Abstract
Lysosomes and lysosomal proteases have been found to participate during several forms of cell death pathways including apoptosis. A critical step in the mediation of apoptotic signaling is the release of cathepsins to the cytosol, a process known as lysosomal membrane permeabilization (LMP). In this chapter, we describe immunofluorescence detection of LMP in cell cultures stained for cathepsin B and LAMP-2 using three confocal techniques namely laser scanning, spinning disk, and aperture correlation spinning disk confocal to obtain images. Image analysis is performed using Huygens software for deconvolution. LMP results in a decrease in the fraction of cathepsin B colocalizing with LAMP-2, which is quantified through Manders’ colocalization coefficient. Analysis of the images obtained by the three techniques show the same trend but the magnitude of the decrease differs due to the axial resolution. The observations emphasize the use of highest possible resolution when determining colocalization.
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References
De Duve C (1959) Lysosomes, a new group of cytoplasmic particles. In: Hayashi T (ed) Subcellular Particles. The Ronald Press Co, New York, pp 128–159
Turk B, Turk V (2009) Lysosomes as ‘suicide bags’ in cell death: myth or reality? J Biol Chem 284:21783–21787
Roberg K, Öllinger K (1998) Oxidative stress causes relocation of the lysosomal enzyme cathepsin D with ensuing apoptosis in neonatal rat cardiomyocytes. Am J Pathol 152(5):1151–1156
Boya P, Kroemer G (2008) Lysosomal membrane permeabilization in cell death. Oncogene 27(50):6434–6451. doi:10.1038/onc.2008.310
Bivik CA, Larsson PK, Kågedal KM, Rosdahl IK, Öllinger KM (2006) UVA/B-induced apoptosis in human melanocytes involves translocation of cathepsins and Bcl-2 family members. J Invest Dermatol 126(5):1119–1127
Roberg K, Kågedal K, Öllinger K (2002) Microinjection of cathepsin d induces caspase-dependent apoptosis in fibroblasts. Am J Pathol 161(1):89–96
Schestkowa O, Geisel D, Jacob R, Hasilik A (2007) The catalytically inactive precursor of cathepsin D induces apoptosis in human fibroblasts and HeLa cells. J Cell Biochem 101(6):1558–1566
Scheffler JM, Schiefermeier N, Hube LA (2014) Mild fixation and permeabilization protocol for preserving structures of endosomes, focal adhesions, and actin filaments during immunofluorescence analysis. Methods Enzymol 535:93–102
Stadler C, Skogs M, Brismar H, Uhlén M, Lundberg E (2010) A single fixation protocol for proteome-wide immunofluorescence localization studies. J Proteomics 73(6):1067–1078
Donaldson J (2001) Immunofluorescence staining. Curr Protoc Cell Biol 4. doi:10.1002/0471143030.cb0403s00
Jamur MC, Oliver C (2010) Permeabilization of cell membranes. Methods Mol Biol 588:63–66
Bauer CR (2014) Labeling and use of monoclonal antibodies in immunofluorescence: protocols for cytoskeletal and nuclear antigens. Methods Mol Biol 1131:543–548
Yamanaka M, Smith NI, Fujita K (2014) Introduction to super-resolution microscopy. Microscopy 63:177–192
Cox S, Jones GE (2013) Imaging cells at hte nanoscale. Int J Biochem Cell Biol 45:1669–1678
Conchello JA, Lichtman JW (2005) Optical sectioning microscopy. Nat Methods 2:920–231
Sanderson MJ, Smith I, Parker I, Bootman MD (2014) Fluorescence microscopy. Cold Spring Harb Protoc 2014(10):pdb.top071795. doi:10.1101/pdb.top071795
Oreopoulos J, Berman R, Browne M (2014) Spinning-disk confocal microscopy: present technology and future trends. Methods Cell Biol 123:153–175
Karlsson T, Lagerholm BC, Vikström E, Loitto VM, Magnusson KE (2013) Water fluxes through aquaporin-9 prime epithelial cells for rapid wound healing. Biochem Biophys Res Commun 430(3):993–998
Karlsson T, Bolshakova A, Magalhães MA, Loitto VM, Magnusson KE (2013) Fluxes of water through aquaporin 9 weaken membrane-cytoskeleton anchorage and promote formation of membrane protrusions. PLoS One 8(4):e59901
Wang YL (2007) Computational restoration of fluorescence images: noise reduction, deconvolution, and pattern recognition. Methods Cell Biol 81:435–445
Dunn WD, Kamocka MM, McDonald JH (2011) A practical guide to evaluating colocalization in biological microscopy. Am J Physiol Cell Physiol 300:C723–C742
Jež M, Bas T, Veber M, Košir A, Dominko T, Page R, Rožman P (2013) The hazards of DAPI photoconversion: effects of dye, mounting media and fixative, and how to minimize the problem. Histochem Cell Biol 139:195–204
Acknowledgement
This work was supported by grants from the Swedish Research Council, Swedish Cancer Society, County Council of Östergötland, Konung Gustav V och Drottning Victorias Frimurarestiftelse and Stiftelsen Olle Engkvist Byggmästare.
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Giraldo, A.M.V., Öllinger, K., Loitto, V. (2017). Microscopic Analysis of Lysosomal Membrane Permeabilization. In: Öllinger, K., Appelqvist, H. (eds) Lysosomes. Methods in Molecular Biology, vol 1594. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6934-0_5
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DOI: https://doi.org/10.1007/978-1-4939-6934-0_5
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