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J. Cell Biol.,
Volume 143, Number 7, December 28, 1998 1981-1995


* Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom; and Focal adhesion assembly and actin stress fiber formation were studied in serum-starved Swiss 3T3
fibroblasts permeabilized with streptolysin-O. Permeabilization in the presence of GTP
Department of Physiology,
University College London, London WC1E 6JJ, United Kingdom
S stimulated rho-dependent formation of stress fibers, and the redistribution of vinculin and paxillin from a perinuclear
location to focal adhesions. Addition of GTP
S at
8 min after permeabilization still induced paxillin recruitment to focal adhesion-like structures at the ends
of stress fibers, but vinculin remained in the perinuclear region, indicating that the distributions of these two
proteins are regulated by different mechanisms. Paxillin recruitment was largely rho-independent, but could
be evoked using constitutively active Q71L ADP-ribosylation factor (ARF1), and blocked by NH2-terminally truncated
17ARF1. Moreover, leakage of endogenous
ARF from cells was coincident with loss of GTP
S-
induced redistribution of paxillin to focal adhesions,
and the response was recovered by addition of ARF1.
The ability of ARF1 to regulate paxillin recruitment to
focal adhesions was confirmed by microinjection of
Q71LARF1 and
17ARF1 into intact cells. Interestingly, these experiments showed that V14RhoA-
induced assembly of actin stress fibers was potentiated
by Q71LARF1. We conclude that rho and ARF1 activate complimentary pathways that together lead to the
formation of paxillin-rich focal adhesions at the ends of
prominent actin stress fibers.
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