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Recombinant production and biochemical analysis of actin mutants has been hampered by the fact that actin has an absolute requirement for the eukaryotic chaperone CCT to reach its native state. We therefore have developed a method to rapidly screen the folding capacity and functionality of actin variants, by combining in vitro expression of labelled actin with analysis on native gels, band shift assays or copolymerization tests. Additionally, we monitor, using immuno-fluorescence, incorporation of actin variants in cytoskeletal structures in transfected cells. We illustrate the method by two examples. In one we show that tagged versions of actin do not always behave native-like and in the other we study some of the molecular defects of three &bgr;-actin mutants that have been associated with diseases.

The heterohexameric chaperone protein prefoldin can capture unfolded actin and bind with cytosolic chaperonin (c-cpn) wherein target proteins are being transferred to. The prefoldin gene also has a role in chaperonin-mediated folding. Is also promotes folding in an environment with various pathways by directing target proteins to c-cpn. All of the said pathways rely on the binding and release of prefoldin by chaperones.

In vitro experiments involving sequence and mass analysis are performed to study the role played by the protein cofactor A in the protein folding of the beta-tubulin chain. Results indicate that cofactor A interacts with a folding intermediate of beta-tubulin released by the chaperon containing TCP-1. This finding suggests that cofactor A is also a chaperone which induces tubulin folding.

The complete pathway leading to the generation of exchange-competent beta-tubulin is identified. ATP-dependent interaction with cytosolic chaperonin results in the production of folding intermediates which interact sequentially with four proteins. Cofactors A and D capture and stabilize beta-tubulin in a quasi-native conformation. Cofactor E, when binded to the cofactor D-beta-tubulin, interacts with cofactor C, causing the release of beta-tubulin peptides that are committed to the native state. Yeast homologs of cofactors D and E are identified through sequence analysis.

Abstract Background Nemaline myopathy is a neuromuscular disorder characterized by the presence of nemaline bodies in patient muscles. 20% of the cases are associated with α-skeletal muscle actin mutations. We previously showed that actin mutations can cause four different biochemical phenotypes and that expression of NM associated actin mutants in fibroblasts, myoblasts and myotubes induces a range of cellular defects. Findings We conducted the same biochemical experiments for twelve new actin mutants associated with nemaline myopathy. We observed folding and polymerization defects. Immunostainings of these and eight other mutants in transfected cells revealed typical cellular defects such as nemaline rods or aggregates, decreased incorporation in F-actin structures, membrane blebbing, the formation of thickened actin fibres and cell membrane blebbing in myotubes. Conclusion Our results confirm that NM associated α-actin mutations induce a range of defects at the biochemical level as well as in cultured fibroblasts and muscle cells.

Abstract Background About 20 % of nemaline myopathies are thus far related to skeletal muscle alpha-actin. Seven actin mutants located in different parts of the actin molecule and linked to different forms of the disease were selected and expressed as EGFP-tagged constructs in differentiated C2C12 mytoubes. Results were compared with phenotypes in patient skeletal muscle fibres and with previous expression studies in fibroblasts and C2C12 myoblasts/myotubes. Results Whereas EGFP wt-actin nicely incorporated into endogenous stress fibres and sarcomeric structures, the mutants showed a range of phenotypes, which generally changed upon differentiation. Many mutants appeared delocalized in myoblasts but integrated into endogenous actin structures after 4–6 days of differentiation, demonstrating a poor correlation between the appearance in myotubes and the severity of the disease. However, for some mutants, integration into stress fibres induced aberrant structures in differentiated cells, like thickening or fragmentation of stress fibres. Other mutants almost failed to integrate but formed huge aggregates in the cytoplasm of myotubes. Those did not co-stain with alpha-actinin, a main component of nemaline bodies found in patient muscle. Interestingly, nuclear aggregates as formed by two of the mutants in myoblasts were found less frequently or not at all in differentiated cells. Conclusion Myotubes are a suitable system to study the capacity of a mutant to incorporate into actin structures or to form or induce pathological changes. Some of the phenotypes observed in undifferentiated myoblasts may only be in vitro effects. Other phenotypes, like aberrant stress fibres or rod formation may be more directly correlated with disease phenotypes. Some mutants did not induce any changes in the cellular actin system, indicating the importance of additional studies like functional assays to fully characterize the pathological impact of a mutant.

Research was conducted to examine the binding behavior of truncated target molecules to cytosolic chaperonin containing TCP-1 by characterizing the features of actin and tubulin target proteins. Results demonstrate that the deletion of three delimited regions in beta-actin reduces or abolishes the interaction with CCT. Findings also indicate that actin and tubulin contact similar CCT subunits as shown by peptide mimetics that represent the binding regions and inhibit target polypeptide binding to CCT.

