Advertisement

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

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.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 199

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. Auguet T, Molina JC, Lorenzo A, Vila J, Sirvent JJ, Richart C (2000) Synchronus renal cell carcinoma and Bellini duct carcinoma: a case report on a rare coincidence. World J Urol 18:449–451

  2. Baggett AW, Cournia Z, Han MS, Patargias G, Glass AC, Liu SY, Nolen BJ (2012) Structural characterization and computer-aided optimization of a small-molecule inhibitor of the arp2/3 complex, a key regulator of the actin cytoskeleton. ChemMedChem 7:1286–1294

  3. Bosse T, Ehinger J, Czuchra A, Benesch S, Steffen A, Wu X, Schloen K, Niemann HH, Scita G, Stradal TE, Brakebusch C, Rottner K (2007) Cdc42 and phosphoinositide 3-kinase drive Rac-mediated actin polymerization downstream of c-Met in distinct and common pathways. Mol Cell Biol 27:6615–6628

  4. Bryant DM, Mostov KE (2008) From cells to organs: building polarized tissue. Nat Rev Mol Cell Biol 9:887–901

  5. Cantarella C, Sepe L, Fioretti F, Ferrari MC, Paolella G (2009) Analysis and modelling of motility of cell populations with MotoCell. BMC Bioinforma 10(Suppl 12):S12

  6. Carter MD, Tha S, McLoughlin MG, Owen DA (1992) Collecting duct carcinoma of the kidney: a case report and review of the literature. J Urol 147:1096–1098

  7. Chen Z, Borek D, Padrick SB, Gomez TS, Metlagel Z, Ismail AM, Umetani J, Billadeau DD, Otwinowski Z, Rosen MK (2010) Structure and control of the actin regulatory WAVE complex. Nature 468:533–538

  8. Collins A, Warrington A, Taylor KA, Svitkina T (2011) Structural organization of the actin cytoskeleton at sites of clathrin-mediated endocytosis. Curr Biol 21:1167–1175

  9. Di Nardo A, Cicchetti G, Falet H, Hartwig JH, Stossel TP, Kwiatkowski DJ (2005) Arp2/3 complex-deficient mouse fibroblasts are viable and have normal leading-edge actin structure and function. Proc Natl Acad Sci USA 102:16263–16268

  10. Dobronski P, Czaplicki M, Kozminska E, Pykalo R (1999) Collecting (Bellini) duct carcinoma of the kidney—clinical, radiologic and immunohistochemical findings. Int Urol Nephrol 31:601–609

  11. Duleh SN, Welch MD (2010) WASH and the Arp2/3 complex regulate endosome shape and trafficking. Cytoskeleton (Hoboken) 67:193–206

  12. Firat-Karalar EN, Welch MD (2011) New mechanisms and functions of actin nucleation. Curr Opin Cell Biol 23:4–13

  13. Gomez TS, Kumar K, Medeiros RB, Shimizu Y, Leibson PJ, Billadeau DD (2007) Formins regulate the actin-related protein 2/3 complex-independent polarization of the centrosome to the immunological synapse. Immunity 26:177–190

  14. Gu J, Tamura M, Pankov R, Danen EH, Takino T, Matsumoto K, Yamada KM (1999) Shc and FAK differentially regulate cell motility and directionality modulated by PTEN. J Cell Biol 146:389–403

  15. Gupton SL, Anderson KL, Kole TP, Fischer RS, Ponti A, Hitchcock-DeGregori SE, Danuser G, Fowler VM, Wirtz D, Hanein D, Waterman-Storer CM (2005) Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin. J Cell Biol 168:619–631

  16. Hetrick B, Han MS, Helgeson LA, Nolen BJ (2013) Small molecules CK-666 and CK-869 inhibit actin-related protein 2/3 complex by blocking an activating conformational change. Chem Biol 20:701–712

  17. Hotulainen P, Lappalainen P (2006) Stress fibers are generated by two distinct actin assembly mechanisms in motile cells. J Cell Biol 173:383–394

  18. Ilatovskaya DV, Pavlov TS, Levchenko V, Negulyaev YA, Staruschenko A (2011) Cortical actin binding protein cortactin mediates ENaC activity via Arp2/3 complex. FASEB J 25:2688–2699

  19. Karpushev AV, Ilatovskaya DV, Staruschenko A (2010) The actin cytoskeleton and small G protein RhoA are not involved in flow-dependent activation of ENaC. BMC Res Notes 3:210

