Kimihide Hayakawa

Quantitative analysis of low molecular weight G-actin-binding proteins, cofilin, ADF and profilin, expressed in developing and degenerating chicken skeletal muscles

SummaryA large amount of G-actin is pooled in the cytoplasm of young embryonic skeletal muscle and, although its concentration is reduced as muscle develops, the total amount of actin in muscle cells increases remarkably. Three G-actin-binding proteins, cofilin, ADF and profilin, are known to be involved in creating the G-actin pool in the embryonic muscle. To better understand how they are responsible for the regulation of assembly and disassembly of actin in developing and degenerating muscles, we measured the amounts of the three G-actin-binding proteins by means of quantitative immunoblotting and compared them with that of G-actin. The sum of the amounts of the three actin-binding proteins was insufficient at early developmental stages but sufficient at later stages to account for the pool of G-actin in young muscle cells. It decreased in parallel with the decrease in the G-actin pool as muscle developed. Expression of thymosin β4, which is known to be extremely important for G-actin-sequestering in a variety of non-muscle cells, was detected at a considerable level in young embryonic but not in adult skeletal muscles according to Northern and Western blotting. In degenerating denervated and dystrophic muscles, cofilin and profilin, but not ADF, were significantly increased in amount. From these results, we conclude that the G-actin pool in young embryonic skeletal muscle is mainly due to cofilin, ADF, profilin and thymosin β4, but thymosin β4 as well as ADF becomes less important as muscle develops. Cofilin and profilin may also be involved in the redistribution of actin during myofibrillogenesis and in the process of actin disassembly in degenerating muscles.

Expression of cofilin isoforms during development of mouse striated muscles

Cofilin (CF) is an actin regulatory protein that plays a critical role in actin filament dynamics in a variety of cells. Two cofilin isoforms, muscle-type (M-CF) and nonmuscle-type (NM-CF) encoded by different genes, exist in mammals; in the adult, the former is predominantly expressed in muscle tissues, while the latter is distributed in various non-muscle tissues (Ono et al., 1994). In this study, we examined cofilin isoform expression during skeletal and cardiac muscle development in mice using cDNA probes and antibodies which distinguish the isoforms. We found that the expression of M-CF was initiated in terminally differentiated myogenic cells in both the myotome and limb buds. In myogenic cell cultures, its expression occurred coupled with myotube formation. NM-CF was expressed in developing skeletal and cardiac muscles but disappeared from skeletal muscle during postnatal development, while its expression persisted in the heart, even in adult mice. A similar situation was observed in the heart of other mammals. Thus, it is likely that the both cofilin isoforms are involved in the regulation of actin assembly during myofibrillogenesis. Only M-CF could be involved in actin dynamics in mature skeletal muscle, while both isoforms could be in the mature heart.

Differential Assembly of Cytoskeletal and Sarcomeric Actins in Developing Skeletal Muscle Cells In Vitro

Abstract Monoclonal antibodies (McAb) to actin were prepared to analyze the assembly of actin isoforms in developing muscle cells in vitro. One of the antibodies (SkA-06) was specific for &agr;-sarcomeric actin isoforms in skeletal and cardiac muscles, while the others recognized cytoskeletal (&bgr;, &ggr;) actin isoforms in smooth muscle and non-muscle tissues as well as the sarcomeric (&agr;) actins. Using SkA-06 and a polyclonal antibody (PcAb) specific for cytoskeletal actins, the subcellular localization of the actin isoforms was examined by immunocytochemical methods. While in developing young myotubes, cytoskeletal and sarcomeric actins were co-localized in nascent myofibrils or stress-fiber-like structures, sarcomeric actins predominated in striated myofibrils in more developed myotubes. When FITC-labeled cytoskeletal and sarcomeric actins were introduced into young myotubes by a microinjection method, the latter became detectable in striated structures sooner than the former but they were finally incorporated into striated myofibrils. These results suggest that &agr;-actin(s) as well as &bgr;- and &ggr;-actins can be incorporated into myofibrils, but &agr;-actin(s) is assembled preferentially into myofibrils in developing muscle cells.

