Journal
CELLULAR AND MOLECULAR LIFE SCIENCES
Volume 69, Issue 7, Pages 1137-1151Publisher
SPRINGER BASEL AG
DOI: 10.1007/s00018-011-0812-5
Keywords
Adaptor protein; Cytoskeleton; Matrix degradation; Cell invasion; c-Src activation
Categories
Funding
- Canadian Institutes of Health Research [MOP-13270, MOP-42546]
- Peterborough K. M. Hunter Graduate Studentship for Cancer Research
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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. AFAP Delta ABD, 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 AFAP Delta ABD 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 AFAP Delta ABD and c-Src. When AFAP Delta ABD-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 AFAP Delta ABD, 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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