Abstract
Although PTEN has been widely described as a nuclear and cytosolic protein, in the last 2 years, alternative organelles, such as the endoplasmic reticulum (ER), pure mitochondria, and mitochondria-associated membranes (MAMs), have been recognized as pivotal targets of PTEN activity.
Here, we describe different methods that have been used to highlight PTEN subcellular localization.
First, a protocol to extract nuclear and cytosolic fractions has been described to assess the “canonical” PTEN localization. Moreover, we describe a protocol for mitochondria isolation with proteinase K (PK) to further discriminate whether PTEN associates with the outer mitochondrial membrane (OMM) or resides within the mitochondria. Finally, we focus our attention on a subcellular fractionation protocol of cells that permits the isolation of MAMs containing unique regions of ER membranes attached to the outer mitochondrial membrane (OMM) and mitochondria without contamination from other organelles. In addition to biochemical fractionations, immunostaining can be used to determine the subcellular localization of proteins; thus, a detailed protocol to obtain good immunofluorescence (IF) is described. The employment of these methodological approaches could facilitate the identification of different PTEN localizations in several physiopathological contexts.
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References
Pulido R, Baker SJ, Barata JT, Carracedo A, Cid VJ, Chin-Sang ID, Dave V, den Hertog J, Devreotes P, Eickholt BJ, Eng C, Furnari FB, Georgescu MM, Gericke A, Hopkins B, Jiang X, Lee SR, Losche M, Malaney P, Matias-Guiu X, Molina M, Pandolfi PP, Parsons R, Pinton P, Rivas C, Rocha RM, Rodriguez MS, Ross AH, Serrano M, Stambolic V, Stiles B, Suzuki A, Tan SS, Tonks NK, Trotman LC, Wolff N, Woscholski R, Wu H, Leslie NR (2014) A unified nomenclature and amino acid numbering for human PTEN. Sci Signal 7(332):pe15. doi:10.1126/scisignal.2005560
Salmena L, Carracedo A, Pandolfi PP (2008) Tenets of PTEN tumor suppression. Cell 133(3):403–414. doi:10.1016/j.cell.2008.04.013
Bononi A, Bonora M, Marchi S, Missiroli S, Poletti F, Giorgi C, Pandolfi PP, Pinton P (2013) Identification of PTEN at the ER and MAMs and its regulation of Ca(2+) signaling and apoptosis in a protein phosphatase-dependent manner. Cell Death Differ 20(12):1631–1643. doi:10.1038/cdd.2013.77
Bassi C, Ho J, Srikumar T, Dowling RJ, Gorrini C, Miller SJ, Mak TW, Neel BG, Raught B, Stambolic V (2013) Nuclear PTEN controls DNA repair and sensitivity to genotoxic stress. Science 341(6144):395–399. doi:10.1126/science.1236188
Li P, Wang D, Li H, Yu Z, Chen X, Fang J (2014) Identification of nucleolus-localized PTEN and its function in regulating ribosome biogenesis. Mol Biol Rep 41(10):6383–6390. doi:10.1007/s11033-014-3518-6
Trotman LC, Wang X, Alimonti A, Chen Z, Teruya-Feldstein J, Yang H, Pavletich NP, Carver BS, Cordon-Cardo C, Erdjument-Bromage H, Tempst P, Chi SG, Kim HJ, Misteli T, Jiang X, Pandolfi PP (2007) Ubiquitination regulates PTEN nuclear import and tumor suppression. Cell 128(1):141–156. doi:10.1016/j.cell.2006.11.040
Zhu Y, Hoell P, Ahlemeyer B, Krieglstein J (2006) PTEN: a crucial mediator of mitochondria-dependent apoptosis. Apoptosis 11(2):197–207. doi:10.1007/s10495-006-3714-5
Liang H, He S, Yang J, Jia X, Wang P, Chen X, Zhang Z, Zou X, McNutt MA, Shen WH, Yin Y (2014) PTENalpha, a PTEN isoform translated through alternative initiation, regulates mitochondrial function and energy metabolism. Cell Metab 19(5):836–848. doi:10.1016/j.cmet.2014.03.023
Wieckowski MR, Giorgi C, Lebiedzinska M, Duszynski J, Pinton P (2009) Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells. Nat Protoc 4(11):1582–1590. doi:10.1038/nprot.2009.151
Giorgi C, Baldassari F, Bononi A, Bonora M, De Marchi E, Marchi S, Missiroli S, Patergnani S, Rimessi A, Suski JM, Wieckowski MR, Pinton P (2012) Mitochondrial Ca(2+) and apoptosis. Cell Calcium 52(1):36–43. doi:10.1016/j.ceca.2012.02.008
Marchi S, Lupini L, Patergnani S, Rimessi A, Missiroli S, Bonora M, Bononi A, Corra F, Giorgi C, De Marchi E, Poletti F, Gafa R, Lanza G, Negrini M, Rizzuto R, Pinton P (2013) Downregulation of the mitochondrial calcium uniporter by cancer-related miR-25. Curr Biol 23(1):58–63. doi:10.1016/j.cub.2012.11.026
