Cellular titin localization in stress fibers and interaction with myosin II filaments in vitro.
We previously discovered a cellular isoform of titin (originally named T-protein) colocalized with myosin II in the terminal web domain of the chicken intestinal epithelial cell brush border cytoskeleton (Eilertsen, K.J., and T.C.S. Keller. 1992. J. Cell Biol. 119:549-557). Here, we demonstrate that cellular titin also colocalizes with myosin II filaments in stress fibers and organizes a similar array of myosin II filaments in vitro. To investigate interactions between cellular titin and myosin in vitro, we purified both proteins from isolated intestinal epithelial cell brush borders by a combination of gel filtration and hydroxyapatite column chromatography. Electron microscopy of brush border myosin bipolar filaments assembled in the presence and absence of cellular titin revealed a cellular titin-dependent side-by-side and end-to-end alignment of the filaments into highly ordered arrays. Immunogold labeling confirmed cellular titin association with the filament arrays. Under similar assembly conditions, purified chicken pectoralis muscle titin formed much less regular aggregates of muscle myosin bipolar filaments. Sucrose density gradient analyses of both cellular and muscle titin-myosin supramolecular arrays demonstrated that the cellular titin and myosin isoforms coassembled with a myosin/titin ratio of approximately 25:1, whereas the muscle isoforms coassembled with a myosin:titin ratio of approximately 38:1. No coassembly aggregates were found when cellular myosin was assembled in the presence of muscle titin or when muscle myosin was assembled in the presence of cellular titin. Our results demonstrate that cellular titin can organize an isoform-specific association of myosin II bipolar filaments and support the possibility that cellular titin is a key organizing component of the brush border and other myosin II-containing cytoskeletal structures including stress fibers.
Eilertsen
K J
KJ
Molecular Biophysics Program, Florida State University, Tallahassee 32306-3050.
Kazmierski
S T
ST
Keller
T C
TC
D000818:Animals; D002451:Cell Compartmentation; D002642:Chick Embryo; D064211:Connectin; D003599:Cytoskeleton / Q000648:ultrastructure*; D005347:Fibroblasts; D007158:Immunologic Techniques; D046911:Macromolecular Substances; D008871:Microvilli / Q000648:ultrastructure; D009124:Muscle Proteins / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D011494:Protein Kinases
D064211:Connectin; D046911:Macromolecular Substances; D009124:Muscle Proteins; D011494:Protein Kinases; D009218:Myosins