Abstract
Calpains are a family of calcium-dependent intracellular cysteine proteases that regulate important physiological processes by substrate cleavage. Despite the fact that the role of calpains in cell migration and other processes has been extensively studied in vitro, the same does not apply to cell migration and morphogenetic events during embryogenesis, in vivo. Herein, we describe the use of three different methods to selectively block calpain activity in vivo in order to investigate the impact on Xenopus gastrulation and neurulation, namely, a calpain inhibitor, a dominant negative, and a morpholino antisense oligonucleotide (MO). We also provide methods to determine the effectiveness of the calpain inhibition and effect on cell fate specification and morphogenetic movements, during embryogenesis in vivo.
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References
Wells A, Huttenlocher A, Lauffenburger DA (2005) Calpain proteases in cell adhesion and motility. Int Rev Cytol 245:1–16. https://doi.org/10.1016/S0074-7696(05)45001-9
Croall DE, DeMartino GN (1991) Calcium-activated neutral protease (calpain) system: structure, function, and regulation. Physiol Rev 71(3):813–847
Sorimachi H, Tsukahara T, Okada-Ban M, Sugita H, Ishiura S, Suzuki K (1995) Identification of a third ubiquitous calpain species--chicken muscle expresses four distinct calpains. Biochim Biophys Acta 1261(3):381–393
Zanardelli S, Christodoulou N, Skourides PA (2013) Calpain2 protease: a new member of the Wnt/Ca(2+) pathway modulating convergent extension movements in Xenopus. Dev Biol 384(1):83–100. https://doi.org/10.1016/j.ydbio.2013.09.017
Antoniades I, Stylianou P, Christodoulou N, Skourides PA (2017) Addressing the functional determinants of FAK during ciliogenesis in multiciliated cells. J Biol Chem 292(2):488–504. https://doi.org/10.1074/jbc.M116.767111
Antoniades I, Stylianou P, Skourides PA (2014) Making the connection: ciliary adhesion complexes anchor basal bodies to the actin cytoskeleton. Dev Cell 28(1):70–80. https://doi.org/10.1016/j.devcel.2013.12.003
Charalambous A, Andreou M, Antoniades I, Christodoulou N, Skourides PA (2012) In vivo, site-specific, covalent conjugation of quantum dots to proteins via split-intein splicing. Methods Mol Biol 906:157–169. https://doi.org/10.1007/978-1-61779-953-2_11
Charalambous A, Antoniades I, Christodoulou N, Skourides PA (2011) Split-inteins for simultaneous, site-specific conjugation of quantum dots to multiple protein targets in vivo. J Nanobiotechnol 9:37. https://doi.org/10.1186/1477-3155-9-37
Charalambous A, Koyioni M, Antoniades I, Pegeioti D, Eleftheriou I, Michaelidou SS, Amelichev SA, Konstantinov LS, Rakitin OA, Koutentis PA, Skourides PA (2015) 1,2,3-Dithiazoles – new reversible melanin synthesis inhibitors: a chemical genomics study. Medchemcomm 6(5):935–946. https://doi.org/10.1039/c5md00052a
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Antoniades, I., Charalambous, A., Christodoulou, N., Zanardelli, S., Skourides, P.A. (2019). Methods of Calpain Inhibition to Determine the Role of Calpains in Embryo Development in Amphibians. In: Messer, J. (eds) Calpain. Methods in Molecular Biology, vol 1915. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-8988-1_19
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DOI: https://doi.org/10.1007/978-1-4939-8988-1_19
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