Abstract
Confocal microscopy permits the analysis of the subcellular distribution of proteins. Colocalization between target proteins and specific markers of differential cell compartments provides an efficient approach to studying protein traffic. In this chapter, we describe an automated method to denoise confocal microscopy images and assess the colocalization of their stainings using ImageJ software. As a step further from conventional single colocalization measurements, in the proposed method, we analyze stacks of three different stainings using two-by-two comparisons. To demonstrate the reliability and usefulness of our proposal, the method was used to compare the traffic of the voltage-gated Kv1.3 potassium channel, which is a well-defined plasma membrane protein, in the presence and absence of KCNE4, a regulatory subunit that strongly retains the channel intracellularly.
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Alberts B, Johnson A, Lewis J, Morgan D, Raff M, Roberts K, Walter P (2015) Molecular biology of the cell, 6th edn. Garland Science, Taylor & Francis Group, LLC, New York
Armstrong CM, Hille B (1998) Voltage-gated ion channels and electrical excitability. Neuron 20:371–380
Pérez-Verdaguer M, Capera J, Serrano-Novillo C, Estadella I, Sastre D, Felipe A (2016) The voltage-gated potassium channel Kv1.3 is a promising multitherapeutic target against human pathologies. Expert Opin Ther Targets 20:577–591. https://doi.org/10.1517/14728222.2016.1112792
Serrano-Albarrás A, Estadella I, Cirera-Rocosa S, Navarro-Pérez M, Felipe A (2018) Kv1.3: a multifunctional channel with many pathological implications. Expert Opin Ther Targets 22:101–105. https://doi.org/10.1080/14728222.2017.1420170
Martínez-Mármol R, Styrczewska K, Pérez-Verdaguer M, Vallejo-Gracia A, Comes N, Sorkin A, Felipe A (2017) Ubiquitination mediates Kv1.3 endocytosis as a mechanism for protein kinase C-dependent modulation. Sci Rep 7:42395. https://doi.org/10.1038/srep42395
Sole L, Roura-Ferrer M, Perez-Verdaguer M, Oliveras A, Calvo M, Fernandez-Fernandez JM, Felipe A (2009) KCNE4 suppresses Kv1.3 currents by modulating trafficking, surface expression and channel gating. J Cell Sci 122:3738–3748. https://doi.org/10.1242/jcs.056689
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. https://doi.org/10.1038/nmeth.2019
Bolte S, Cordelières FP (2006) A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 224:213–232. https://doi.org/10.1111/j.1365-2818.2006.01706.x
Manders EMM, Stap J, Brakenhoff GJ, Van Driel R, Aten A (1992) Dynamics of three-dimensional replication patterns during the S-phase, analysed by double labelling of DNA and confocal microscopy. J Cell Sci 103(Pt 3):857–862
Pearson K (1895) Note on regression and inheritance in the case of two parents. Proc R Soc London 58:240–242. https://doi.org/10.1098/rspl.1895.0041
(2018) Manel Bosch ijm-Macros. https://github.com/manelbosch76/ijm-Macros. Accessed 5 Jun 2018
Rueden C (2013) Plugins—ImageJ. https://imagej.net/index.php?title=Plugins&oldid=24930. Accessed 2 Apr 2018
Cordelières FP, Bolte S (2008) JACoP v2.0: improving the user experience with co-localization studies. ImageJ User Dev Conf 174–181
Cordelières FP (2008) JACoP [ImageJ Documentation Wiki]. http://imagejdocu.tudor.lu/doku.php?id=plugin:analysis:jacop_2.0:just_another_colocalization_plugin:start. Accessed 2 Apr 2018
Collins TJ (2007) ImageJ for microscopy. BioTechniques 43:25–30. https://doi.org/10.2144/000112505
Collins TJ (2007) MBF plugin collection. http://imagej.net/plugins/mbf/index.html. Accessed 2 Apr 2018
Kota Miura ijm-Macros. https://github.com/miura/IJ_BCautoMacro. Accessed 2 Apr 2018
Schindelin J, Cardona A, Eglinger J, Rueden C, Brocher J, Hiner M, Arena ET, Arganda-Carreras I (2010) Introduction to macro programming—ImageJ. https://imagej.net/Introduction_into_Macro_Programming#Installing_macros. Accessed 2 Apr 2018
ImageJ Wiki Subtract background [ImageJ Documentation Wiki]. http://imagejdocu.tudor.lu/doku.php?id=gui:process:subtract_background. Accessed 14 Jan 2018
Ridler TW, Calvard S (1978) Picture thresholding using an iterative selection method. IEEE Trans Syst Man Cybern 8:630–632. https://doi.org/10.1109/TSMC.1978.4310039
Kota Miura ijm-Macros (2014). https://github.com/miura/IJ_BCautoMacro. Accessed 5 Jun 2018
ImageJ Wiki (2010) Binary [ImageJ Documentation Wiki]. http://imagejdocu.tudor.lu/doku.php?id=gui:process:binary. Accessed 1 May 2018
ImageJ Wiki (2008) Image calculator [ImageJ Documentation Wiki]. http://imagejdocu.tudor.lu/doku.php?id=gui:process:image_calculator. Accessed 1 May 2018
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Sastre, D., Estadella, I., Bosch, M., Felipe, A. (2019). Triple-Colocalization Approach to Assess Traffic Patterns and Their Modulation. In: Rebollo, E., Bosch, M. (eds) Computer Optimized Microscopy. Methods in Molecular Biology, vol 2040. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9686-5_11
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DOI: https://doi.org/10.1007/978-1-4939-9686-5_11
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