Hiromi Takano-Ohmuro

Autophagy in embryonic erythroid cells: its role in maturation

Yolk sac-derived embryonic erythroid cells differentiate synchronously in the peripheral blood of Syrian hamster. The stage of differentiation on day 10 of gestation is equivalent to polychromatophilic erythroblast stage and that on day 13 is equivalent to the reticulocyte stage in adult animals. The cytoplasm of embryonic erythroid cells became scant and devoid of most organelles on day 12 of gestation. In addition, there were very few non-erythroid cells in circulation before day 13. Thus the embryonic erythroid cells serve a pure and synchronous system to study the mechanisms of terminal differentiation. The number of mitochondria in the embryonic erythroid cells decreased to about 10% of the initial number during the period between day 10 and day 12 of gestation. In contrast, the frequency of autophagy of mitochondria increased 4.6-fold in the same period. The cytochrome c content of the cell decreased as the mitochondria became extinct. However, release of cytochrome c into the cytoplasm was not detectable through day 10-13 of gestation, suggesting that the mitochondria were digested within a closed compartment. Decomposed mitochondria and ferritin particles were detected in lysosomes by electron microscopy on and after day 12 of gestation, which also suggested digestion in a closed compartment. Mitochondrial ATP synthase subunit c, which is known to be a protease-refractory protein, was retained in the cells even after the disappearance of mitochondria, indicating that most of the mitochondria were not extruded from the cells. The digestion of mitochondria in autolysosomes may allow the cells to escape from rapid apoptotic cell death through concomitant removal of mitochondrial death-promoting factors such as cytochrome c.

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.

Autophosphorylation of β-connectin (titin 2) in vitro

Abstract Phosphorylation of β-connectin (titin 2), an elastic protein of chicken breast muscle, occurred in the presence of [γ-32P] ATP, 0.2 mM CaCl2 and 25 mM phosphate buffer, pH 7.0. Addition of 3 mM MgCl2 did not affect the phosphorylation. However, Ca2+ ions were required for the phosphorylation and EGTA inhibited it even if MgCl2 were present. Myosin light chain kinase (gizzard MLCK) cAMP dependent protein kinase (A kinase), and protein kinase C (C kinase) did not phosphorylate β-connectin in vitro under optimal conditions. Thus it appears that β-connectin, possibly containing a domain homologous with MLCK, has an autophosphorylating action.

Enhancement of Ca2+-induced Ca2+ release in calpain treated rabbit skinned muscle fibers

Calpain treatment of rabbit skinned muscle fibers resulted in proteolysis of junctional foot protein or Ca2+ release channel of the sarcoplasmic reticulum. Electrophoretic and immunoblot analyses indicate that calpain cleaves off approximately 130 kDa peptide from the N-terminus. After such treatment, Ca2+ capacity of the sarcoplasmic reticulum remained normal and both Ca2+ and adenine nucleotide dependence of Ca2+-induced Ca2+ release mechanism were retained. However, the Ca2+-activated Ca2+ release rate was increased by two fold after the proteolysis. The results suggest the presence of functional domains in the junctional foot protein, and the N-terminus domain controls the activity of the Ca2+ channel without changing Ca2+ and nucleotide sensitivities.

Sea anemone (Actinia equina) myosin

Abstract 1. 1. Myosin was purified to homogeneity from both tentacle and body of the sea anemone, Actinia equina . 2. 2. Sea anemone myosin was two-headed myosin (Type II), approximately 160 nm long. 3. 3. The tail tended to bend at about 60 nm from the tip of the tail. 4. 4. Sea anemone myosin consisted of heavy chain (about 230 kDa) and two species of light chains: 18 and 19 kDa for tentacle myosin; 17 and 22 kDa for body myosin. 5. 5. The K + -EDTA-activated and Ca 2+ -activated ATPase activities were approximately 0.40 and 0.07 (tentacle), 0.39 and 0.06 (body) μmol mg −1 min −1 , respectively. 6. 6. In both myosins, the Mg 2+ -actin activated ATPase activities were as low as 0.02 μmol mg −1 min −1 . 7. 7. However, it was elevated to 0.055 μmol mg −1 min −1 , when myosin light chain was phosphorylated by myosin light chain kinase from chicken gizzard. 8. 8. It appears that sea anemone myosin-actin interaction was regulated by myosin light chain phosphorylation.