T. Hijikata et al., UNANTICIPATED TEMPORAL AND SPATIAL EFFECTS OF SARCOMERIC ALPHA-ACTININ PEPTIDES EXPRESSED IN PTK2 CELLS, Cell motility and the cytoskeleton, 38(1), 1997, pp. 54-74
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.