Abstract
Many essential functions in eukaryotic cells like phagocytosis, division, and motility rely on the dynamical properties of the actin cytoskeleton. A central player in the actin system is the Arp2/3 complex. Its activity is controlled by members of the WASP (Wiskott–Aldrich syndrome protein) family. In this work, we investigated the effect of the carbazole derivative wiskostatin, a recently identified N-WASP inhibitor, on actin-driven processes in motile cells of the social ameba Dictyostelium discoideum. Drug-treated cells exhibited an altered morphology and strongly reduced pseudopod formation. However, TIRF microscopy images revealed that the overall cortical network structure remained intact. We probed the mechanical stability of wiskostatin-treated cells using a microfluidic device. While the total amount of F-actin in the cells remained constant, their stiffness was strongly reduced. Furthermore, wiskostatin treatment enhanced the resistance to fluid shear stress, while spontaneous motility as well as chemotactic motion in gradients of cAMP were reduced. Our results suggest that wiskostatin affects the mechanical integrity of the actin cortex so that its rigidity is reduced and actin-driven force generation is impaired.
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References
Annesley SJ, Fisher PR (2009) Dictyostelium discoideum—a model for many reasons. Mol Cell Biochem 329(1–2):73–91
Bear JE, Rawls JF, Saxe CLS III (1998) SCAR, a WASP-related protein, isolated as a suppressor of receptor defects in late Dictyostelium development. J Cell Biol 142:1325–1335
Bompard G, Rabeharivelo G, Morin N (2008) Inhibition of cytokinesis by wiskostatin does not rely on N-WASP/Arp2/3 complex pathway. BMC Cell Biol 9:42
Bosgraaf L, Waijer A, Engel R, Visser AJWG, Wessels D, Soll D, Haastert PJMV (2005) RasGEF-containing proteins GbpC and GbpD have differential effects on cell polarity and chemotaxis in Dictyostelium. J Cell Sci 118:1899–1910
Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 72:248–254
Braunmüller S, Schmid L, Franke T (2011) Dynamics of red blood cells and vesicles in microchannels of oscillating width. J Phys 23(184116):184111–184118
Bretschneider T, Diez S, Anderson K, Heuser J, Clarke M, Muller-Taubenberger A, Kohler J, Gerisch G (2004) Dynamic actin patterns and Arp2/3 assembly at the substrate-attached surface of motile cells. Curr Biol 14(1):1–10
Buenemann M, Levine H, Rappel W-J, Sander LM (2010) The role of cell contraction and adhesion in Dictyostelium motility. Biophys J 99:50–58
Campellone KG, Welch MD (2010) A nucleator arms race: cellular control of actin assembly. Nat Rev Mol Cell Biol 11:237–251
Co C, Wong DT, Gierke S, Chang V, Taunton J (2007) Mechanism of actin network attachment to moving membranes: barbed end capture by N-WASP WH2 domains. Cell 128(5):901–913
Crocker JC, Grier DG (1996) Methods of digital video microscopy for colloidal studies. J Colloid Interface Sci 179(1):298–310
Diez S, Gerisch G, Anderson K, Muller-Taubenberger A, Bretschneider T (2005) Subsecond reorganization of the actin network in cell motility and chemotaxis. Proc Natl Acad Sci USA 102(21):7601–7606
Fletcher DA, Mullins RD (2010) Cell mechanics and the cytoskeleton. Nature 463:485–492
Gautreau A, Ho HYH, Li JX, Steen H, Gygi SP, Kirschner MW (2004) Purification and architecture of the ubiquitous wave complex. Proc Natl Acad Sci USA 101(13):4379–4383
Gerisch G, Bretschneider T, Müller-Taubenberger A, Simmeth E, Ecke M, Diez S, Anderson K (2004) Mobile actin clusters and traveling waves in cells recovering from actin depolymerization. Biophys J 87:3493–3503
Goley ED, Welch MD (2006) The Arp2/3 complex: an actin nucleator comes of age. Nat Rev Mol Cell Biol 7:713–726
Guerriero CJ, Weisz OA (2007) N-WASP inhibitor wiskostatin nonselectively perturbs membrane transport by decreasing cellular ATP levels. Am J Physiol Cell Physiol 292(4):C1562–C1566
Harwood A, Coates JC (2004) A prehistory of cell adhesion. Curr Opin Cell Biol 16:470–476
Ibarra N, Pollitt A, Insall RH (2005) Regulation of actin assembly by SCAR/WAVE proteins. Biochem Soc Trans 33:1243–1246
Kosta A, Laporte C, Lam D, Tresse E, Luciani M-F, Goldstein P (2006) How to assess and study cell death in Dictyostelium discoideum. Dictyostelium discoideum protocols. Methods Mol Biol 346:535–550
Kunda P, Craig G, Dominguez V, Baum B (2003) Abi, Sra1, and Kette control the stability and localization of SCAR/WAVE to regulate the formation of actin-based protrusions. Curr Biol 13(21):1867–1875
Leung DW, Morgan DM, Rosen MK (2006) Biochemical properties and inhibitors of N-WASP. Methods Enzymol 406:281–296
Li R, Gundersen GG (2008) Beyond polymer polarity: how the cytoskeleton builds a polarized cell. Nat Rev Mol Cell Biol 9:860–873
