A rapid degradation of calponin 2 is required for cytokinesis.

Calponin 2 is an actin cytoskeleton-associated protein and plays a role in regulating cell motility-related functions such as phagocytosis, migration, and division. We previously reported that overexpression of calponin 2 inhibits the rate of cell proliferation. To investigate the underlying mechanism, our present study found that the levels of endogenous calponin 2 in NIH3T3 and HEK293 cells rapidly decreased before cell division characterized by an absence at the actin contractile ring. In cells lacking endogenous calponin 2, transfective expression of GFP-fusion calponin 2 inhibited cell proliferation similar to that of nonfusion calponin 2. Fluorescent imaging studies of mitotic cells indicated that a proper level of calponin 2 expression and effective degradation during cytokinesis are necessary for normal cell division. Computer-assisted dynamic image analysis of dividing cells revealed that overexpression of calponin 2 significantly affects motility and shape behaviors of cells only on the interval from the start of anaphase to the start of cytokinesis, i.e., the pre-cytokinesis phase, but not on the interval from the start of cytokinesis to 50% completion of cytokinesis. The pre-cytokinesis degradation of calponin 2 was attenuated by MG132 inhibition of the ubiquitin proteasome and inhibitor of protein kinase C (PKC), suggesting that PKC phosphorylation-triggered degradation of calponin 2 could determine the rate of cytokinesis. The novel role of calponin 2 in regulating the rate of cytokinesis may be targeted for therapeutic applications such as in an inhibition of malignant tumor growth.

Mechanoregulation of SM22α/Transgelin.

SM22α, also named transgelin, is an actin filament-associated protein in smooth muscle and fibroblasts. Three decades after its discovery, the biological function of SM22α remains under investigation. Here we report a novel finding that the expression and degradation of SM22α/transgelin are regulated by mechanical tension. Following a mass spectrometry identification of SM22α degradation in isolated and tension-unloaded mouse aorta, we developed specific monoclonal antibodies to study the regulation of SM22α in human fetal lung myofibroblast line MRC-5 and primary cultures of neonatal mouse skin fibroblasts. The level of SM22α is positively related to the mechanical tension in the cytoskeleton produced by the myosin II motor in response to the stiffness of the culture matrix. Quantitative reverse transcription polymerase chain reaction demonstrated that the expression of SM22α is regulated at the transcriptional level. This mechanical regulation resembles that of calponin 2, another actin filament-associated protein. Immunofluorescent staining co-localized SM22α with F-actin, myosin, and calponin 2 in mouse skin fibroblasts. The close phylogenetic relationship between SM22α and the calponin family supports that SM22α is a calponin-like regulatory protein. The level of SM22α is decreased in skin fibroblasts isolated from calponin 2 knockout mice, suggesting interrelated regulation and function of the two proteins. On the other hand, SM22α expression was maximized at a matrix stiffness higher than that for calponin 2 in the same cell type, indicating differentiated regulation and tension responsiveness. The novel mechanoregulation of SM22α/transgelin lays the groundwork for understanding its cellular functions.

Deletion of calponin 2 in macrophages attenuates the severity of inflammatory arthritis in mice.

Calponin is an actin cytoskeleton-associated protein that regulates motility-based cellular functions. Three isoforms of calponin are present in vertebrates, among which calponin 2 encoded by the Cnn2 gene is expressed in multiple types of cells, including blood cells from the myeloid lineage. Our previous studies demonstrated that macrophages from Cnn2 knockout (KO) mice exhibit increased migration and phagocytosis. Intrigued by an observation that monocytes and macrophages from patients with rheumatoid arthritis had increased calponin 2, we investigated anti-glucose-6-phosphate isomerase serum-induced arthritis in Cnn2-KO mice for the effect of calponin 2 deletion on the pathogenesis and pathology of inflammatory arthritis. The results showed that the development of arthritis was attenuated in systemic Cnn2-KO mice with significantly reduced inflammation and bone erosion than that in age- and stain background-matched C57BL/6 wild-type mice. In vitro differentiation of calponin 2-null mouse bone marrow cells produced fewer osteoclasts with decreased bone resorption. The attenuation of inflammatory arthritis was confirmed in conditional myeloid cell-specific Cnn2-KO mice. The increased phagocytotic activity of calponin 2-null macrophages may facilitate the clearance of autoimmune complexes and the resolution of inflammation, whereas the decreased substrate adhesion may reduce osteoclastogenesis and bone resorption. The data suggest that calponin 2 regulation of cytoskeleton function plays a novel role in the pathogenesis of inflammatory arthritis, implicating a potentially therapeutic target.

