Elsevier

Experimental Cell Research

Volume 316, Issue 7, 15 April 2010, Pages 1234-1240
Experimental Cell Research

Research Article
Regulation of cellular actin architecture by S100A10

https://doi.org/10.1016/j.yexcr.2010.01.022Get rights and content

Abstract

Actin structures are involved in several biological processes and the disruption of actin polymerisation induces impaired motility of eukaryotic cells. Different factors are involved in regulation and maintenance of the cytoskeletal actin architecture. Here we show that S100A10 participates in the particular organisation of actin filaments. Down-regulation of S100A10 by specific siRNA triggered a disorganisation of filamentous actin structures without a reduction of the total cellular actin concentration. In contrast, the formation of cytoskeleton structures containing tubulin was unhindered in S100A10 depleted cells. Interestingly, the cellular distribution of annexin A2, an interaction partner of S100A10, was unaffected in S100A10 depleted cells. Cells lacking S100A10 showed an impaired migration activity and were unable to close a scratched wound. Our data provide first insights of S100A10 function as a regulator of the filamentous actin network.

Introduction

Dynamic alterations of the actin network are involved in several physiological processes in eukaryotic cells. Actin is associated with cell migration and cell adhesions as well as in the formation of cellular structures like cytoskeleton and membrane architecture. It has been proposed that some members of the S100 protein family are engaged in regulation of the cytoskeleton dynamics. The S100 protein family is considered as a group of multitasking proteins involved in several biological processes including the Ca2+ signalling network, cell growth and motility, cell cycle progression as well as cell differentiation [1], [2]. In several studies, a complex composed of annexin A2 (ANX2) and S100A10 was detected which regulates cellular cytoskeleton [3], [4], [5], [6]. S100A10 is expressed in several different cells and tissues such as the epidermis where it is present in both, the cytosol and in membranes [7]. In contrast to other members of the S100 protein family, S100A10 is insensitive towards Ca2+ because the protein always shows the equivalent of a Ca2+-loaded structure and thus S100A10 is in a permanently active status [8]. Hence, S100A10 achieves biological functions, i.e. the well-described interaction with ANX2, in a calcium-independent manner [9]. S100A10 can be targeted to cell membranes due to the complex formation with ANX2. This interaction induces the formation of specific membrane domains accompanied by modulation of the actin cytoskeleton [10], [11]. It is conceivable that S100A10 functions not only as a structural scaffold protein connecting the two ANX2 chains in sub-membranous regions but may also achieve protein interactions itself in the absence of ANX2 [12]. Recently, it was shown that S100A10 is able to interact with several subunits of different ion channels and that it is involved in the regulation of the traffic of plasma membrane proteins [13], [14], [15], [16]. Additional interactions of S100A10 with cytosolic and membrane-associated proteins have also been described [17], [18]. S100A10 seems to have an influence in formation of actin containing structures. It was shown that an interaction between ANX2/S100A10 and the actin-binding protein AHNAK is able to regulate cell membrane architecture [5]. Furthermore, the ANX2/S100A10 complex is recruited by phosphatidylinositol (4,5)-biphosphate to membrane areas which function as actin assembly sites [6]. It should be noted, however, that ANX2 does not generally co-localise with actin in cells [19]. Recently, it was shown that ANX2 is not required for association of S100A10 with the extracellular plasma membrane as detected in the colorectal cancer cell line CCL-222. Interestingly, S100A10 seems to be involved in the regulation of invasive processes in colorectal cancer as down-regulation of S100A10 by specific siRNA attenuates the invasiveness of CCL-222 cells [20].

In the present study, to get a deeper insight in an S100A10 mediated regulation of actin arrangement we knocked down S100A10 by RNA interference to assess its contribution in actin dynamics as well as in cellular migration.

Section snippets

Cell culture

The human epithelial squamous carcinoma cell line A431 was cultured in DMEM supplemented with 10% fetal bovine serum. Cells were grown to 80% confluence and were passaged at a split ratio of 1:4. Cells were harvested at 70–90% confluence and lysed in a buffer containing 100 mM sodium phosphate pH 7.5, 5 mM EDTA, 2 mM MgCl2, 0.1% CHAPS, 500 μM leupeptin, and 0.1 mM PMSF. After centrifugation (15 min; 15,000 rpm) the supernatant was immediately processed further for Western blotting.

siRNA mediated knockdown of S100A10

Small

Down-regulation of S100A10 by specific siRNA

At first, we used RNA interference to manipulate the S100A10 protein level. Specific S100A10 siRNA was able to effectively deplete S100A10 in A431 cells 96 h after the first transfection (Fig. 1). The used S100A10 siRNA was specific since the protein level of S100A11, a further member of the S100 protein family, persisted unaffected by S100A10 down-regulation as assessed by Western blotting. Additionally, a signal derived from S100A10 was completely absent in A431 cells treated with the

Discussion

In the present study we investigated the ability of S100A10 to regulate different factors of the cytoskeleton. We showed that distinguishable components of specific cytoskeleton structures like actin fibres and microtubuli, respectively, are differently influenced by S100A10. In contrast to the cytoskeletal tubulin network, it seems that the formation of the proper cellular actin filament structures is dependent on the occurrence of S100A10. In cells lacking S100A10 by treatment with specific

Acknowledgments

This study was supported by a grant of the Wilhelm Sander-Stiftung to C.M. C.M. thanks F. von Eggeling for continuous support.

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    1

    Present address: Department of Tumor Biology, Fritz Lipmann Institut (FLI) - Leibniz Institute for Age Research, 07708 Jena, Germany.

    2

    Contributed equally to this work.

    3

    Department of Molecular Biology, Fritz Lipmann Institut (FLI) - Leibniz Institute for Age Research, 07708 Jena, Germany.

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