Machesky LM, Mullins RD, Higgs HN, Kaiser DA, Blanchoin L, May RC, Hall ME, Pollard TD (1999) Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex. Proc Natl Acad Sci USA 96:3739–3744
Maniak M, Rauchenberger R, Albrecht R, Murphy J, Gerisch G (1995) Coronin involved in phagocytosis: dynamics of particle-induced relocalization visualized by a green fluorescent protein tag. Cell 83:915–924
Morton WM, Ayscough KR, McLaughlin PJ (2000) Latrunculin alters the actin-monomer subunit interface to prevent polymerization. Nat Cell Biol 2:376–378
Myers SA, Han JW, Lee Y, Firtel RA, Chung CY (2005) A Dictyostelium homologue of WASP is required for polarized F-actin assembly during chemotaxis. Mol Biol Cell 16:2191–2206
Myers SA, Leeper LR, Chung CY (2006) WASP-interacting protein is important for actin filament elongation and prompt pseudopod formation in response to a dynamic chemoattractant gradient. Mol Biol Cell 17:4564–4575
Noguchi H, Gompper G, Schmid L, Wixforth A, Franke T (2010) Dynamics of fluid vesicles in flow through structured microchannels. EPL 89:28002
Osserman R (1978) The isoperimetric inequality. Bull Am Math Soc 84:1182–1238
Peterson JR, Mitchison TJ (2002) Small molecules, big impact: a history of chemical inhibitors and the cytoskeleton. Chem Biol 9:1275–1285
Peterson JR, Bickford LC, Morgan D, Kim AS, Ouerfelli O, Kirschner MW, Rosen MK (2004) Chemical inhibition of N-WASP by stabilization of a native autoinhibited conformation. Nat Struct Mol Biol 11:747–755
Pollard TD, Borisy GG (2003) Cellular motility driven by assembly and disassembly of actin filaments. Cell 112:453–465
Pollitt AY, Insall RH (2009) WASP and SCAR/WAVE proteins: the drivers of actin assembly. J Cell Sci 122:2575–2578
Prassler J, Murr A, Stocker S, Faix J, Murphyand J, Marriott G (1998) DdLIM is a cytoskeleton-associated protein involved in the protrusion of lamellipodia in Dictyostelium. Mol Biol Cell 9:545–559
Ridler TW, Calvard S (1978) Thresholding using an iterative selection method. IEEE Trans Syst Man Cybern 8:630–632
Roth U, Müller S, Hanisch F-G (2006) Proteomic analysis of Dictyostelium discoideum. Dictyostelium discoideum protocols. Methods Mol Biol 346:95–109
Schindl M, Wallraff E, Deubzer B, Witke W, Gerisch G, Sackmann E (1995) Cell-substrate interactions and locomotion of dictyostelium wild-type and mutants defective in three cytoskeletal proteins: a study using quantitative reflection interference contrast microscopy. Biophys J 68:1177–1190
Schneider N, Weber I, Faix J, Prassler J, Muller-Taubenberger A, Kohler J, Burghardt E, Gerisch G, Marriott G (2003) A LIM protein involved in the progression of cytokinesis and regulation of the mitotic spindle. Cell Motil Cytoskelet 56(2):130–139
Schnitzler W (2004) Abbildung von Biomolekülen im kontinuierlichen Fluss. Diplomarbeit, Universität Ulm, Ulm
Small JV (2010) Dicing with dogma: de-branching the lamellipodium. Trends Cell Biol 20(11):628–633
Steffen A, Faix J, Resch GP, Linkner J, Wehland J, Small JV, Rottner K, Stradal TEB (2006) Filopodia formation in the absence of functional WAVE- and Arp2/3-complexes. Mol Biol Cell 17(6):2581–2591
Takenawa T, Suetsugu S (2007) The WASP-WAVE protein network: connecting the membrane to the cytoskeleton. Nat Rev Mol Cell Biol 8:37–48
Thrasher AJ, Burns SO (2010) WASP: a key immunological multitasker. Nat Rev Immunol 10:182–192
Urban E, Jacob S, Nemethova M, Resch GP, Small JV (2010) Electron tomography reveals unbranched networks of actin filaments in lamellipodia. Nat Cell Biol 12(5):429–436
Veltman DM, Insall RH (2010) WASP family proteins—their evolution and its physiological implications. Mol Biol Cell 21:2880–2893
Whitesides GM, Ostuni E, Takayama S, Jiang X, Ingber DE (2001) Soft lithography in biology and biochemistry. Annu Rev Biomed Eng 3:335–373
Acknowledgments
We thank Achim Quaas for assistance with the MATLAB algorithms, Christian Fiedler for support on the F-actin assay, Kirsten Krüger for assistance in cell culture, and Douwe Veltman for discussions. We thank Prof. Bernd Walz for generous access to his Zeiss LSM 710 confocal microscope and Prof. Otto Baumann for continuous help in operating the microscope. Financial support by the Deutsche Forschungsgemeinschaft (DFG BE 3978/3-1) is gratefully acknowledged.
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Pfannes, E.K.B., Theves, M., Wegner, C. et al. Impact of the carbazole derivative wiskostatin on mechanical stability and dynamics of motile cells. J Muscle Res Cell Motil 33, 95–106 (2012). https://doi.org/10.1007/s10974-012-9287-8
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DOI: https://doi.org/10.1007/s10974-012-9287-8