Functional Basis of Three New Recessive Mutations of Slow Skeletal Muscle Troponin T Found in Non-Amish TNNT1 Nemaline Myopathies.

Troponin T (TnT) is the tropomyosin (Tm)-binding and thin filament-anchoring subunit of troponin and plays a central role in striated muscle contraction. A nonsense mutation in exon 11 of the TNNT1 gene encoding slow skeletal muscle troponin T (ssTnT) truncating the polypeptide chain at Glu(180) causes a lethal recessive nemaline myopathy (NM) in the Amish (ANM). More TNNT1 NM mutations have been reported recently with similar recessive phenotypes. A nonsense mutation in exon 9 causes truncation at Ser(108), and a splicing site mutation causes truncation at Leu(203). Another splicing site mutation causes an internal deletion of the 39 exon 8-encoded amino acids. We engineered and characterized these ssTnT mutants to demonstrate that the Ser(108) truncation exhibits a Tm binding affinity lower than that of the ANM Glu(180) truncation, indicating a partial loss of Tm-binding site 1. Despite the presence of Tm-binding sites 1 and 2, ssTnT truncated at Leu(203) binds Tm with decreased affinity, consistent with its recessive NM phenotype and the requirement of troponin complex formation for high-affinity binding of TnT to Tm. The exon 8-deleted ssTnT has a partial loss of Tm-binding site 1 but retains high-affinity Tm-binding site 2. However, exon 8-deleted ssTnT exhibits a dramatically diminished Tm binding affinity, indicating a long-range conformational effect of this middle region deletion. Predicted from the TnT structure-function relationship, removal of the N-terminal variable region partially rescued this negative impact. These novel findings lay a foundation for understanding the pathogenesis of TNNT1 myopathies and provide insights into the development of targeted treatment.

Deletion of Calponin 2 in Mouse Fibroblasts Increases Myosin II-Dependent Cell Traction Force.

Cell traction force (CTF) plays a critical role in controlling cell shape, permitting cell motility, and maintaining cellular homeostasis in many biological processes such as angiogenesis, development, wound healing, and cancer metastasis. Calponin is an actin filament-associated cytoskeletal protein in smooth muscles and multiple types of non-muscle cells. An established biochemical function of calponin is the inhibition of myosin ATPase in smooth muscle cells. Vertebrates have three calponin isoforms. Among them, calponin 2 is expressed in epithelial cells, endothelial cells, macrophages, myoblasts, and fibroblasts and plays a role in regulating cytoskeleton activities such as cell adhesion, migration, and cytokinesis. Knockout (KO) of the gene encoding calponin 2 (Cnn2) in mice increased cell motility, suggesting a function of calponin 2 in modulating CTF. In this study, we examined fibroblasts isolated from Cnn2 KO and wild-type (WT) mice using CTF microscopy. Primary mouse fibroblasts were cultured on polyacrylamide gel substrates embedded with fluorescent beads to measure root-mean-square traction, total strain energy, and net contractile movement. The results showed that calponin 2-null fibroblasts exhibit traction force greater than that of WT cells. Adherent calponin 2-null fibroblasts de-adhered faster than the WT control during mild trypsin treatment, consistent with an increased CTF. Blebbistatin, an inhibitor of myosin II ATPase, is more effective upon an alteration in cell morphology when calponin 2 is present in WT fibroblasts than that on Cnn2 KO cells, indicating their additive effects in inhibiting myosin motor activity. The novel finding that calponin 2 regulates myosin-dependent CTF in non-muscle cells demonstrates a mechanism for controlling cell motility-based functions.

Mechanoregulation of h2-calponin gene expression and the role of Notch signaling.