  20. Karpushev AV, Levchenko V, Ilatovskaya DV, Pavlov TS, Staruschenko A (2011) Novel role of Rac1/WAVE signaling mechanism in regulation of the epithelial Na+ channel. Hypertension 57:996–1002

  21. Kinoshita T, Nohata N, Watanabe-Takano H, Yoshino H, Hidaka H, Fujimura L, Fuse M, Yamasaki T, Enokida H, Nakagawa M, Hanazawa T, Okamoto Y, Seki N (2012) Actin-related protein 2/3 complex subunit 5 (ARPC5) contributes to cell migration and invasion and is directly regulated by tumor-suppressive microRNA-133a in head and neck squamous cell carcinoma. Int J Oncol 40:1770–1778

  22. Lebensohn AM, Kirschner MW (2009) Activation of the WAVE complex by coincident signals controls actin assembly. Mol Cell 36:512–524

  23. Machesky LM, Insall RH (1998) Scar1 and the related Wiskott-Aldrich syndrome protein, WASP, regulate the actin cytoskeleton through the Arp2/3 complex. Curr Biol 8:1347–1356

  24. Machesky LM, Atkinson SJ, Ampe C, Vandekerckhove J, Pollard TD (1994) Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose. J Cell Biol 127:107–115

  25. Magdalena J, Millard TH, Etienne-Manneville S, Launay S, Warwick HK, Machesky LM (2003) Involvement of the Arp2/3 complex and Scar2 in Golgi polarity in scratch wound models. Mol Biol Cell 14:670–684

  26. Nakamura Y, Wood CL, Patton AP, Jaafari N, Henley JM, Mellor JR, Hanley JG (2011) PICK1 inhibition of the Arp2/3 complex controls dendritic spine size and synaptic plasticity. EMBO J 30:719–730

  27. Naray-Fejes-Toth A, Helms MN, Stokes JB, Fejes-Toth G (2004) Regulation of sodium transport in mammalian collecting duct cells by aldosterone-induced kinase, SGK1: structure/function studies. Mol Cell Endocrinol 217:197–202

  28. Nicholson-Dykstra SM, Higgs HN (2008) Arp2 depletion inhibits sheet-like protrusions but not linear protrusions of fibroblasts and lymphocytes. Cell Motil Cytoskeleton 65:904–922

  29. Nolen BJ, Tomasevic N, Russell A, Pierce DW, Jia Z, McCormick CD, Hartman J, Sakowicz R, Pollard TD (2009) Characterization of two classes of small molecule inhibitors of Arp2/3 complex. Nature 460:1031–1034

  30. Peleg B, Disanza A, Scita G, Gov N (2011) Propagating cell-membrane waves driven by curved activators of actin polymerization. PLoS ONE 6:e18635

  31. Pollard TD (2007) Regulation of actin filament assembly by Arp2/3 complex and formins. Annu Rev Biophys Biomol Struct 36:451–477

  32. Robinson RC, Turbedsky K, Kaiser DA, Marchand JB, Higgs HN, Choe S, Pollard TD (2001) Crystal structure of Arp2/3 complex. Science 294:1679–1684

  33. Rouiller I, Xu XP, Amann KJ, Egile C, Nickell S, Nicastro D, Li R, Pollard TD, Volkmann N, Hanein D (2008) The structural basis of actin filament branching by the Arp2/3 complex. J Cell Biol 180:887–895

  34. Saarikangas J, Zhao H, Lappalainen P (2010) Regulation of the actin cytoskeletonplasma membrane interplay by phosphoinositides. Physiol Rev 90:259–289

  35. Sarmiento C, Wang W, Dovas A, Yamaguchi H, Sidani M, El-Sibai M, Desmarais V, Holman HA, Kitchen S, Backer JM, Alberts A, Condeelis J (2008) WASP family members and formin proteins coordinate regulation of cell protrusions in carcinoma cells. J Cell Biol 180:1245–1260

  36. Shao D, Forge A, Munro PM, Bailly M (2006) Arp2/3 complex-mediated actin polymerisation occurs on specific pre-existing networks in cells and requires spatial restriction to sustain functional lamellipod extension. Cell Motil Cytoskeleton 63:395–414

  37. Soderling SH (2009) Grab your partner with both hands: cytoskeletal remodeling by Arp2/3 signaling. Sci Signal 2:e5

  38. Spillane M, Ketschek A, Jones SL, Korobova F, Marsick B, Lanier L, Svitkina T, Gallo G (2011) The actin nucleating Arp2/3 complex contributes to the formation of axonal filopodia and branches through the regulation of actin patch precursors to filopodia. Dev Neurobiol 32:17671–17689

