Unanticipated temporal and spatial effects of sarcomeric α-actinin peptides expressed in PtK2 cells

Academic Article

Abstract

  • To understand the multiple roles of α-actinin in the assembly of (1) Z bands in muscle, and (2) a variety of cytoskeletal structures in non-muscle cells, 4 sarcomeric α-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/MYC); and (4) minus actin- binding site (-A/MYC). These four cDNAs were individually transfected into PtK2 cells. The exogenous sarcomeric α-actinin (s-α-actinin/MYC) was followed with labeled anti-MYC, the endogenous non-sarcomeric α-actinin (non-s-α-actinin) with labeled anti-non-s-α-actinin. The salient findings were: (1) the selective intracellular localizations of each expressed MYC- tagged peptide differed one from the other; (2) their respective localizations in the 10-24-h post-transfection (p.t.) period differed from their localizations in the 48-72-h p.t. period; (3) each MYC-positive peptide was cytotoxic, but each in a distinctive way; and (4) while the selective targeting of FL/MYC to dense bodies, adhesion plaques, adherens junctions, and ruffled membranes was consistent with binding studies in cell-free systems, the incorporation of the mutated peptides, particularly +A/MYC and -A/MYC was not. Changes in localization over time and the distinctive cytopathologies probably reflect domain-specific targeting. They also suggest unexpected cooperative involvement of multiple domains of α-actinin with specific receptors in distal cytoskeletal structures. To date, such qualitative in vivo interactions have not been described either in in vitro binding studies, or in short-term experiments involving localization and/or fate of microinjected labeled molecules into living cells.
  • Authors

    Published In

  • Cytoskeleton  Journal
  • Author List

  • Hijikata T; Lin ZX; Holtzer S; Choi J; Sweeney HL; Holtzer H
  • Start Page

  • 54
  • End Page

  • 74
  • Volume

  • 38
  • Issue

  • 1