Abstract
Proximity ligation assay (PLA) appears as a quick and easy technique to visualize within fixed cells the occurrence and in situ distribution of protein complexes. PLA has been validated to detect protein-protein interactions within the nuclear compartment. Here, we describe a protocol which allows the detection of interactions between A-type nuclear lamins and either LEM-domain proteins (such as emerin, integrated within the inner nuclear membrane, and LAP2α which accumulates within the nucleoplasm) or gene regulatory factors (e.g., the transcription factor SREBP1). The distinct amounts and patterns of PLA signals obtained for various complexes highlight the pertinence of using PLA to reveal in situ where and to which extent nuclear envelope proteins bind specific partners.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Simon DN, Wilson KL (2013) Partners and post-translational modifications of nuclear lamins. Chromosoma 122:13–31
Oldenburg AR, Delbarre E, Thiede B, Vigouroux C, Collas P (2014) Deregulation of Fragile X-related protein 1 by the lipodystrophic lamin A p.R482W mutation elicits a myogenic gene expression program in preadipocytes. Hum Mol Genet 23:1151–1162
Liu B, Ghosh S, Yang X, Zheng H, Liu X, Wang Z, Jin G, Zheng B, Kennedy BK, Suh Y, Kaeberlein M, Tryggvason K, Zhou Z (2012) Resveratrol rescues SIRT1-dependent adult stem cell decline and alleviates progeroid features in laminopathy-based progeria. Cell Metab 16:738–750
Liu B, Wang Z, Zhang L, Ghosh S, Zheng H, Zhou Z (2013) Depleting the methyltransferase Suv39h1 improves DNA repair and extends lifespan in a progeria mouse model. Nat Commun 4(1868):1–12
Krohne G (2004) Lamins. Methods Cell Biol 78:573–596
Shin JY, Mendez-Lopez I, Wang Y, Hays AP, Tanji K, Lefkowitch JH, Schulze PC, Worman HJ, Dauer WT (2013) Lamina-associated polypeptide-1 interacts with the muscular dystrophy protein emerin and is essential for skeletal muscle maintenance. Dev Cell 26:591–603
Delbarre E, Tramier M, Coppey-Moisan M, Gaillard C, Courvalin JC, Buendia B (2006) The truncated prelamin A in Hutchinson-Gilford progeria syndrome alters segregation of A-type and B-type lamin homopolymers. Hum Mol Genet 15:1113–1122
Ivorra C, Kubicek M, Gonzalez JM, Sanz-Gonzalez SM, Alvarez-Barrientos A, O’Connor JE, Burke B, Andres V (2006) A mechanism of AP-1 suppression through interaction of c-Fos with lamin A/C. Genes Dev 20:307–320
Vadrot N, Duband-Goulet I, Cabet E, Attanda W, Barateau A, Vicart P, Gerbal F, Briand N, Vigouroux C, Oldenburg AR, Lund EG, Collas P, Buendia B (2015) The p.R482W substitution in A-type lamins deregulates SREBP1 activity in Dunnigan-type familial partial lipodystrophy. Hum Mol Genet 24:2096–2109
Mahen R, Hattori H, Lee M, Sharma P, Jeyasekharan AD, Venkitaraman AR (2013) A-type lamins maintain the positional stability of DNA damage repair foci in mammalian nuclei. PLoS One 8:e61893
Barascu A, Le Chalony C, Pennarun G, Genet D, Imam N, Lopez B, Bertrand P (2012) Oxidative stress induces an ATM-independent senescence pathway through p38 MAPK-mediated lamin B1 accumulation. EMBO J 31:1080–1094
Fredriksson S, Gullberg M, Jarvius J, Olsson C, Pietras K, Gustafsdottir SM, Ostman A, Landegren U (2002) Protein detection using proximity-dependent DNA ligation assays. Nat Biotechnol 20:473–477
Söderberg O, Gullberg M, Jarvius M, Ridderstrale K, Leuchowius KJ, Jarvius J, Wester K, Hydbring P, Bahram F, Larsson LG, Landegren U (2006) Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods 3:995–1000
Lievens S, Tavernier J (2006) Single protein complex visualization: seeing is believing. Nat Methods 3:971–972
Wilson KL, Foisner R (2010) Lamin-binding Proteins. Cold Spring Harb Perspect Biol 2:a000554
Duband-Goulet I, Woerner S, Gasparini S, Attanda W, Konde E, Tellier-Lebegue C, Craescu CT, Gombault A, Roussel P, Vadrot N, Vicart P, Östlund C, Worman HJ, Zinn-Justin S, Buendia B (2011) Subcellular localization of SREBP1 depends on its interaction with the C-terminal region of wild-type and disease related A-type lamins. Exp Cell Res 317:2800–2813
Acknowledgment
This work was supported by CNRS, University Paris Diderot Paris 7, and the Association Française contre les Myopathies.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Barateau, A., Buendia, B. (2016). In Situ Detection of Interactions Between Nuclear Envelope Proteins and Partners. In: Shackleton, S., Collas, P., Schirmer, E. (eds) The Nuclear Envelope. Methods in Molecular Biology, vol 1411. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3530-7_9
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3530-7_9
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3528-4
Online ISBN: 978-1-4939-3530-7
eBook Packages: Springer Protocols