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The actin-binding domain of actin filament-associated protein (AFAP) is involved in the regulation of cytoskeletal structure

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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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Acknowledgments

We thank Dr. Daniel C. Flynn (West Virginia University) for providing AFAP antibody and plasmids. We thank Ms. Serisha Moodley for technical assistance and Ms. Lauren Turrell for proofreading the manuscript. This work was supported by Canadian Institutes of Health Research operating grants MOP-13270 and MOP-42546. HX was supported by Peterborough K. M. Hunter Graduate Studentship for Cancer Research.

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Authors and Affiliations

  1. Division of Cellular and Molecular Biology, University Health Network Toronto General Research Institute, Toronto, ON, Canada

    Helan Xiao, Bing Han, Monika Lodyga, Xiao-Hui Bai, Yingchun Wang & Mingyao Liu

  2. Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, ON, Canada

    Helan Xiao & Mingyao Liu

  3. Department of Surgery, Faculty of Medicine, University of Toronto, Room TMDT 2-814, 101 College Street, Toronto, ON, M5G 1L7, Canada

    Mingyao Liu

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  1. Helan Xiao
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Correspondence to Mingyao Liu.

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Xiao, H., Han, B., Lodyga, M. et al. The actin-binding domain of actin filament-associated protein (AFAP) is involved in the regulation of cytoskeletal structure. Cell. Mol. Life Sci. 69, 1137–1151 (2012). https://doi.org/10.1007/s00018-011-0812-5

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  • DOI: https://doi.org/10.1007/s00018-011-0812-5

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