UNANTICIPATED TEMPORAL AND SPATIAL EFFECTS OF SARCOMERIC ALPHA-ACTININ PEPTIDES EXPRESSED IN PTK2 CELLS

To understand the multiple roles of alpha-actinin in the assembly of ( 1) Z bands in muscle, and (2) a variety of cytoskeletal structures in non-muscle cells, 4 sarcomeric alpha-actinin derived cDNAs tagged with a MYC epitope were constructed. The constructs were: (1) full-length (FL/MYC); (2) minus EF-hands (-EF/MYC); (3) actin-binding site ((+)A/M YC); and (4) minus actin-binding site ((-)A/MYC). These four cDNAs wer e individually transfected into PtK2 cells. The exogenous sarcomeric a lpha-actinin (s-alpha-actinin/MYC) was followed with labeled anti-MYC, the endogenous non-sarcomeric alpha-actinin (non-s-alpha-actinin) wit h labeled anti-non-s-alpha-actinin. The salient findings were: (1) the selective intracellular localizations of each expressed MYC-tagged pe ptide differed one from the other; (2) their respective localizations in the 10-24-h post-transfection (p.t.) period differed from their loc alizations in the 48-72-h p.t. period; (3) each MYC-positive peptide w as cytotoxic, but each in a distinctive way; and (4) while the selecti ve targeting of FL/MYC to dense bodies, adhesion plaques, adherens jun ctions, and ruffled membranes was consistent with binding studies in c ell-free systems, the incorporation of the mutated peptides, particula rly (+)A/MYC and -A/MYC was not. Changes in localization over time and the distinctive cytopathologies probably reflect domain-specific targ eting. They also suggest unexpected cooperative involvement of multipl e domains of alpha-actinin with specific receptors in distal cytoskele tal structures. To date, such qualitative in vivo interactions have no t been described either in in vitro binding studies, or in short-term experiments involving localization and/or fate of microinjected labele d molecules into living cells. (C) 1997 Wiley-Liss, Inc.