Marchi S, Patergnani S, Pinton P (2014) The endoplasmic reticulum-mitochondria connection: one touch, multiple functions. Biochim Biophys Acta 1837(4):461–469. doi:10.1016/j.bbabio.2013.10.015
Patergnani S, Suski JM, Agnoletto C, Bononi A, Bonora M, De Marchi E, Giorgi C, Marchi S, Missiroli S, Poletti F, Rimessi A, Duszynski J, Wieckowski MR, Pinton P (2011) Calcium signaling around Mitochondria Associated Membranes (MAMs). Cell Commun Signal 9:19. doi:10.1186/1478-811X-9-19
Giorgi C, Missiroli S, Patergnani S, Duszynski J, Wieckowski M, Pinton P (2015) Mitochondria-associated membranes (MAMs): composition molecular mechanisms and physiopathological implications. Antioxid Redox Signal. doi:10.1089/ars.2014.6223
Marchi S, Marinello M, Bononi A, Bonora M, Giorgi C, Rimessi A, Pinton P (2012) Selective modulation of subtype III IP(3)R by Akt regulates ER Ca(2)(+) release and apoptosis. Cell Death Dis 3:e304. doi:10.1038/cddis.2012.45
Bononi A, Pinton P (2014) Study of PTEN subcellular localization. Methods. doi:10.1016/j.ymeth.2014.10.002
Dunn KW, Kamocka MM, McDonald JH (2011) A practical guide to evaluating colocalization in biological microscopy. Am J Physiol Cell Physiol 300(4):C723–C742. doi:10.1152/ajpcell.00462.2010
Bonora M, De Marchi E, Patergnani S, Suski JM, Celsi F, Bononi A, Giorgi C, Marchi S, Rimessi A, Duszynski J, Pozzan T, Wieckowski MR, Pinton P (2014) Tumor necrosis factor-alpha impairs oligodendroglial differentiation through a mitochondria-dependent process. Cell Death Differ 21(8):1198–1208. doi:10.1038/cdd.2014.35
Friedman JR, Webster BM, Mastronarde DN, Verhey KJ, Voeltz GK (2010) ER sliding dynamics and ER-mitochondrial contacts occur on acetylated microtubules. J Cell Biol 190(3):363–375. doi:10.1083/jcb.200911024
Wu X, Hepner K, Castelino-Prabhu S, Do D, Kaye MB, Yuan XJ, Wood J, Ross C, Sawyers CL, Whang YE (2000) Evidence for regulation of the PTEN tumor suppressor by a membrane-localized multi-PDZ domain containing scaffold protein MAGI-2. Proc Natl Acad Sci U S A 97(8):4233–4238
Vazquez F, Matsuoka S, Sellers WR, Yanagida T, Ueda M, Devreotes PN (2006) Tumor suppressor PTEN acts through dynamic interaction with the plasma membrane. Proc Natl Acad Sci U S A 103(10):3633–3638. doi:10.1073/pnas.0510570103
Lachyankar MB, Sultana N, Schonhoff CM, Mitra P, Poluha W, Lambert S, Quesenberry PJ, Litofsky NS, Recht LD, Nabi R, Miller SJ, Ohta S, Neel BG, Ross AH (2000) A role for nuclear PTEN in neuronal differentiation. J Neurosci 20(4):1404–1413
Howitt J, Lackovic J, Low LH, Naguib A, Macintyre A, Goh CP, Callaway JK, Hammond V, Thomas T, Dixon M, Putz U, Silke J, Bartlett P, Yang B, Kumar S, Trotman LC, Tan SS (2012) Ndfip1 regulates nuclear Pten import in vivo to promote neuronal survival following cerebral ischemia. J Cell Biol 196(1):29–36. doi:10.1083/jcb.201105009
Bonora M, Bononi A, De Marchi E, Giorgi C, Lebiedzinska M, Marchi S, Patergnani S, Rimessi A, Suski JM, Wojtala A, Wieckowski MR, Kroemer G, Galluzzi L, Pinton P (2013) Role of the c subunit of the FO ATP synthase in mitochondrial permeability transition. Cell Cycle 12(4):674–683. doi:10.4161/cc.23599
Song MS, Salmena L, Carracedo A, Egia A, Lo-Coco F, Teruya-Feldstein J, Pandolfi PP (2008) The deubiquitinylation and localization of PTEN are regulated by a HAUSP-PML network. Nature 455(7214):813–817. doi:10.1038/nature07290
Acknowledgements
The financial support of Telethon GGP11139B to P.P., the Italian Association for Cancer Research (IG-14442 to P.P. and MFAG-13521 to C.G.), the Italian Cystic Fibrosis Research Foundation (grant #19/2014), and the Italian Ministry of Education, University and Research (COFIN, FIRB, and Futuro in Ricerca) to P.P. are gratefully acknowledged.
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Missiroli, S., Morganti, C., Giorgi, C., Pinton, P. (2016). Methods to Study PTEN in Mitochondria and Endoplasmic Reticulum. In: Salmena, L., Stambolic, V. (eds) PTEN. Methods in Molecular Biology, vol 1388. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3299-3_13
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DOI: https://doi.org/10.1007/978-1-4939-3299-3_13
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