  39. Steffen A, Faix J, Resch GP, Linkner J, Wehland J, Small JV, Rottner K, Stradal TE (2006) Filopodia formation in the absence of functional. Mol Biol Cell 17:2581–2591

  40. Strasser GA, Rahim NA, VanderWaal KE, Gertler FB, Lanier LM (2004) Arp2/3 is a negative regulator of growth cone translocation. Neuron 43:81–94

  41. Sun CX, Magalhaes MA, Glogauer M (2007) Rac1 and Rac2 differentially regulate actin free barbed end formation downstream of the fMLP receptor. J Cell Biol 179:239–245

  42. Sun SC, Wang ZB, Xu YN, Lee SE, Cui XS, Kim NH (2011) Arp2/3 complex regulates asymmetric division and cytokinesis in mouse oocytes. PLoS ONE 6:e18392

  43. Suraneni P, Rubinstein B, Unruh JR, Durnin M, Hanein D, Li R (2012) The Arp2/3 complex is required for lamellipodia extension and directional fibroblast cell migration. J Cell Biol 197:239–251

  44. Verdorfer I, Culig Z, Hobisch A, Bartsch G, Hittmair A, Duba HC, Erdel M (1998) Characterisation of a collecting duct carcinoma by cytogenetic analysis and comparative genomic hybridisation. Int J Oncol 13:461–464

  45. Volkmann N, Amann KJ, Stoilova-McPhie S, Egile C, Winter DC, Hazelwood L, Heuser JE, Li R, Pollard TD, Hanein D (2001) Structure of Arp2/3 complex in its activated state and in actin filament branch junctions. Science 293:2456–2459

  46. Wang W, Goswami S, Lapidus K, Wells AL, Wyckoff JB, Sahai E, Singer RH, Segall JE, Condeelis JS (2004) Identification and testing of a gene expression signature of invasive carcinoma cells within primary mammary tumors. Cancer Res 64:8585–8594

  47. Warren DT, Andrews PD, Gourlay CW, Ayscough KR (2002) Sla1p couples the yeast endocytic machinery to proteins regulating actin dynamics. J Cell Sci 115:1703–1715

  48. Watanabe S, Okawa K, Miki T, Sakamoto S, Morinaga T, Segawa K, Arakawa T, Kinoshita M, Ishizaki T, Narumiya S (2010) Rho and anillin-dependent control of mDia2 localization and function in cytokinesis. Mol Biol Cell 21:3193–3204

  49. Weed SA, Parsons JT (2001) Cortactin: coupling membrane dynamics to cortical actin assembly. Oncogene 20:6418–6434

  50. Weed SA, Karginov AV, Schafer DA, Weaver AM, Kinley AW, Cooper JA, Parsons JT (2000) Cortactin localization to sites of actin assembly in lamellipodia requires interactions with F-actin and the Arp2/3 complex. J Cell Biol 151:29–40

  51. Wilson PD (2011) Apico-basal polarity in polycystic kidney disease epithelia. Biochim Biophys Acta 1812:1239–1248

  52. Wu C, Asokan SB, Berginski ME, Haynes EM, Sharpless NE, Griffith JD, Gomez SM, Bear JE (2012) Arp2/3 is critical for lamellipodia and response to extracellular matrix cues but is dispensable for chemotaxis. Cell 148:973–987

  53. Yamaguchi H, Condeelis J (2007) Regulation of the actin cytoskeleton in cancer cell migration and invasion. Biochim Biophys Acta 1773:642–652

  54. Yamaguchi H, Lorenz M, Kempiak S, Sarmiento C, Coniglio S, Symons M, Segall J, Eddy R, Miki H, Takenawa T, Condeelis J (2005) Molecular mechanisms of invadopodium formation: the role of the N-WASP-Arp2/3 complex pathway and cofilin. J Cell Biol 168:441–452

  55. Zagoria RJ, Wolfman NT, Karstaedt N, Hinn GC, Dyer RB, Chen YM (1990) CT features of renal cell carcinoma with emphasis on relation to tumor size. Investig Radiol 25:261–266

Download references

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.).

Author information

Correspondence to Daria V. Ilatovskaya.

Additional information

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

Electronic supplementary material

Below is the link to the electronic supplementary material.

Fig. S1
figure7

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
figure8

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
figure9

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)

(AVI 9 mb)

(AVI 9 mb)

High resolution image (TIFF 23 kb)

High resolution image (TIFF 147 kb)

High resolution image (TIFF 41 kb)

(AVI 9 mb)

(AVI 9 mb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Keywords

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