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 t he chicken intestinal epithelial cell brush border cytoskeleton (Eiler tsen, K. J., and T. C. S. Keller. 1992. J. Cell Biol. 119:549-557). He re, 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 titi n and myosin in vitro, we purified both proteins from isolated intesti nal epithelial eel brush borders by a combination of gel filtration an d hydroxyapatite column chromatography. Electron microscopy of brush b order myosin bipolar filaments assembled in the presence and absence o f cellular titin revealed a cellular titin-dependent side-by-side and end-to-end alignment of the filaments into highly ordered arrays. Immu nogold labeling confirmed cellular titin association with the filament arrays. Under similar assembly conditions, purified chicken pectorali s muscle titin formed much less regular aggregates of muscle myosin bi polar filaments. Sucrose density gradient analyses of both cellular an d muscle titin-myosin supramolecular arrays demonstrated that the cell ular titin and myosin isoforms coassembled with a myosin/titin ratio o f similar to 25:1, whereas the muscle isoforms coassembled with a myos in:titin ratio of similar to 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. O ur results demonstrate that cellular titin can organize an isoform-spe cific association of myosin II bipolar filaments and support the possi bility that cellular titin is a key organizing component of the brush border and other myosin II-containing cytoskeletal structures includin g stress fibers.