A sarcomeric alpha-actinin truncated at the carboxyl end induces the breakdown of stress fibers in PtK2 cells and the formation of nemaline-like bodies and breakdown of myofibrils in myotubes.

Abstract

In many nonmuscle cells, nonsarcomeric a-actinin is distributed in the dense bodies of stress fibers, adhesion plaques, and adherens junctions. In-striated muscle, a sarcomeric isoform of a-actinin (s-a-wactlnn) Is found in the Z-bands of myofibrils and subsarcolemmal adhesion plaques. To understand the role(s) of the a-actinin isoforms in the assembly and maintenance of such cytoskeletal structures, full-length or truncated s-a-actinin cDNAs were expressed in PtK2 cells and in primary skeletal myogenic cells. We found the following. (i) In transfected PtK2 cells the truncated s-aactinin was rapidly incorporated into preexisting dense bodies, adhesion plaques, and adherens junctions. With time these structures collapsed, and the affected cells detached from the substrate. (ii) In myotubes the truncated s-a-actinin was incorporated into nascent Z-bands. Many of these progressively hypertrophied, forming nemaline-lke bodies. With time the affected myofibrils fagmented, and the myotubes detached from the substrate. (iii) In both cell types the truncated s-a-actinin was significntly more disruptive of the cytoskeletal structures than the full-length molecule. (iv) Pools of "overexpressed" full-length or truncated protein did not selfaggregate into homogeneous, amorphous complexes; rather the exogenous proteins selectively colocalized with the same cohort of cytoskeletal proteins with which the endogenous a-actinin normally associates. The similarity among the hypertrophied Z-bands in transfected myotubes, the nemaline bodies in patients with nemaline myopathies, and the streaming Z-bands seen in various muscle pathologies raises the possibility that the genetically determined nemaline bodies and the pathologically induced Z-band alterations may reflect primary and/or posttranslational modifications of s-a-actinin.a-Actinin appears to occupy a strategic role in the assembly and maintenance of stress fibers of nonmuscle cells and the myofibrils of muscle cells (1, 2). Two isoforms of a-actinin have been identified, a muscle-specific sarcomeric isoform, s-a-actinin, and a nonsarcomeric isoform, non-s-a-actinin (3-5). In cell-free systems, binding affinities have been demonstrated between both a-actinins and many of the proteins comprising stress fibers, myofibrils, and their respective membrane-attachment sites. Protein sequence analyses of the isoforms reveal a globular head that links actin filaments, a rod-shaped central portion thought important for anti-parallel dimer formation, and a carboxyl terminus with consensus sequences for two EF-hands with a calciumbinding potential (6-8).Cytoimmunofluorescence studies on nonmuscle cells have localized non-s-a-actinin to dense bodies in stress fibers and to the vinculin-positive adhesion plaques and adherens junctions into which stress fibers insert (9-11). Comparable studies on skeletal and cardiac cells have localized s-a-actinin to Z-bands in striated myofibrils, to precursor I-Z-I-like complexes in muscle undergoing myofibrillogenesis, and to vinculin-positive...

Dynamics of Z-band based proteins in developing skeletal muscle cells

“…The YFP-27-kD construct also caused disruption of stress fibers in transfected fibroblasts (data not shown), and the premyofibrils and myofibrils in transfected muscle cells. Similar disruptions of stress fibers and myofibrils were also found with expression of a truncated alpha-actinin that was much larger (aa 1-728, Schultheiss et al, 1992). Taken together these results suggest that a pool of dynamic alpha-actinin molecules is important for the maintenance and formation of stress fibers, premyofibrils and myofibrils.…”

Myopathy mutations in α-skeletal-muscle actin cause a range of molecular defects

“…A previous study found that expression of truncated α-actinin in myoblasts disrupted normal cross-linking of the thin filaments and induced the formation of aggregates and nemaline bodies. In later stage myotubes, it induced hypertrophied Z-bands and nemaline bodies composed of α-actinin and actin, whereas, in PtK2 cells, it induced the breakdown of the stress fibers into clumps containing α-actinin and actin (Schultheiss et al, 1992).…”

Isoproterenol Activates Extracellular Signal–Regulated Protein Kinases in Cardiomyocytes Through Calcineurin

“…In cardiomyocytes, the Z band is close to the T tubules. 36,37 Because voltage-dependent Ca 2ϩ channels are abundant in the T tubules and Ca 2ϩ -releasing channels are also localized in the membrane of sarcoplasmic reticulum, which is close to the T tubules, 37,38 the local Ca 2ϩ concentration around the Z band might be quite high. Calcineurin has been reported to be activated by sustained increase in Ca 2ϩ .…”