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

Cell Motil Cytoskeleton. 1997;38(1):54-74. doi: 10.1002/(SICI)1097-0169(1997)38:1<54::AID-CM6>3.0.CO;2-H.


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/MYC); and (4) minus actin-binding site (-A/MYC). These four cDNAs were individually transfected into PtK2 cells. The exogenous sarcomeric alpha-actinin (s-alpha-actinin/MYC) was followed with labeled anti-MYC, the endogenous non-sarcomeric alpha-actinin (non-s-alpha-actinin) with labeled anti-non-s-alpha-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 alpha-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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actinin / analysis*
  • Actinin / chemistry
  • Actinin / genetics
  • Actinin / physiology
  • Animals
  • Base Sequence
  • Binding Sites
  • Cell Death
  • Cell Line
  • Cytoskeleton / chemistry*
  • Dimerization
  • Epithelial Cells / metabolism
  • Kidney / cytology
  • Macropodidae
  • Molecular Sequence Data
  • Peptides / analysis
  • Peptides / physiology
  • Recombinant Fusion Proteins / analysis
  • Sarcomeres / chemistry*
  • Time Factors
  • Transfection


  • Peptides
  • Recombinant Fusion Proteins
  • Actinin