Arp2/3 complex inhibitors adversely affect actin cytoskeleton remodeling in the cultured murine kidney collecting duct M-1 cells
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Dynamic remodeling of the actin cytoskeleton plays an essential role in cell migration and various signaling processes in living cells. One of the critical factors that controls the nucleation of new actin filaments in eukaryotic cells is the actin-related protein 2/3 (Arp2/3) complex. Recently, two novel classes of small molecules that bind to different sites on the Arp2/3 complex and inhibit its ability to nucleate F-actin have been discovered and described. The current study aims at investigating the effects of CK-0944666 (CK-666) and its analogs (CK-869 and inactive CK-689) on the reorganization of the actin microfilaments in the cortical collecting duct cell line, M-1. We show that treatment with CK-666 and CK869 results in the reorganization of F-actin and drastically affects cell motility rate. The concentrations of the compounds used in this study (100–200 μM) neither cause loss of cell viability nor influence cell shape or monolayer integrity; hence, the effects of described compounds were not due to structural side effects. Therefore, we conclude that the Arp2/3 complex plays an important role in cell motility and F-actin reorganization in M-1 cells. Furthermore, CK-666 and its analogs are useful tools for the investigation of the Arp2/3 complex.
KeywordsArp2/3 complex Actin filaments CK-0944666 Cell motility
The authors would like to acknowledge Dr. Grigoriy Stein (Institute of Cytology RAS) for help with microscopy experiments and sincerely thank Vladislav Levchenko (Medical College of Wisconsin) for help with the MTT cytotoxicity assay and critical reading of the manuscript. We also acknowledge the help of Glenn Slocum and Bradley Endres (Medical College of Wisconsin) for helpful discussion and correction of the manuscript. The laboratory of Dr. A.N. Tomilin (Institute of Cytology RAS, St. Petersburg) is recognized for sharing the antibodies for immunofluorescence. This research was supported by R01HL108880 from the National Heart, Lung, and Blood Institute and the American Diabetes Association grant 1-10-BS-168 (to A.S.), Russian Foundation for Basic Research grant RFBR-13-04-00700 and the Molecular and Cell Biology Program of the Russian Academy of Sciences (to Y.A.N. and D.V.I.), and the OPTEC research grant (to D.V.I. and L.S.S.).
Effects of CK-666 on wound healing in M-1 cells monolayer. (a), Woundhealing montage of M-1 cells’ treated with DMSO (control, S1a) and 200 μM of CK-666 for up to 14 hrs (S1b). Shown is a movie created from images taken every 3 min during 14 hours of treatment. Scale bar is shown. (AVI 8.77 mb) (AVI 8.77 mb)
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