Catherine Astier

Calpain 3 deficiency is associated with myonuclear apoptosis and profoundperturbation of the IκBα/NF-κB pathway in limb-girdle musculardystrophy type 2A

Nature Med. 5, 503– 511 (1999). The top left corner of Fig. 1b on page 505 was cropped so that you could not view the calpain 3-stained nuclei in endomysia space. The corrected figure is shown below. We regret this error.

Alpha actinin–CapZ, an anchoring complex for thin filaments in Z-line

CapZ is a widely distributed and highly conserved, heterodimeric protein, that nucleates actin polymerization and binds to the barbed ends of actin filaments, preventing the addition or loss of actin monomers. CapZ interaction with actin filaments was shown to be of high affinity and decreased in the presence of PIP2. CapZ was located in nascent Z-lines during skeletal muscle myofibrillogenesis before the striated appearance of thin filaments in sarcomers. In this study, the stabilization and the anchorage of thin filaments were explored through identification of CapZ partners in the Z-line. Fish (sea bass) striated white muscle and its related Z-line proteins were selected since they correspond to the simplest Z-line organization. We report here the interaction between purified CapZ and α-actinin, a major component of Z filaments and polar links in Z-discs. Affinity of CapZ for α-actinin, estimated by fluorescence and immunochemical assays, is in the μ m range. This association was found to be independent of actin and shown to be weakened in the presence of phosphoinositides. Binding contacts on the α-actinin molecule lie in the 55 kDa repetitive domain. A model including CapZ/α-actinin/titin/actin interactions is proposed considering Luthers 3D Z-line reconstruction.

Fish muscle cytoskeleton integrity is not dependent on intact thin filaments

Striated musclecytoskeleton was studied by ultrastructure and electrophoresis.Treatment of sea bass white muscle myofibrils and glycerinatedfibres with calpain caused disruption of costameres, intermediatefilaments, and Z-line, without altering sarcomeres. V8 proteasealso caused loss of costameres and Z-line, and disruptedsarcomeres without affecting the intermediate filaments.Recombinant lipase caused loss of Z-lines and also sarcolemmadetachment, without changing sarcomeres or intermediatefilaments. DNase-1 removed thin filaments and partially removedZ-lines while leaving intact the sarcolemma attachments andintermediate filaments. Calpain, V8 protease, lipase and DNase-1treatments induced extensive loss of α-actinin from the Z-line, which could be related to titin cleavage (calpain, V8),phosphoinositide hydrolysis (lipase), and actin depolymerisation(DNase-1). These results show that the cytoskeletal componentsare independent of intact thin filaments

The identification of a second cofilin binding site on actin suggests a novel, intercalated arrangement of F-actin binding.

The cofilins are members of a protein family that binds monomeric and filamentous actin, severs actin filaments, and increases monomer off-rate from the pointed end. Here, we characterize the cofilin-actin interface. We confirm earlier work suggesting the importance of the lower region of subdomain 1 encompassing the N and C termini (site 1) in cofilin binding. In addition, we report the discovery of a new cofilin binding site (site 2) from residues 112–125 that form a helix toward the upper, rear surface of subdomain 1 in the standard actin orientation (Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., and Holmes, K. C. (1990) Nature 347, 37–44). We propose that cofilin binds “behind” one monomer and “in front” of the other longitudinally associated monomer, accounting for the fact that cofilin alters the twist in the actin (McGough, A., Pope, B., Chiu, W., and Weeds, A. (1997) J. Cell Biol. 138, 771–781). The characterization of the cofilin-actin interface will facilitate an understanding of how cofilin severs and depolymerizes filaments and may shed light on the mechanism of the gelsolin family because they share a similar fold with the cofilins (Hatanaka, H., Ogura, K., Moriyama, K., Ichikawa, S., Yahara, I., and Inagiki, F. (1996) Cell 85, 1047–1055).

Sarcomeric disorganization in post-mortem fish muscles.

1. The post-mortem evolution of protein pattern in fish striated muscle was followed by SDS-PAGE, after different conditions of storage time and temperature. 2. Sarcoplasmic and sarcomeric fractions were analyzed respectively by low and high ionic strength extractions of fish muscle samples. 3. No evident modification of electrophoretic patterns was observed during the pre-rigor mortis period. 4. The high mol. wt proteins titin and nebulin were highly sensitive to proteolysis during the rigor mortis period. 5. Myosin extraction was predominantly influenced by the storage temperature. The myosin content of the extracts decreased during the rigor mortis period at storage temperatures greater than 8 degrees C. 6. alpha-Actinin was very resistant to proteolysis, but could be released from Z-disc structure during post-mortem aging.

Binding of a native titin fragment to actin is regulated by PIP2

Titin is a giant protein which extends from Z‐line to M‐line in striated muscles. We report here the purification of a 150‐kDa titin fragment, obtained after V8 protease treatment of myofibrils. This polypeptide was located at the N1‐line level, in a titin part known to exhibit stiff properties correlated to an association with actin. By solid or liquid phase binding assays and cosedimentation, we have clearly demonstrated a direct, saturable and relative high affinity binding of the native titin fragment to F‐actin. The 150‐kDa titin fragment was also shown to accelerate actin polymerization. Furthermore, the actin‐titin interaction was found to be inhibited by phosphoinositides.

