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/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.
Hijikata
T
T
Department of Anatomy, Medical School, University of Gunma, Maebashi, Japan.
Lin
Z X
ZX
Holtzer
S
S
Choi
J
J
Sweeney
H L
HL
Holtzer
H
H
D000185:Actinin / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016923:Cell Death; D002460:Cell Line; D003599:Cytoskeleton / Q000737:chemistry*; D019281:Dimerization; D004847:Epithelial Cells / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D007614:Macropodidae; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012518:Sarcomeres / Q000737:chemistry*; D013997:Time Factors; D014162:Transfection
D010455:Peptides; D011993:Recombinant Fusion Proteins; D000185:Actinin