Orientation of Smooth Muscle-Derived A10 Cells in Culture by Cyclic Stretching: Relationship between Stress Fiber Rearrangement and Cell Reorientation

Abstract Mechanical stress causes various responses in cells both in vivo and in vitro. Realignment of cells and stress fibers is one of the remarkable phenomena that are induced by the stress. However, the mechanism by which their realignment is controlled is largely unknown. In this study, effects of mechanical stretch on the morphology of cultured cells were examined using a cyclic and reciprocal cell stretching apparatus. A10 cells, a cell line derived from rat aortic smooth muscle, were used as a model, since they are spindle-shaped and have remarkable stress fibers aligned along the longitudinal cell axis. Therefore, the orientation of the cell and stress fibers could be easily identified. When the cells were cultured on elastic silicone membranes and subjected to cyclic and reciprocal stretch with an amplitude of 20% at a frequency of 60 cycles per minute, actin stress fibers were aligned obliquely to the direction of stretching with angles of 50 to 70 degrees within about 15 min after the onset of stretching. Then, after 1–3 hr of cyclic stretching, the long axes of a majority of the cells were also reoriented to similar directions to the stress fibers. The stretch-induced cell reorientation was blocked by 1 μM cytochalasin B, but not by colcemid. These results indicate that the orientation of cells and actin filaments are closely related and actin filaments play a critical role in the early step of the cell reorientation.

Subcellular localization of dystrophin and vinculin in cardiac muscle fibers and fibers of the conduction system of the chicken ventricle.

Abstractu2002The subcellular localization of dystrophin and vinculin was investigated in cardiac muscle fibers and fibers of the conduction system of the chicken ventricle by immunofluorescence confocal microscopy. In ventricular cardiac muscle fibers, strong staining with antibody against dystrophin appeared as regularly arranged transverse striations at the sarcolemmal surface, and faint but uniform staining was seen in narrow strips between these striations. In fibers of the ventricular conduction system, the sarcolemma was stained uniformly with this antibody, but strong staining was found as regular striations in many areas and as scattered patches in other areas of the sarcolemma. These intensely stained striations and scattered patches of dystrophin were colocalized with those of vinculin. Because dystrophin striations were located at the level of Z bands of the underlying myofibrils, they were regarded as the concentration of this protein at costameres together with vinculin. In fibers of the conduction system, myofibrils were close to the sarcolemma where dystrophin and vinculin assumed a striated pattern, at some distance from the cell membrane where these proteins exhibited a patchy distribution, and distant from the sarcolemma where dystrophin was uniformly distributed. These data suggest that the distribution patterns of dystrophin reflect the degree of association between the sarcolemma and underlying myofibrils.

Effects of Exogenous 0-Actinin (CapZ) on Actin Filamentous Structures in Cultured Muscle Cells

Abstract β-Actinin (CapZ) is a heterodimeric actin-binding protein which is localized in the Z-bands of myofibril. It caps the barbed end of actin filaments and nucleates actin-polymerization in a Ca2+-independent manner. As judged by these properties of β-actinin, it is conceivable that β-actinin is involved in the regulation of actin assembly, especially in the formation of I-Z-I complex during myofibrillogenesis. In this study, to examine the function of β-actinin in myofibrillogenesis, recombinant β-actinin (r-β-actinin) produced in an E. coli expression system was introduced into cultured myogenic cells by a microinjection method. Stress fibers in C2 myoblasts were disrupted soon after microinjection of recombinant β-actinin, but nascent as well as well-organized myofibrils were scarcely affected by exogenous β-actinin. Based on these observations, we suggest that in myoblasts where actin filaments are dynamically reorganized, reassembly process of actin filaments may be affected by the exogenous β-actinin, whereas actin filaments become more stable and less sensitive to exogenous β-actinin, when they are organized into myofibrillar structures and anchored to Z-lines in myotubes.