The essential role of mechanical signals in regulating the function of living cells is universally observed. However, how mechanical signals are transduced in cells to regulate gene expression is largely unknown. We previously demonstrated that the gene encoding h2-calponin (Cnn2) is sensitively regulated by mechanical tension. In the present study, mouse genomic DNA containing the Cnn2 promoter was cloned, and a nested set of 5' truncations was studied. Transcriptional activity of the Cnn2 promoter-reporter constructs was examined in transfected NIH/3T3, HEK293, and C2C12 cells for their responses to the stiffness of culture substrate. The results showed significant transcriptional activities of the -1.00- and -1.24-kb promoter constructs, whereas the -0.61-kb construct was inactive. The -1.38-, -1.57-, and -2.12-kb constructs showed higher transcriptional activity, whereas only the -1.57- and -2.12-kb constructs exhibited repression of expression when the host cells were cultured on low stiffness substrate. Internal deletion of the segment between -1.57 and -1.38 kb in the -2.12-kb promoter construct abolished the low substrate stiffness-induced repression. Site-specific deletion or mutation of an HES-1 transcription factor binding site in this region also abolished this repression effect. The level of HES-1 increased in cells cultured under a low tension condition, corresponding to the down-regulation of h2-calponin. h2-Calponin gene expression is further affected by the treatment of cells with Notch inhibitor and activator, suggesting an upstream signaling mechanism.

Toad heart utilizes exclusively slow skeletal muscle troponin T: an evolutionary adaptation with potential functional benefits.

The three isoforms of vertebrate troponin T (TnT) are normally expressed in a muscle type-specific manner. Here we report an exception that the cardiac muscle of toad (Bufo) expresses exclusively slow skeletal muscle TnT (ssTnT) together with cardiac forms of troponin I and myosin as determined using immunoblotting, cDNA cloning, and/or LC-MS/MS. Using RT-PCR and 3'- and 5'-rapid amplification of cDNA ends on toad cardiac mRNA, we cloned full-length cDNAs encoding two alternatively spliced variants of ssTnT. Expression of the cloned cDNAs in Escherichia coli confirmed that the toad cardiac muscle expresses solely ssTnT, predominantly the low molecular weight variant with the exon 5-encoded NH(2)-terminal segment spliced out. Functional studies were performed in ex vivo working toad hearts and compared with the frog (Rana) hearts. The results showed that toad hearts had higher contractile and relaxation velocities and were able to work against a significantly higher afterload than that of frog hearts. Therefore, the unique evolutionary adaptation of utilizing exclusively ssTnT in toad cardiac muscle corresponded to a fitness value from improving systolic function of the heart. The data demonstrated a physiological importance of the functional diversity of TnT isoforms. The structure-function relationship of TnT may be explored for the development of new treatment of heart failure.

Nonmyofilament-associated troponin T fragments induce apoptosis.

Troponin T (TnT) is a striated muscle-specific protein and an abundant component of the myofilaments. Nonmyofilament-associated TnT is rapidly degraded in myocytes, implying an importance in the maintenance of the cellular environment. However, if the level of nonmyofilament-associated TnT or TnT fragments exceeds the degradation capacity, it may cause cytotoxicity. To investigate this hypothesis, we constructed bicistronic vectors to express different portions of TnT polypeptide chain, together with nonfusion green fluorescent protein as a tracer for the transfection. Cytotoxicity of the TnT fragments was studied through forced expression in C(2)C(12) myoblasts and human embryonic kidney-293 nonmuscle cells and examination of the viability of the transfected cells. The results demonstrated that, in the absence of myofilaments, the conserved COOH-terminal and middle fragments of TnT were highly effective on inducing cell death via apoptosis, whereas the NH(2)-terminal variable region was not. As combined effects, nonmyofilament-associated intact cardiac TnT and a COOH-terminal truncated slow TnT fragment found in Amish nemaline myopathy exhibited intermediate cytotoxicity. A particular significance of this finding is that peak releases of TnT or TnT fragments from decomposition of a large number of myofibrils in acute myocardial infarction may breach the cellular protection of proteolytic degradation and result in apoptosis as a potential cause for the loss of cardiomyocytes.

