projection is 340 Å in length and has 12 density peaks that probably correspond to protein domains. A pair of peaks is found at each end of the molecule, these probably correspond to the actin binding region. Eight peaks are observed in the central, rod-shaped region, these may correspond to the spectrin-like repeats predicted from the amino acid sequence. However, these eight central peaks are not arranged in four pairs but, instead, consist of three central pairs flanked at either end by a single peak, which appears larger and denser in projection than the three central pairs. The individual α-actinin molecules in projection lack 2-fold symmetry suggesting that either smooth muscle α-actinin lacks a molecular 2-fold symmetry axis or that the molecular 2-fold is not parallel with the crystallographic 2-fold axis. The ends of the molecule have different appearance in projection, suggesting that the molecule is twisted about the long axis. A hypothesis is proposed to explain the variations in molecular length and Ca2+ sensitivity between α-actinin isoforms.