Ca2+-sensitive gelation of actin filaments by a new protein factor

Cytoplasmic pH in single living specimens of Chaos carolinensis is determined microfluorometrically by measuring the ratio of fluorescence intensity of microinjected fluorescein-thiocarbamyl (FTC)-ovalbumin at two different excitation wavelengths. The probe is evenly distributed throughout, and confined to, the cytoplasm, and the fluorescence intensity ratio depends only upon pH . It is independent of pathlength, concentration of probe, divalent cations, and ionic strength. Ratios are calibrated with a standard curve generated in situ by adjusting internal pH of FTC-ovalbumincontaining amebae with weak acid and weak base or by injection of strong buffers . With this technique, the average cytoplasmic pH of freely moving amebae is found to be 6.75 (SD ± 0.3) . The pH of a given spot relative to the morphology of a moving ameba remains fairly constant (± 0.05 U), whereas the pH of two different spots in the same cell may differ by as much as 0.4 U, and average pH in different amebae ranges from 6.3 to 7.4, with a suggestion of clustering about pH 6.5 and 6.8. During wound healing, there is a local, transient drop in pH (as great as 0.35 U) at the wound site upon puncture, proportional in extent to the degree of damage . Comparison of tails and advancing pseudopod tips reveals no significant difference in cytoplasmic pH at this level of spatial (50 ,urn diameter spot) and temporal (1 .3 s) resolution . Fluctuations in intracellular pH and/or intracellular free Ca" may be involved in regulation of cytoplasmic structure and contractility.

Section: Comparison Of Tails and Pseudopodsmentioning

confidence: 99%

Intracellular pH in single motile cells.

Heiple¹,

Taylor²

1980

“…Its native structure is a homodimer with a subunit molecular weight of 103 kD (Singh et al ., 1977;Suzuki et al ., 1979;Baron et al, 1987) and is visualized in the electron microscope as a dumbbell-shaped molecule, 30-40 tun long and 3-4 nm wide with an enlarged head domain at each end (Podlubnaya et al ., 1975;Bretscher et al, 1979;Pollard, 1981) . Distinct a-actinin isoforms have been identified in muscle (Endo and Masaki, 1982) and nonmuscle cells (Burridge and Feramisco, 1981;Duhaiman and Bamburg, 1984;Bennett et al, 1984;Landon et al, 1985) ; however, the only known functional difference between the isoforms is that binding of muscle a-actinin to actin is calcium insensitive, whereas the binding of nonmuscle a-actinins to actin is inhibited by calcium (Mimura and Asano, 1979;Burridge and Feramisco, 1981;Duhaiman and Bamburg, 1984 ;Bennet et al ., 1984 ;Landon et al, 1985) .…”

mentioning

confidence: 99%

Disruption of the actin cytoskeleton after microinjection of proteolytic fragments of alpha-actinin.

Pavalko

Burridge

1991

151

102

“…In smooth muscle, it is present in cytoplasmic dense bodies and membrane-associated dense plaques (21,41). Proteins immunologically and biochemically related to a-actinin have been isolated from several nonmuscle tissues or cells: bovine brain (42), Ehrlich tumor cells (36,37), plasma membranes of sarcoma 180 ascites cells (46), HeLa cells (6), human platelets (40), rat liver (29), and porcine kidney (28). Many of these proteins, if not all, are Ca 2+ sensitive, i.e., are inhibited from cross-linking actin filaments by Ca 2+, different from any of the muscle a-actinins.…”

mentioning

confidence: 99%

Differential expression and distribution of chicken skeletal- and smooth-muscle-type alpha-actinins during myogenesis in culture.

Endo¹,

Takao²

1984

Antibodies to chicken fast skeletal muscle (pectoralis) a-actinin and to smooth muscle (gizzard) a-actinin were absorbed with opposite antigens by affinity chromatography, and four antibody fractions were thus obtained: common antibodies reactive with both pectoralis and gizzard a-actinins ([C]anti-P a-An and [C]anti-G a-An), antibody specifically reactive with pectoralis a-actinin ([S]anti-P a-An), and antibody specifically reactive with gizzard a-actinin ([S]anti-G a-An). In indirect immunofluorescence microscopy, (C)anti-P aAn, (S)anti-P a-An, and (C)anti-G a-An stained Z bands of skeletal muscle myofibrils, whereas (S)anti-G a-An did not. Although (S)anti-G a-An and two common antibodies stained smooth muscle cells, (S)anti-P u-An did not. We used (S)anti-P a-An and (S)anti-G a-An for immunofluorescence microscopy to investigate the expression and distribution of skeletal-and smoothmuscle-type a-actinins during myogenesis of cultured skeletal muscle cells. Skeletal-muscletype a-actinin was found to be absent from myogenic cells before fusion but present in them after fusion, restricted to Z bodies or Z bands. Smooth-muscle-type ~-actinin was present diffusely in the cytoplasm and on membrane-associated structures of mononucleated and fused myoblasts, and then confined to membrane-associated structures of myotubes. Immunoblotting and peptide mapping by limited proteolysis support the above results that skeletalmuscle-type a-actinin appears at the onset of fusion and that smooth-muscle-type a-actinin persists throughout the myogenesis. These results indicate (a) that the timing of expression of skeletal-muscle-type a-actinin is under regulation coordinate with other major skeletal muscle proteins; (b) that, with respect to expression and distribution, skeletal-muscle-type a-actinin is closely related to a-actin, whereas smooth-muscle-type a-actinin is to 3'-and ~-actins; and (c) that skeletal-and smooth-muscle4ype a-actinins have complementary distribution and do not co-exist in situ.a-Actinin, which was discovered by Ebashi and Ebashi (12), is a structural protein of Z bands in skeletal muscle myofibrils (33). It is also located near the fascia adherens of intercalated disks as well as in Z bands in cardiac muscle (44). In smooth muscle, it is present in cytoplasmic dense bodies and membrane-associated dense plaques (21,41). Proteins immunologically and biochemically related to a-actinin have been isolated from several nonmuscle tissues or cells: bovine brain (42), Ehrlich tumor cells (36,37), plasma membranes of sarcoma 180 ascites cells (46), HeLa cells (6), human platelets (40), rat liver (29), and porcine kidney (28). Many of these proteins, if not all, are Ca 2+ sensitive, i.e., are inhibited from cross-linking actin filaments by Ca 2+, different from any of the muscle a-actinins. Immunofluorescence microscopy, immunoelectron microscopy, and microinjection using fluorescence-labeled a-actinin have revealed that a-actinin or proteins immunologically related to a-actinin are also present in cultur...