Abstract
Clathrin-mediated endocytosis (CME) is a universal and evolutionarily conserved process that enables the internalization of numerous cargo proteins, including receptors for nutrients and signaling molecules, as well as synaptic vesicle reformation. Multiple genetic and chemical approaches have been developed to interfere with this process. However, many of these tools do not selectively block CME, for example by targeting components shared with clathrin-independent endocytosis pathways or by interfering with other cellular processes that indirectly affect CME.
Clathrin, via interactions of endocytic proteins with its terminal domain (TD), serves as a central interaction hub for coat assembly in CME. Here, we describe an ELISA-based, high-throughput screening method used to identify small molecules that inhibit these interactions. In addition, we provide protocols for the purification of recombinant protein domains used for screening, e.g., the clathrin TD and the amphiphysin B/C domain. The screen has been applied successfully in the past, and ultimately led to the discovery of the Pitstop® family of inhibitors, but remains in use to evaluate the inhibitory potency of derivatives of these compounds, and to screen for completely novel inhibitor families.
The original version of this chapter was revised. A correction to this chapter can be found at https://doi.org/10.1007/978-1-4939-8719-1_19
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Change history
24 October 2018
This book was inadvertently published with the incorrect title as Clathrin-Mediated Endoytosis: Methods and Protocols. This has now been corrected throughout the book to Clathrin-Mediated Endocytosis: Methods and Protocols.
References
van der Bliek AM, Redelmeier TE, Damke H, Tisdale EJ, Meyerowitz EM, Schmid SL (1993) Mutations in human dynamin block an intermediate stage in coated vesicle formation. J Cell Biol 122:553–563
Evergren E, Marcucci M, Tomilin N, Low P, Slepnev V, Andersson F, Gad H, Brodin L, De Camilli P, Shupliakov O (2004) Amphiphysin is a component of clathrin coats formed during synaptic vesicle recycling at the lamprey giant synapse. Traffic 5:514–528
Slepnev VI, Ochoa GC, Butler MH, De Camilli P (2000) Tandem arrangement of the clathrin and AP-2 binding domains in amphiphysin 1 and disruption of clathrin coat function by amphiphysin fragments comprising these sites. J Biol Chem 275:17583–17589
Zhao X, Greener T, Al-Hasani H, Cushman SW, Eisenberg E, Greene LE (2001) Expression of auxilin or AP180 inhibits endocytosis by mislocalizing clathrin: evidence for formation of nascent pits containing AP1 or AP2 but not clathrin. J Cell Sci 114:353–365
Hinrichsen L, Harborth J, Andrees L, Weber K, Ungewickell EJ (2003) Effect of clathrin heavy chain- and alpha-adaptin-specific small inhibitory RNAs on endocytic accessory proteins and receptor trafficking in HeLa cells. J Biol Chem 278:45160–45170
Motley A, Bright NA, Seaman MN, Robinson MS (2003) Clathrin-mediated endocytosis in AP-2-depleted cells. J Cell Biol 162:909–918
Macia E, Ehrlich M, Massol R, Boucrot E, Brunner C, Kirchhausen T (2006) Dynasore, a cell-permeable inhibitor of dynamin. Dev Cell 10:839–850
Hill TA, Gordon CP, McGeachie AB, Venn-Brown B, Odell LR, Chau N, Quan A, Mariana A, Sakoff JA, Chircop M et al (2009) Inhibition of dynamin mediated endocytosis by the dynoles--synthesis and functional activity of a family of indoles. J Med Chem 52:3762–3773
McCluskey A, Daniel JA, Hadzic G, Chau N, Clayton EL, Mariana A, Whiting A, Gorgani NN, Lloyd J, Quan A et al (2013) Building a better dynasore: the dyngo compounds potently inhibit dynamin and endocytosis. Traffic 14:1272–1289
Park RJ, Shen H, Liu L, Liu X, Ferguson SM, De Camilli P (2013) Dynamin triple knockout cells reveal off target effects of commonly used dynamin inhibitors. J Cell Sci 126:5305–5312
Preta G, Cronin JG, Sheldon IM (2015) Dynasore - not just a dynamin inhibitor. Cell Commun Signal 13:24
Drake MT, Traub LM (2001) Interaction of two structurally distinct sequence types with the clathrin terminal domain beta-propeller. J Biol Chem 276:28700–28709
Kang DS, Kern RC, Puthenveedu MA, von Zastrow M, Williams JC, Benovic JL (2009) Structure of an arrestin2-clathrin complex reveals a novel clathrin binding domain that modulates receptor trafficking. J Biol Chem 284:29860–29872
Kern RC, Kang DS, Benovic JL (2009) Arrestin2/clathrin interaction is regulated by key N- and C-terminal regions in arrestin2. Biochemistry 48:7190–7200
Miele AE, Watson PJ, Evans PR, Traub LM, Owen DJ (2004) Two distinct interaction motifs in amphiphysin bind two independent sites on the clathrin terminal domain beta-propeller. Nat Struct Mol Biol 11:242–248
von Kleist L, Stahlschmidt W, Bulut H, Gromova K, Puchkov D, Robertson MJ, MacGregor KA, Tomilin N, Pechstein A, Chau N et al (2011) Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition. Cell 146:471–484
Merrifield CJ, Feldman ME, Wan L, Almers W (2002) Imaging actin and dynamin recruitment during invagination of single clathrin-coated pits. Nat Cell Biol 4:691–698
Stahlschmidt W, Robertson MJ, Robinson PJ, McCluskey A, Haucke V (2014) Clathrin terminal domain-ligand interactions regulate sorting of mannose 6-phosphate receptors mediated by AP-1 and GGA adaptors. J Biol Chem 289:4906–4918
Acknowledgments
This work has been supported by grants from the Deutsche Forschungsgemeinschaft (SFB765/b4 to V.H. and SFB958/A11 to M.K.).
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Haucke, V., Krauß, M. (2018). Identifying Small-Molecule Inhibitors of the Clathrin Terminal Domain. In: Swan, L. (eds) Clathrin-Mediated Endocytosis. Methods in Molecular Biology, vol 1847. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8719-1_5
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DOI: https://doi.org/10.1007/978-1-4939-8719-1_5
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