The actin-binding domain of actin filament-associated protein (AFAP) is involved in the regulation of cytoskeletal structure |
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Authors: | Helan?Xiao Bing?Han Monika?Lodyga Xiao-Hui?Bai Yingchun?Wang Email author" target="_blank">Mingyao?LiuEmail author |
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Institution: | (1) Division of Cellular and Molecular Biology, University Health Network Toronto General Research Institute, Toronto, ON, Canada;(2) Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, ON, Canada;(3) Department of Surgery, Faculty of Medicine, University of Toronto, Room TMDT 2-814, 101 College Street, Toronto, ON, M5G 1L7, Canada; |
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Abstract: | Actin filament-associated protein (AFAP) plays a critical role in the regulation of actin filament integrity, formation and
maintenance of the actin network, function of focal contacts, and cell migration. Here, we show that endogenous AFAP was present
not only in the cytoskeletal but also in the cytosolic fraction. Depolymerization of actin filaments with cytochalasin D or
latrunculin A increased AFAP in the cytosolic fraction. AFAP harbors an actin-binding domain (ABD) in its C-terminus. AFAPΔABD,
an AFAP mutant with selective ABD deletion, was mainly in the cytosolic fraction when overexpressed in the cells, which was
associated with a disorganized cytoskeleton with reduced stress fibers, accumulation of F-actin on cellular membrane, and
formation of actin-rich small dots. Cortactin, a well-known podosome marker, was colocalized with AFAPΔABD in these small
dots at the ventral surface of the cell, indicating that these small dots fulfill certain criteria of podosomes. However,
these podosome-like small dots did not digest gelatin matrix. This may be due to the reduced interaction between AFAPΔABD
and c-Src. When AFAPΔABD-transfected cells were stimulated with phorbol ester, they formed podosome-like structures with larger
sizes, less numerous and longer life span, in comparison with wild-type AFAP-transfected cells. These results indicate that
the association of AFAP with F-actin through ABD is crucial for AFAP to regulate cytoskeletal structures. The AFAPΔABD, as
cytosolic proteins, may be more accessible to the cellular membrane, podosome-like structures, and thus be more interactive
for the regulation of cellular functions. |
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