Myofilament incorporation determines the stoichiometry of troponin I in transgenic expression and the rescue of a null mutation.

The highly organized contractile machinery in skeletal and cardiac muscles requires an assembly of myofilament proteins with stringent stoichiometry. To understand the maintenance of myofilament protein stoichiometry under dynamic protein synthesis and catabolism in muscle cells, we investigated the equilibrium of troponin I (TnI) in mouse cardiac muscle during developmental isoform switching and in under- and over-expression models. Compared with the course of developmental TnI isoform switching in normal hearts, the postnatal presence of slow skeletal muscle TnI lasted significantly longer in the hearts of cardiac TnI (cTnI) knockout (cTnI-KO) mice, in which the diminished synthesis was compensated by prolonging the life of myofilamental TnI. Transgenic postnatal expression of an N-terminal truncated cTnI (cTnI-ND) using alpha-myosin heavy chain promoter effectively rescued the lethality of cTnI-KO mice and shortened the postnatal presence of slow TnI in cardiac muscle. cTnI-KO mice rescued with different levels of cTnI-ND over-expression exhibited similar levels of myocardial TnI comparable to that in wild type hearts, demonstrating that excessive synthesis would not increase TnI stoichiometry in the myofilaments. Consistently, haploid under-expression of cTnI in heterozygote cTnI-KO mice was sufficient to sustain the normal level of myocardial cTnI, indicating that cTnI is synthesized in excess in wild type cardiomyocytes. Altogether, these observations suggest that under wide ranges of protein synthesis and turnover, myofilament incorporation determines the stoichiometry of troponin subunits in muscle cells.

Cytoskeletal Tropomyosin as a Biomarker in Clostridium difficile Infection.

BACKGROUND: Current diagnostics of Clostridium difficile infection (CDI) heavily relies on detection of the disease-causing organism. The objective of this study was to investigate a cytoskeletal protein, tropomyosin (Tpm), as a CDI biomarker. METHODS: Fecal Tpm was tested by monoclonal antibodies (mAbs) in a 12-month prospective study. Remnant diarrheal clinical specimens and relevant clinical data were collected. The CDI positive (CDI+, n = 230) and CDI negative (CDI-, n = 228) groups were composed of samples testing positive or negative by polymerase chain reaction (PCR) (Xpert® C. difficile/Epi, Cepheid), respectively. The other enteric pathogen (OEP) group (n = 52) was composed of specimens tested for the presence of other enteric pathogens or parasites by routine testing methods. Extracted fecal Tpm was detected by Western blot and the results were correlated with CDI based on clinical and microbiology laboratory data. RESULTS: A total of 510 stool specimens were tested. Tpm is not stable in stool, suggesting the utility of fresh specimens. In the CDI+ group, specificity and sensitivity of Tpm detection in correlation with a CDI were 93.2% and 53.7%, respectively, when only "true CDI" and "not CDI" were analyzed (110 samples). For CDI+ samples, 23% did not satisfy CDI clinical signs. Tpm positives in the CDI- group (8.3%) had inflammatory bowel diseases. CONCLUSION: Tpm has a potential role as a CDI biomarker in combination with C. difficile PCR and an appropriate clinical evaluation. However, non-muscle Tpm, as a biomarker for CDI, suffers from a low sensitivity in our study. Therefore further investigation using larger cohorts is needed.

Restricted N-terminal truncation of cardiac troponin T: a novel mechanism for functional adaptation to energetic crisis.