Isolation and properties of white skeletal muscle α-actinin from sea-trout (Salmo trutta) and bass (Dicentrarchus labrax)

Abstract Fish a-actinin purified from sea-trout and bass white muscle by means of two different extraction procedures was used to investigate the eventual presence of different muscle isoforms in Z-disks. These fish α-actinins have the same apparent molecular weight (100 kDa) and the same isoelectric point (pI = 5.6), and also have a total antigenic identity towards anti-bass and anti-chicken α-actinin antibodies, suggesting a single molecular species. The role of fish α-actinin as an anchorage site for thin actin filaments and elastic titin filaments in Z-bands was studied. Despite conservation of the actin-binding site, fish α-actinin has a better actin-binding ability (kD = 0.3 μM), than chicken smooth muscle a-actinin (kD = 1.6 μM). Several other structural and functional characteristics of fish α-actinin were also studied: conservation of sequence and domain structure, the role of divalent ions (Ca2+, Mg2+) and the dielectric constant of the medium in α-actinin-actin interaction. Although the reason for fish white muscle a-actinins close affinity to actin was not clearly established, our results suggested that the physicochemical environment of the Z-filaments in Z-disks might be crucial.

Coevolution of actin and associated proteins: an α-actinin-like protein in a cyanobacterium (Spirulina platensis)

Actin, together with associated proteins, such as myosin, cross-linking or capping proteins, has been observed in all eukaryotic cells. Presence of actin or actin-like proteins has also been reported in prokaryotic organisms belonging to the cyanobacteria. Our aim was first to extend the characterization of an actin-like protein to another prokaryotic cell, i.e. Spirulina, then to compare the antigenic reactivity of this new protein with that of Synechocystis and skeletal actins. We observed that some of the conserved antigenic epitopes corresponded to actin regions known to interact with cross-linking proteins. We also report for the first time that alpha-actinin and filamin purified from chicken gizzard both interact with a prokaryotic actin-like protein. Finally, we searched for the occurrence of a cross-linking protein in these cyanobacteria and identified a 105-kDa protein as an alpha-actinin-like protein using specific antibodies.

Alpha-actinin in different invertebrate muscle cell types of Drosophila melanogaster, the earthworm Eisenia foetida, and the snail Helix aspersa.

The presence and distribution of α-actinin has been studied in several invertebrate muscle cell types. These comprised transversely striated muscle (flight muscle) from the fruit fly Drosophila melanogaster, transversely striated muscle (heart muscle) from the snail Helix aspersa, obliquely striated muscle (body wall muscle) from the earthworm Eisenia foetida, smooth muscle (retractor muscle) from H. aspersa, and smooth muscle (outer muscular layer of the pseudoheart) from E. foetida. The study was carried by means of Western blot analysis, ELISA, and immunohistochemical electron microscopy, using anti α-actinin antibody. Immunoreaction for a protein with the same molecular weight as that of mammalian α- actinin was detected in all muscle types studied, although the amount and intensity of immunoreaction varied among them. In the insect muscle, immunolabelling was found along the whole Z-line. In both the transversely striated muscle from the snail and the obliquely striated muscle from the earthworm, immunolabelling did not occupy the whole Z-line but showed discontinuous, orderly arranged patches along the Z-line course. In the two smooth muscles studied (snail and earthworm), immunolabelling was limited to small patches which did not show an apparently ordered distribution. Since it is assumed that α-actinin is located at the anchorage sites for actin filaments, present observations suggest that, only in the Drosophila muscle, actin filaments areparallely arranged in all their course, whereas in the other invertebrate muscles studied these filaments converge on discontinuously distributed anchorage sites.

Analysis of long-range structural effects induced by DNase-I interaction with actin monomeric form or complexed to CapZ.

Two fundamental properties of monomeric actin were examined in this study, ie its interaction with DNase-I, and the inhibition of endonuclease activity consecutive to the association of the two molecules. In particular, the topological independence between catalytic site of DNase-I and interface with actin, structural changes in actin monomer and the absence of conformational changes in DNase-I were described. We demonstrated a loss of flexibility of antigenic structures in actin subdomain I (ie epitopes 18-28 and 95-105) as well as modification in the exposure of Cys10 and Cys374 after DNase-I binding. Furthermore, the conformational changes induced by DNase-I into the actin molecule weakened the interaction of CapZ to its binding site located in the C-terminal region of actin monomer. These structural changes were time-dependent. When actin was cleaved in the DNase-I binding loop (sequence 38-52) at position 42 by E coli A2 strain protease, a tight DNase-I binding to split actin and the conformational changes were still observed, whereas the DNase-I inhibition activity was completely abolished. Finally, when we substitute Ca2+ by Mg2+ (ATP-Mg2+ monomeric actin) which induces a tighter conformation of actin and partially restores the inhibitory ability of split actin, long-range conformational effects of DNase-I are prevented and the ternary complex DNase-I-actin-CapZ is obtained.