Cofilin overexpression affects actin cytoskeleton organization and migration of human colon adenocarcinoma cells

Histochemistry and Cell Biology, Nov 2012

The dynamic reorganization of actin cytoskeleton is regulated by a large number of actin-binding proteins. Among them, the interaction of ADF/cofilin with monomeric and filamentous actin is very important, since it severs actin filaments. It also positively influences actin treadmilling. The activity of ADF/cofilin is reversibly regulated by phosphorylation and dephosphorylation at Ser-3, with the phosphorylated form (P-cofilin) being inactive. Here, we studied the effects of overexpression of cofilin and two cofilin variants in the human colon adenocarcinoma LS180 cell line. We have generated the LS180 cells expressing three different cofilin variants: WT (wild type), Ser 3 Ala (S3A) (constitutively active) or Ser 3 Asp (S3D) (constitutively inactive cofilin). The cells expressing WT cofilin were characterized by abundant cell spreading and colocalization of cofilin with the submembranous F-actin. Similar effects were observed in cells expressing S3A cofilin. In contrast, LS180 cells expressing S3D cofilin remained longitudinal in morphology and cofilin was equally distributed within the cell body. Furthermore, the migration ability of LS180 cells expressing different cofilin mutants was analyzed. In comparison to control cells, we have noticed a significant, approximately fourfold increase in the migration factor value of cells overexpressing WT type cofilin. The overexpression of S3D cofilin resulted in an almost complete inhibition of cell motility. The estimation of actin pool in the cytosol of LS180 cells expressing S3A cofilin has shown a significantly lower level of total actin in reference to control cells. The opposite effect was observed in LS180 cells overexpressing S3D cofilin. In summary, the results of our experiments indicate that phosphorylation “status” of cofilin is a factor affecting the actin cytoskeleton organization and migration abilities of colon adenocarcinoma LS180 cells.

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Cofilin overexpression affects actin cytoskeleton organization and migration of human colon adenocarcinoma cells

Agnieszka Popow-Wozniak 0 1 2 Antonina Joanna Mazur 0 1 2 Hans Georg Mannherz 0 1 2 Maria Malicka-Baszkiewicz 0 1 2 Dorota Nowak 0 1 2 0 M. Malicka-Baszkiewicz 1 A. Popow-Wozniak (&) A. J. Mazur M. Malicka-Baszkiewicz D. Nowak Department of Cell Pathology, Faculty of Biotechnology, University of Wrocaw , Przybyszewskiego 63, 51-148 Wrocaw, Poland 2 A. J. Mazur H. G. Mannherz Department of Anatomy and Molecular Embryology, Ruhr-University , 44780 Bochum, Germany The dynamic reorganization of actin cytoskeleton is regulated by a large number of actin-binding proteins. Among them, the interaction of ADF/cofilin with monomeric and filamentous actin is very important, since it severs actin filaments. It also positively influences actin treadmilling. The activity of ADF/cofilin is reversibly regulated by phosphorylation and dephosphorylation at Ser-3, with the phosphorylated form (P-cofilin) being inactive. Here, we studied the effects of overexpression of cofilin and two cofilin variants in the human colon adenocarcinoma LS180 cell line. We have generated the LS180 cells expressing three different cofilin variants: WT (wild type), Ser 3 Ala (S3A) (constitutively active) or Ser 3 Asp (S3D) (constitutively inactive cofilin). The cells expressing WT cofilin were characterized by abundant cell spreading and colocalization of cofilin with the submembranous F-actin. Similar effects were observed in cells expressing S3A cofilin. In contrast, LS180 cells expressing S3D cofilin remained longitudinal in morphology and cofilin was equally distributed within the cell body. Furthermore, the migration ability of LS180 cells expressing different cofilin mutants was analyzed. In comparison to control cells, we have noticed a significant, approximately fourfold increase in the migration factor value of cells overexpressing WT type cofilin. The overexpression of S3D cofilin resulted in an almost complete inhibition of cell motility. The estimation of actin pool in the cytosol of LS180 cells expressing S3A cofilin has shown a significantly lower level of total actin in reference to control cells. The opposite effect was observed in LS180 cells overexpressing S3D cofilin. In summary, the results of our experiments indicate that phosphorylation ''status'' of cofilin is a factor affecting the actin cytoskeleton organization and migration abilities of colon adenocarcinoma LS180 cells. - Tumor cell motility, migration to distant locations and invasion crucially depend on the reorganization of the actin cytoskeleton (Sheterline et al. 1998; Webb and Horwitz 2003; Lambrechts et al. 2004). Through the regulation of actin dynamics cells can coordinate these different functions. Cellular activity and behavior are mediated by internal and external cues, which activate a number of small GTP-binding proteins of the Rho family (Hall 2005) and thus orchestrate actin filament dynamics by coordinative activation of a number of actin-binding proteins (ABPs) (Pantaloni et al. 2001). Among them, the interaction of cofilin and actin-depolymerizing factor (ADF) with monomeric and filamentous actin is of paramount importance, since they stimulate the dynamic behavior of actin filaments (Condeelis et al. 2001b). Both cofilin and ADF are able to sever existing F-actin filaments. It also positively influences actin treadmilling, however the exact mechanism of this process is described by several models and hypotheses (Carlier et al. 1997; Chen et al. 2000; Condeelis 2001; Gurniak et al. 2005; Andrianantoandro and Pollard 2006; Pavlov et al. 2007; van Rheenen et al. 2007; Kuchi et al. 2007; van Rheenen et al. 2009). The activity of cofilin is reversibly regulated by phosphorylation and dephosphorylation at Ser-3, with the phosphorylated form (P-cofilin) being inactive. It is known that LIM-kinases (LIMK 1 and LIMK 2) and TES-kinase (TESK), that inactivate cofilin, are activated by RhoROCK and PAK kinases (Mizuno et al. 1994; Okano et al. 1995). The corresponding phosphatase Slingshot (SSH) (Okano et al. 1995) and a member of haloacid dehalogenases, Chronofin (Gohla et al. 2005), regulate the activity of cofilin by dephosphorylation (Mizuno et al. 1994). Furthermore, the biological pathways leading to cofilin activation are stimulated by epidermal growth factor (EGF) (Mouneimne et al. 2004, 2006). EGF is an important chemoattractant, which plays a crucial role in metastasis of mammary tumors (Wyckoff et al. 2004; Kedrin et al. 2007). Upon EGF stimulation of carcinoma cells cofilin is mobilized and activated to act locally under the cell membrane leading to a reorganization of actin cytoskeleton resulting in formation of cellular protrusions, such as lamellipodia and invadopodia. These processes together with chemotactic cues determine the direction of migration (Ghosh et al. 2004; Condeelis et al. 2005). It was previously shown that transfection of carcinoma cells with anticofilin siRNA (Hotulainen et al. 2005) or overexpression of a constitutively ac (...truncated)


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Agnieszka Popow-Woźniak, Antonina Joanna Mazur, Hans Georg Mannherz, Maria Malicka-Błaszkiewicz, Dorota Nowak. Cofilin overexpression affects actin cytoskeleton organization and migration of human colon adenocarcinoma cells, Histochemistry and Cell Biology, 2012, pp. 725-736, Volume 138, Issue 5, DOI: 10.1007/s00418-012-0988-2