The N-terminal variable region of cardiac troponin T (TnT) is a regulatory structure that can be selectively removed during myocardial ischaemia reperfusion by mu-calpain proteolysis. Here we investigated the pathophysiological significance of this post-translational modification that removes amino acids 1-71 of cardiac TnT. Working heart preparations were employed to study rat acute myocardial infarction and transgenic mouse hearts over-expressing the N-terminal truncated cardiac TnT (cTnT-ND). Ex vivo myocardial infarction by ligation of the left anterior descending coronary artery induced heart failure and produced cTnT-ND not only in the infarct but also in remote zones, including the right ventricular free wall, indicating a whole organ response in the absence of systemic neurohumoral mechanisms. Left ventricular pressure overload in mouse working hearts produced increased cTnT-ND in both ventricles, suggesting a role of haemodynamic stress in triggering an acute whole organ proteolytic regulation. Transgenic mouse hearts in which the endogenous intact cardiac TnT was partially replaced by cTnT-ND showed lowered contractile velocity. When afterload increased from 55 mmHg to 90 mmHg, stroke volume decreased in the wild type but not in the transgenic mouse hearts. Correspondingly, the left ventricular rapid-ejection time of the transgenic mouse hearts was significantly longer than that of wild type hearts, especially at high afterload. The restricted deletion of the N-terminal variable region of cardiac troponin T demonstrates a novel mechanism by which the thin filament regulation adapts to sustain cardiac function under stress conditions.

Cytoskeletal tension regulates both expression and degradation of h2-calponin in lung alveolar cells.

Calponin is an actin filament-associated regulatory protein, and its h2 isoform is expressed in lung alveolar epithelial cells under postnatal upregulation during lung development corresponding to the commencement of respiratory expansion. Consistent with this correlation to mechanical tension, the expression of h2-calponin in alveolar cells is dependent on substrate stiffness and cytoskeleton tension. The function of h2-calponin in the stability of actin cytoskeleton implicates a role in balancing the strength and compliance of alveoli. An interesting finding is a rapid degradation of h2-calponin in lung after prolonged deflation, which is prevented by inflation of the lung to the in situ expanded volume. Decreasing mechanical tension in cultured alveolar cells by reducing the dimension of culture matrix reproduced the degradation of h2-calponin. Inhibition of myosin II ATPase also resulted in the degradation of h2-calponin in alveolar cells, showing a determining role of the tension in the actin cytoskeleton. Alveolar cells statically cultured on silicon rubber membrane build high tension in the cytoskeleton corresponding to a high expression of h2-calponin. Chronic cyclic stretching of cells on the membrane did not increase but decreased the expression of h2-calponin. This finding suggests that when cellular structure adapts to the stretched dimension, cyclic relaxations periodically release cytoskeleton tension and lower the total amount of tension that the cell senses over time. Therefore, the isometric tension, other than tension dynamics, determines the expression of h2-calponin. The tension regulation of h2-calponin synthesis and degradation demonstrates a novel mechanical regulation of cellular biochemistry.

Role of H2-calponin in regulating macrophage motility and phagocytosis.

The actin cytoskeleton plays a major role in cell motility that is essential for the function of phagocytes. Calponin is an actin-associated regulatory protein. Here we report the finding of significant levels of the h2 isoform of calponin in peripheral blood cells of myeloid lineage. To study the functional significance, h2-calponin gene (Cnn2) interrupted mice were constructed. Germ line transmission of the Cnn2-flox-neo allele was obtained in chimeras from two independent clones of targeted embryonic stem cells. The insertion of the neo(R) cassette into intron 2 of the Cnn2 gene resulted in a significant knockdown of h2-calponin expression. Removing the frt-flanked neo(R) cassette by FLP1 recombinase rescued the knockdown effect. Cre recombinase-induced deletion of the loxP-flanked exon 2 eliminated the expression of h2-calponin protein. H2-calponin-free mice showed reduced numbers of peripheral blood neutrophils and monocytes. H2-calponin-free macrophages demonstrated a higher rate of proliferation and faster migration than that of h2-calponin-positive cells, consistent with a faster diapedesis of peripheral monocytes and neutrophils. H2-calponin-free macrophages showed reduced spreading in adhesion culture together with decreased tropomyosin in the actin cytoskeleton. The lack of h2-calponin also significantly increased macrophage phagocytotic activity, suggesting a novel mechanism to regulate phagocyte functions.

Microtiter plate monoclonal antibody epitope analysis of Ca2+- and Mg2+-induced conformational changes in troponin C.

