The organization of the nuclear periphery is crucial for many nuclear functions. Nuclear lamins form dense network at the nuclear periphery and play a substantial role in chromatin organization, transcription regulation and in organization of nuclear pore complexes (NPCs). Here, we show that TPR, the protein located preferentially within the nuclear baskets of NPCs, associates with lamin B1. The depletion of TPR affects the organization of lamin B1 but not lamin A/C within the nuclear lamina as shown by stimulated emission depletion microscopy. Finally, reduction of TPR affects the distribution of NPCs within the nuclear envelope and the effect can be reversed by simultaneous knock-down of lamin A/C or the overexpression of lamin B1. Our work suggests a novel role for the TPR at the nuclear periphery: the TPR contributes to the organization of the nuclear lamina and in cooperation with lamins guards the interphase assembly of nuclear pore complexes.

CLIP Laboratories Department of Paediatric Haematology and Oncology 2nd Faculty of Medicine Charles University and University Hospital Motol Prague 5 Úvalu 84 150 06 Prague Czech Republic

Department of Biology of the Cell Nucleus Institute of Molecular Genetics CAS v v i Vídeňská 1083 142 00 Prague Czech Republic

Department of Cybernetics Faculty of Electrical Engineering Czech Technical University Prague Prague Czech Republic

Division BIOCEV Institute of Molecular Genetics CAS v v i Průmyslová 595 Vestec 252 50 Prague Czech Republic

Microscopy Centre LM and EM Institute of Molecular Genetics CAS v v i Vídeňská 1083 142 00 Prague Czech Republic

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Gruenbaum Y, Foisner R. Lamins: nuclear intermediate filament proteins with fundamental functions in nuclear mechanics and genome regulation. Annu Rev Biochem. 2015;84:131–164. doi: 10.1146/annurev-biochem-060614-034115. PubMed DOI

Naetar N, Ferraioli S, Foisner R. Lamins in the nuclear interior—life outside the lamina. J Cell Sci. 2017;130(13):2087–2096. doi: 10.1242/jcs.203430. PubMed DOI

de Leeuw R, Gruenbaum Y, Medalia O. Nuclear lamins: thin filaments with major functions. Trends Cell Biol. 2017 doi: 10.1016/j.tcb.2017.08.004. PubMed DOI

Schermelleh L, Carlton PM, Haase S, Shao L, Winoto L, Kner P, Burke B, Cardoso MC, Agard DA, Gustafsson MG, Leonhardt H, Sedat JW. Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science. 2008;320(5881):1332–1336. doi: 10.1126/science.1156947. PubMed DOI PMC

Shimi T, Kittisopikul M, Tran J, Goldman AE, Adam SA, Zheng Y, Jaqaman K, Goldman RD. Structural organization of nuclear lamins A, C, B1, and B2 revealed by superresolution microscopy. Mol Biol Cell. 2015;26(22):4075–4086. doi: 10.1091/mbc.E15-07-0461. PubMed DOI PMC

Xie W, Chojnowski A, Boudier T, Lim JS, Ahmed S, Ser Z, Stewart C, Burke B. A-type lamins form distinct filamentous networks with differential nuclear pore complex associations. Curr Biol. 2016;26(19):2651–2658. doi: 10.1016/j.cub.2016.07.049. PubMed DOI

Cho S, Irianto J, Discher DE. Mechanosensing by the nucleus: from pathways to scaling relationships. J Cell Biol. 2017;216(2):305–315. doi: 10.1083/jcb.201610042. PubMed DOI PMC

Lammerding J, Fong LG, Ji JY, Reue K, Stewart CL, Young SG, Lee RT. Lamins A and C but not lamin B1 regulate nuclear mechanics. J Biol Chem. 2006;281(35):25768–25780. doi: 10.1074/jbc.M513511200. PubMed DOI

Swift J, Ivanovska IL, Buxboim A, Harada T, Dingal PC, Pinter J, Pajerowski JD, Spinler KR, Shin JW, Tewari M, Rehfeldt F, Speicher DW, Discher DE. Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed differentiation. Science. 2013;341(6149):1240104. doi: 10.1126/science.1240104. PubMed DOI PMC

Daigle N, Beaudouin J, Hartnell L, Imreh G, Hallberg E, Lippincott-Schwartz J, Ellenberg J. Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells. J Cell Biol. 2001;154(1):71–84. doi: 10.1083/jcb.200101089. PubMed DOI PMC

Lenz-Bohme B, Wismar J, Fuchs S, Reifegerste R, Buchner E, Betz H, Schmitt B. Insertional mutation of the Drosophila nuclear lamin Dm0 gene results in defective nuclear envelopes, clustering of nuclear pore complexes, and accumulation of annulate lamellae. J Cell Biol. 1997;137(5):1001–1016. doi: 10.1083/jcb.137.5.1001. PubMed DOI PMC

Liu J, Rolef Ben-Shahar T, Riemer D, Treinin M, Spann P, Weber K, Fire A, Gruenbaum Y. Essential roles for Caenorhabditis elegans lamin gene in nuclear organization, cell cycle progression, and spatial organization of nuclear pore complexes. Mol Biol Cell. 2000;11(11):3937–3947. doi: 10.1091/mbc.11.11.3937. PubMed DOI PMC

Guo Y, Kim Y, Shimi T, Goldman RD, Zheng Y. Concentration-dependent lamin assembly and its roles in the localization of other nuclear proteins. Mol Biol Cell. 2014;25(8):1287–1297. doi: 10.1091/mbc.e13-11-0644. PubMed DOI PMC

Sullivan T, Escalante-Alcalde D, Bhatt H, Anver M, Bhat N, Nagashima K, Stewart CL, Burke B. Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy. J Cell Biol. 1999;147(5):913–920. doi: 10.1083/jcb.147.5.913. PubMed DOI PMC

Maeshima K, Iino H, Hihara S, Funakoshi T, Watanabe A, Nishimura M, Nakatomi R, Yahata K, Imamoto F, Hashikawa T, Yokota H, Imamoto N. Nuclear pore formation but not nuclear growth is governed by cyclin-dependent kinases (Cdks) during interphase. Nat Struct Mol Biol. 2010;17(9):1065–1071. doi: 10.1038/nsmb.1878. PubMed DOI

Vietri M, Schink KO, Campsteijn C, Wegner CS, Schultz SW, Christ L, Thoresen SB, