An antiactin antibody that distinguishes between cytoplasmic and skeletal muscle actins.

Abstract

We elicited antibodies in rabbits to actin purified from body wall muscle of the marine mollusc, Aplysia californica . We found that this antiactin has an unusual specificity: in addition to reacting with the immunogen, it recognizes cytoplasmic vertebrate actins but not myofibrillar actin . Radioimmunoassay showed little or no cross-reaction with actin purified from either chicken gizzard or rabbit skeletal muscle. Immunocytochemical studies with human fibroblasts and L6 myoblasts revealed intense staining of typical cytoplasmic cables. Myofibrils were not stained after treatment of human and frog skeletal muscle with the antibody, although the distribution of immunofluorescence suggested that cytoplasmic actin is associated with membrane systems in the muscle fiber. The antibody may therefore be especially suited for studying the localization of cytoplasmic actin in skeletal muscle cells even in the presence of a great excess of the myofibrillar form .In addition to its role in muscle contraction, actin is thought to participate in other processes that involve intracellular motility (including fast axonal transport) and to serve as a universal cytoskeletal element in eukaryotic cells (see references 14,35,43). Although greatly conserved during phylogeny, the actin molecule occurs in a variety of forms (24) . In vertebrate muscle, three types have been separated because of differences in charge: a-actin is present in sarcomeres of the skeletal muscle fibril ; ß-and y-actins are found in embryonic and smooth muscle (13,38,45) . Actins have also been isolated from brain and other nonmuscle eukaryotic tissues; these have been called cytoplasmic, and are more closely related to the actins of embryonic muscle than to myofibrillar actin (24) .Immunocytochemical localization has been one of the main tools for obtaining evidence that actin is involved in intracellular motility . The antiactin antibodies previously used, whether raised against actins of smooth muscle or against aactin, have generally reacted with both cytoplasmic and myofibrillar actins (see below) . To determine how actin might be involved in the rapid movement of organelles along axons of identified neurons of the marine mollusc, Aplysia californica, we have raised antibodies in rabbits using Aplysia body wall muscle actin as immunogen . We found that the antibody obtained can be used to localize actin within neurons (reference 29 and Lubit et al ., manuscript in preparation) . We also found that the specificity of the antibody is unlike all previous antiactins because it reacted only with certain forms of cytoplasmic actin and not with myofibrillar actin . This unique THE JOURNAL OF CELL BIOLOGY " VOLUME 86 SEPTEMBER 1980891-897 ©The Rockefeller University Press " 0021-9525/80/09/0891/07 $1 .00 specificity was determined by competitive binding studies and by immunohistochemical examination of vertebrate cells in which the distribution of actin is known . Reactivity of the antibody with cytoplasmic actin was assessed by determining its loc...

Sequencing of proteins from two-dimensional gels by using in situ digestion and transfer of peptides to polyvinylidene difluoride membranes: application to proteins associated with sensitization in Aplysia.

“…Of 52 residues of amino acid sequence obtained for Aplysia actin, only 2, marked with asterisks (*) in Fig. 1, differ from the reported sequence for Strongylocentrotus purpuratus (sea urchin) actin (11). These data are in good agreement with a number of previous reports of a high degree of sequence conservation for actin from a variety of tissue sources and species.…”

Quadriceps myopathy caused by skeletal muscle-specific ablation of βcyto-actin

“…Although we did not detect  cyto -actin on peeled sarcolemma (Rybakova et al, 2000), another study showed sarcolemmal localization of  cytoactin on an in situ skeletal muscle preparation (Lubit and Schwartz, 1980). Differences in antibody sources, tissue preparation, and the documented difficulty imaging cytoplasmic actins (Dugina et al, 2009) might explain these seemingly contradictory results.…”

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

“…Moreover, an antibody that recognizes cytoplasmic actin (probably 3-and 3,-actins) but not myofibriUar actin (a-actin) stains the cytoplasm of fully differentiated skeletal muscle fibers diffusely and the membrane-associated structures strongly (32). Skeletal-muscle-type a-actinin is therefore thought to be closely related to a-actin, and smooth-muscle-type a-actinin to 3-and 7-actins, with respect to timing of expression and distribution.…”