Abstract
Defining the exact role of the actin cytoskeleton in mediating endocytosis through different pathways is a significant challenge. The general consensus is that actin has an important role in organizing the early stages of endocytosis but there is still much to learn. Actin has also been implicated in cell internalization of cell-penetrating peptides (CPPs). It is suggested that CPP variants such as octaarginine (R8) and the HIV Tat peptide induce actin-dependent plasma membrane perturbation and enter via macropinocytosis. Here, we describe confocal microscopy techniques that allow for high-resolution spatial characterization of the actin cytoskeleton in untreated mammalian cells and those incubated with actin-disrupting agents and CPPs. By performing X–Y–Z projection images through different regions of cells to show basal and apical profiles, we initially highlight how these techniques can be used to reveal major differences in cortical and filamentous actin organization between different cell lines. Using these techniques, we demonstrate that the actin-disrupting agent cytochalasin D rapidly changes this framework at concentrations significantly lower than is normally used. Experiments are also performed to highlight that serum starvation significantly sensitizes cells to the effects of R8 on actin-induced ruffling and lamellapodia formation. The techniques described here can be used to gain a higher level of knowledge of the organization of the actin network in individual model cell systems, how this is perturbed using commonly used actin inhibitors, and how plasma membrane reorganization can be induced by the addition of drug delivery vectors such as CPPs.
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References
Cleal K, He L, Watson PD, Jones AT (2013) Endocytosis, intracellular traffic and fate of cell penetrating peptide based conjugates and nanoparticles. Curr Pharm Des 19:2878–2894
Doherty GJ, McMahon HT (2009) Mechanisms of endocytosis. Annu Rev Biochem 78:857–902
Fujimoto LM, Roth R, Heuser JE, Schmid SL (2000) Actin assembly plays a variable, but not obligatory role in receptor-mediated endocytosis in mammalian cells. Traffic 1:161–171
Mooren OL, Galletta BJ, Cooper JA (2012) Roles for actin assembly in endocytosis. Annu Rev Biochem 81:661–686
Robertson AS, Smythe E, Ayscough KR (2009) Functions of actin in endocytosis. Cell Mol Life Sci 66:2049–2065
Anitei M, Hoflack B (2012) Bridging membrane and cytoskeleton dynamics in the secretory and endocytic pathways. Nat Cell Biol 14:11–19
Fretz MM, Penning NA, Al-Taei S, Futaki S, Takeuchi T, Nakase I, Storm G, Jones AT (2007) Temperature-, concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells. Biochem J 403:335–342
Watkins CL, Schmaljohann D, Futaki S, Jones AT (2009) Low concentration thresholds of plasma membranes for rapid energy-independent translocation of a cell-penetrating peptide. Biochem J 420:179–189
Nakase I, Niwa M, Takeuchi T, Sonomura K, Kawabata N, Koike Y, Takehashi M, Tanaka S, Ueda K, Simpson JC, Jones AT, Sugiura Y, Futaki S (2004) Cellular uptake of arginine-rich peptides: roles for macropinocytosis and actin rearrangement. Mol Ther 10:1011–1022
Wadia JS, Stan RV, Dowdy SF (2004) Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat Med 10:310–315
Falcone S, Cocucci E, Podini P, Kirchhausen T, Clementi E, Meldolesi J (2006) Macropino-cytosis: regulated coordination of endocytic and exocytic membrane traffic events. J Cell Sci 119:4758–4769
Jones AT (2007) Macropinocytosis: searching for an endocytic identity and role in the uptake of cell penetrating peptides. J Cell Mol Med 11:670–684
Kerr MC, Teasdale RD (2009) Defining macropinocytosis. Traffic 10:364–371
Lim JP, Gleeson PA (2011) Macropinocytosis: an endocytic pathway for internalising large gulps. Immunol Cell Biol 89:836–843
Haigler HT, McKanna JA, Cohen S (1979) Rapid stimulation of pinocytosis in human carcinoma cells A-431 by epidermal growth factor. J Cell Biol 83:82–90
Nakase I, Tadokoro A, Kawabata N, Takeuchi T, Katoh H, Hiramoto K, Negishi M, Nomizu M, Sugiura Y, Futaki S (2007) Interaction of arginine-rich peptides with membrane-associated proteoglycans is crucial for induction of actin organization and macropinocytosis. Biochemistry 46:492–501
Al Soraj M, He L, Peynshaert K, Cousaert J, Vercauteren D, Braeckmans K, De Smedt SC, Jones AT (2012) siRNA and pharmacological inhibition of endocytic pathways to characterize the differential role of macropinocytosis and the actin cytoskeleton on cellular uptake of dextran and cationic cell penetrating peptides octaarginine (R8) and HIV-Tat. J Control Release 161:132–141
Amand HL, Fant K, Norden B, Esbjorner EK (2008) Stimulated endocytosis in penetratin uptake: effect of arginine and lysine. Biochem Biophys Res Commun 371:621–625
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682
de Curtis I, Meldolesi J (2012) Cell surface dynamics - how Rho GTPases orchestrate the interplay between the plasma membrane and the cortical cytoskeleton. J Cell Sci 125:4435–4444
Ridley AJ (2011) Life at the leading edge. Cell 145:1012–1022
Hewlett LJ, Prescott AR, Watts C (1994) The coated pit and macropinocytic pathways serve distinct endosome populations. J Cell Biol 124:689–703
Swanson JA, Watts C (1995) Macropinocytosis. Trends Cell Biol 5:424–428
McKayed KK, Simpson JC (2013) Actin in action: imaging approaches to study cytoskeleton structure and function. Cells 2:715–731
Peng GE, Wilson SR, Weiner OD (2011) A pharmacological cocktail for arresting actin dynamics in living cells. Mol Biol Cell 22:3986–3994
Ballestrem C, Wehrle-Haller B, Imhof BA (1998) Actin dynamics in living mammalian cells. J Cell Sci 111(Pt 12):1649–1658
Chen BC, Legant WR, Wang K, Shao L, Milkie DE, Davidson MW, Janetopoulos C, Wu XS, Hammer JA 3rd, Liu Z, English BP, Mimori-Kiyosue Y, Romero DP, Ritter AT, Lippincott-Schwartz J, Fritz-Laylin L, Mullins RD, Mitchell DM, Bembenek JN, Reymann AC, Bohme R, Grill SW, Wang JT, Seydoux G, Tulu US, Kiehart DP, Betzig E (2014) Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346:1257998
Lukinavicius G, Reymond L, D’Este E, Masharina A, Gottfert F, Ta H, Guther A, Fournier M, Rizzo S, Waldmann H, Blaukopf C, Sommer C, Gerlich DW, Arndt HD, Hell SW, Johnsson K (2014) Fluorogenic probes for live-cell imaging of the cytoskeleton. Nat Methods 11:731–733
Acknowledgements
This work was supported by a Cardiff University Studentship awarded to LH. This work was supported by a Cardiff University Studentship (LH) and an Engineering and Physical Sciences Research Council (EPSRC) award EP/J021334/1 (ATJ, PW).
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He, L., Watson, P.D., Jones, A.T. (2015). Visualizing Actin Architectures in Cells Incubated with Cell-Penetrating Peptides. In: Langel, Ü. (eds) Cell-Penetrating Peptides. Methods in Molecular Biology, vol 1324. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2806-4_16
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DOI: https://doi.org/10.1007/978-1-4939-2806-4_16
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