Spectroscopic methods such as circular dichroism and Förster resonance energy transfer are current approaches for monitoring protein conformational changes. Those analyses require special equipment and expertise. The need for fluorescence labeling of the protein may interfere with the native structure. We have developed a microtiter plate-based monoclonal antibody (mAb) epitope analysis to detect protein conformational changes in a high throughput manner. This method is based on the concept that the affinity of the antigen-binding site of an antibody for the specific antigenic epitope will change when the 3-D structure of the epitope changes. The effectiveness of this approach was demonstrated in the present study on troponin C (TnC), an allosteric protein in the Ca(2+) regulatory system of striated muscle. Using TnC purified by a highly effective rapid procedure and mAbs developed against epitopes in the N- and C-domains of TnC enzyme-linked immunosorbant assay (ELISA) clearly detected Ca(2+)-induced conformational changes in both the N-terminal regulatory domain and the C-terminal structural domain of TnC. On the other hand, Mg(2+)-binding to the C-domain of TnC resulted in a long-range effect on the N-domain conformation, indicating a functional significance of Ca(2+)-Mg(2+) exchange at the C-domain metal ion-binding sites. In addition to further understanding of the structure-function relationship of TnC, the data demonstrate that the mAb epitope analysis provides a simple high throughput method for monitoring 3-D structural changes in native proteins under physiological condition and has broad applications in protein structure-function relationship studies.

Proteolytic N-terminal truncation of cardiac troponin I enhances ventricular diastolic function.

Besides the core structure conserved in all troponin I isoforms, cardiac troponin I (cTnI) has an N-terminal extension that contains phosphorylation sites for protein kinase A under beta-adrenergic regulation. A restricted cleavage of this N-terminal regulatory domain occurs in normal cardiac muscle and is up-regulated during hemodynamic adaptation (Z.-B. Yu, L.-F. Zhang, and J.-P. Jin (2001) J. Biol. Chem. 276, 15753-15760). In the present study, we developed transgenic mice overexpressing the N-terminal truncated cTnI (cTnI-ND) in the heart to examine its biochemical and physiological significance. Ca(2+)-activated actomyosin ATPase activity showed that cTnI-ND myofibrils had lower affinity for Ca(2+) than controls, similar to the effect of isoproterenol treatment. In vivo and isolated working heart experiments revealed that cTnI-ND hearts had a significantly faster rate of relaxation and lower left ventricular end diastolic pressure compared with controls. The higher baseline relaxation rate of cTnI-ND hearts was at a level similar to that of wild type mouse hearts under beta-adrenergic stimulation. The decrease in cardiac output due to lowered preload was significantly smaller for cTnI-ND hearts compared with controls. These findings indicate that removal of the N-terminal extension of cTnI via restricted proteolysis enhances cardiac function by increasing the rate of myocardial relaxation and lowering left ventricular end diastolic pressure to facilitate ventricular filling, thus resulting in better utilization of the Frank-Starling mechanism.

h2-Calponin is regulated by mechanical tension and modifies the function of actin cytoskeleton.

Calponin is an extensively studied actin-binding protein, but its function is not well understood. Among three isoforms of calponin, h2-calponin is found in both smooth muscle and non-muscle cells. The present study demonstrates that epidermal keratinocytes and fibroblast cells express significant amounts of h2-calponin. The expression of h2-calponin is cell anchorage-dependent. The levels of h2-calponin decrease when cells are rounded up and remain low when cells are prevented from adherence to a culture dish. h2-calponin expression resumes after the floating cells are allowed to form a monolayer in plastic dish. Cell cultures on polyacrylamide gels of different stiffness demonstrated that h2-calponin expression is affected by the mechanical properties of the culture matrix. When cells are cultured on soft gel that applies less traction force to the cell and, therefore, lower mechanical tension in the cytoskeleton, the level of h2-calponin is significantly lower than that in cells cultured on hard gel or rigid plastic dish. Force-expression of h2-calponin enhanced the resistance of the actin filaments to cytochalasin B treatment. Keratinocyte differentiation is accompanied by a mechanical tension-related up-regulation of h2-calponin. Lowering the tension of actin cytoskeleton by inhibiting non-muscle myosin II ATPase decreased h2-calponin expression. In contrast to the mechanical tension regulation of endogenous h2-calponin, the expression of h2-calponin using a cytomegalovirus promotor was independent of the stiffness of culture matrix. The results suggest that h2-calponin represents a novel manifestation of mechanical tension responsive gene regulation that may modify cytoskeleton function.

