Arp2/3 complex inhibitors adversely affect actin cytoskeleton remodeling in the cultured murine kidney collecting duct M-1 cells


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.

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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.).

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Correspondence to Daria V. Ilatovskaya.

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Daria V. Ilatovskaya and Vladislav Chubinskiy-Nadezhdin made equal contributions.

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Fig. S1

200 μM CK-666 does not affect the vitality of M-1 cells. The graph demonstrates data from viability assay that assessed the effects of 100 and 200 μM CK-666 on M-1 cells vitality after 2 h of treatment. ns non-significant (JPEG 28 kb)

Fig. S2

Effect of CK-666 analog, CK-869, on cytoskeletal distribution in M-1 cells. a, M-1 cells were pretreated with CK-869 in concentration of 200 μM for 2 h. Images were taken from the M-1 cells stained with rhodamine-phalloidin to visualize actin microfilaments. Shown are representative images at ×100 (top row, a) and expanded close-up images (bottom row, b). Scale bar 25 μm. c Average motility rates of the M-1 cells before and after treatment with CK-869 normalized to the control level (vehicle). Shown are motility rates for three time periods—control, 1 and 2 h after treatment. *p ≤ 0.005. (JPEG 60 kb)

Fig. S3

Treatment of cells with CK-666 and CK-689 had no effect on the directionality of migration. a The D/T ratio of cell movement. b Polar plots representing random non-directional movement of cells in control and after drug treatment (JPEG 72 kb)

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Ilatovskaya, D.V., Chubinskiy-Nadezhdin, V., Pavlov, T.S. et al. Arp2/3 complex inhibitors adversely affect actin cytoskeleton remodeling in the cultured murine kidney collecting duct M-1 cells. Cell Tissue Res 354, 783–792 (2013) doi:10.1007/s00441-013-1710-y

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  • Arp2/3 complex
  • Actin filaments
  • CK-0944666
  • Cell motility