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Cell and Tissue Research

, Volume 354, Issue 3, pp 783–792 | Cite as

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

  • Daria V. IlatovskayaEmail author
  • Vladislav Chubinskiy-Nadezhdin
  • Tengis S. Pavlov
  • Leonid S. Shuyskiy
  • Viktor Tomilin
  • Oleg Palygin
  • Alexander Staruschenko
  • Yuri A. Negulyaev
Regular Article

Abstract

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.

Keywords

Arp2/3 complex Actin filaments CK-0944666 Cell motility 

Notes

Acknowledgments

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.).

Supplementary material

441_2013_1710_Fig7_ESM.jpg (28 kb)
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)

441_2013_1710_MOESM1_ESM.tif (23 kb)
High resolution image (TIFF 23 kb)
441_2013_1710_Fig8_ESM.jpg (60 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)

441_2013_1710_MOESM2_ESM.tif (147 kb)
High resolution image (TIFF 147 kb)
441_2013_1710_Fig9_ESM.jpg (73 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)

441_2013_1710_MOESM3_ESM.tif (41 kb)
High resolution image (TIFF 41 kb)
Video S1a and Video S1b

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)

(AVI 9 mb)

(AVI 9 mb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Daria V. Ilatovskaya
    • 1
    • 2
    Email author
  • Vladislav Chubinskiy-Nadezhdin
    • 1
  • Tengis S. Pavlov
    • 2
  • Leonid S. Shuyskiy
    • 1
  • Viktor Tomilin
    • 1
  • Oleg Palygin
    • 2
  • Alexander Staruschenko
    • 2
  • Yuri A. Negulyaev
    • 1
  1. 1.Institute of CytologyRussian Academy of SciencesSt. PetersburgRussia
  2. 2.Department of PhysiologyMedical College of WisconsinMilwaukeeUSA

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