Interactions between nebulin-like motifs and thin filament regulatory proteins.

Nebulin (600-900 kDa) and nebulette (107-109 kDa) are two homologous thin filament-associated proteins in skeletal and cardiac muscles, respectively. Both proteins are capped with a unique region at the amino terminus as well as a serine-rich linker domain and SH3 domains at the COOH terminus. Their significant size difference is attributed to the length of the central region wherein both proteins are primarily composed of approximately 35 amino acid repeats termed nebulin-like repeats or motifs. These motifs are marked by a conserved SXXXY sequence and high affinity binding to F-actin. To further characterize the effects that nebulin-like proteins may have on the striated muscle thin filament, we have cloned, expressed, and purified a five-motif chicken nebulette fragment and tested its interaction with the thin filament regulatory proteins. Both tropomyosin and troponin T individually bound the nebulette fragment, although the affinity of this interaction was significantly increased when tropomyosin-troponin T was tested as a binary complex. The addition of troponin I to the tropomyosin-troponin T complex decreased the binding to the nebulette fragment, indicating an involvement of the conserved T2 region of troponin T in this interaction. F-actin cosedimentation demonstrated that the nebulette fragment was able to significantly increase the affinity of the tropomyosin-troponin assembly for F-actin. The relationships provide a means for nebulin-like motifs to participate in the allosteric regulation of striated muscle contraction.

Developmentally regulated expression of calponin isoforms and the effect of h2-calponin on cell proliferation.

h2-calponin is found in both smooth muscle and nonmuscle cells, and its function remains to be established. Western blots with specific monoclonal antibodies detected significant expression of h2-calponin in the growing embryonic stomach and urinary bladder and the early pregnant uterus. Although the expression of h1-calponin is upregulated in the stomach and bladder during postnatal development, the expression of h2-calponin is decreased to low levels in quiescent smooth muscle cells. To investigate a hypothesis that h2-calponin regulates the function of the actin cytoskeleton during cytokinesis, a smooth muscle-originated cell line (SM3) lacking calponin was transfected to express either sense or antisense h2-calponin cDNA and the effects on the rates of cell proliferation were examined. Both stable and transient sense cDNA-transfected cells had a significantly decreased proliferation rate compared with the antisense cDNA-transfected or nontransfected cells. Immunofluorescence microscopy showed that the force-expressed h2-calponin was associated with actin-tropomyosin microfilaments. The number of binuclear cells was significantly greater in the sense cDNA-transfected culture, in which h2-calponin was concentrated in a nuclear ring structure formed by actin filaments. The results suggest that h2-calponin may regulate cytokinesis by inhibiting the activity of the actin cytoskeleton.

Truncation by Glu180 nonsense mutation results in complete loss of slow skeletal muscle troponin T in a lethal nemaline myopathy.

A lethal form of nemaline myopathy, named "Amish Nemaline Myopathy" (ANM), is linked to a nonsense mutation at codon Glu180 in the slow skeletal muscle troponin T (TnT) gene. We found that neither the intact nor the truncated slow TnT protein was present in the muscle of patients with ANM. The complete loss of slow TnT is consistent with the observed recessive pattern of inheritance of the disease and indicates a critical role of the COOH-terminal T2 domain in the integration of TnT into myofibrils. Expression of slow and fast isoforms of TnT is fiber-type specific. The lack of slow TnT results in selective atrophy of type 1 fibers. Slow TnT confers a higher Ca2+ sensitivity than does fast TnT in single fiber contractility assays. Despite the lack of slow TnT, individuals with ANM have normal muscle power at birth. The postnatal onset and infantile progression of ANM correspond to a down-regulation of cardiac and embryonic splice variants of fast TnT in normal developing human skeletal muscle, suggesting that the fetal TnT isoforms complement slow TnT. These results lay the foundation for understanding the molecular pathophysiology and the potential targeted therapy of ANM.