Background: Nemaline myopathy is a neuromuscular disorder characterized by the presence of nemaline bodies in patient muscles. 20% of the cases are associated with a-skeletal muscle actin mutations. We previously showed that actin mutations can cause four different biochemical phenotypes and that expression of NM associated actin mutants in fibroblasts, myoblasts and myotubes induces a range of cellular defects. Findings: We conducted the same biochemical experiments for twelve new actin mutants associated with nemaline myopathy. We observed folding and polymerization defects. Immunostainings of these and eight other mutants in transfected cells revealed typical cellular defects such as nemaline rods or aggregates, decreased incorporation in F-actin structures, membrane blebbing, the formation of thickened actin fibres and cell membrane blebbing in myotubes. Conclusion: Our results confirm that NM associated a-actin mutations induce a range of defects at the biochemical level as well as in cultured fibroblasts and muscle cells. [ABSTRACT FROM AUTHOR]

The article discusses a study which designed a model for the rapid screening of the folding capacity and functionality of actin mutants and tagged actins. The method combines the in vitro expression of labelled actin with the analysis on native gels, band shift assays or copolymerization tests. The incorporation of actin variants in cytoskeletal structures in transferred cells was monitored using immuno-fluorescence. he authors concluded that the method is useful in characterizing actin mutants.

Eukaryotic prefoldin (PFD) is a heterohexameric chaperone with a jellyfish-like structure whose function is to deliver nonnative target proteins, principally actins and tubulins, to the eukaryotic cytosolic chaperonin for facilitated folding. Here we demonstrate that functional PFD can spontaneously assemble from its six constituent individual subunits (PFD1-PFD6), each expressed as a recombinant protein. Using engineered forms of PFD assembled in vitro, we show that the tips of the PFD tentacles are required to form binary complexes with authentic target proteins. We show that PFD uses the distal ends of different but overlapping sets of subunits to form stable binary complexes with different target proteins, namely actin and α- and β-tubulin. We also present data that suggest a model for the order of these six subunits within the hexamer. Our data are consistent with the hypothesis that PFD, like the eukaryotic cytosolic chaperonin, has co-evolved specifically to facilitate the folding of its target proteins. [ABSTRACT FROM AUTHOR]

Nemaline myopathy is a neuromuscular disorder, characterized by muscle weakness and hypotonia and is, in 20% of the cases, caused by mutations in the gene encoding alpha-skeletal muscle actin, ACTA1. It is a heterogeneous disease with various clinical phenotypes and severities. In patients the ultrastructure of muscle cells is often disturbed by nemaline rods and it is thought this is the cause for muscle weakness. To search for possible defects during muscle cell differentiation we expressed alpha-actin mutants in myoblasts and allowed these cells to differentiate into myotubes. Surprisingly, we observed two striking new phenotypes in differentiating myoblasts: rounding up of cells and bleb formation, two features reminiscent of apoptosis. Indeed expression of these mutants induced cell death with apoptotic features in muscle cell culture, using AIF and endonuclease G, in a caspase-independent but calpain-dependent pathway. This is the first report on a common cellular defect induced by NM causing actin mutants, independent of their biochemical phenotypes or rod and aggregate formation capacity. These data suggest that lack of type II fibers or atrophy observed in nemaline myopathy patients may be also due to an increased number of dying muscle cells.

Central core disease (CCD), congenital fibre type disproportion (CFTD), and nemaline myopathy (NM) are earlyonset clinically heterogeneous congenital myopathies, characterized by generalized muscle weakness and hypotonia. All three diseases are associated with alpha-skeletal muscle actin mutations. We biochemically characterized the CCD and CFTD causing actin mutants and show that all mutants fold correctly and are stable. Expression studies in fibroblasts, myoblasts, and myotubes show that these mutants incorporate in filamentous structures. However they do not intercalate between the nascent z-lines in differentiating muscle cell cultures. We also show that the distribution of mitochondria and of the ryanodine receptors, and calcium release properties from ryanodine receptors, are unchanged in myotubes expressing the CCD causing mutants. CFTD causing mutants induce partly similar phenotypes as NM associated ones, such as rods and thickened actin fibers in cell culture. Our results suggest that molecular mechanisms behind CFTD and NM may be partly related.

Mutations in the gene encoding alpha-skeletal-muscle actin, ACTA1, cause congenital myopathies of various phenotypes that have been studied since their discovery in 1999. Although much is now known about the clinical aspects of myopathies resulting from over 60 different ACTA1 mutations, we have very little evidence for how mutations alter the behavior of the actin protein and thus lead to disease. We used a combination of biochemical and cell biological analysis to classify 19 myopathy mutants and found a range of defects in the actin. Using in vitro expression systems, we probed actin folding and actin's capacity to interact with actin-binding proteins and polymerization. Only two mutants failed to fold; these represent recessive alleles, causing severe myopathy, indicating that patients produce nonfunctional actin. Four other mutants bound tightly to cyclase-associated protein, indicating a possible instability in the nucleotide-binding pocket, and formed rods and aggregates in cells. Eleven mutants showed defects in the ability to co-polymerize with wild-type actin. Some of these could incorporate into normal actin structures in NIH 3T3 fibroblasts, but two of the three tested also formed aggregates. Four mutants showed no defect in vitro but two of these formed aggregates in cells, indicating functional defects that we have not yet tested for. Overall, we found a range of defects and behaviors of the mutants in vitro and in cultured cells, paralleling the complexity of actin-based muscle myopathy phenotypes.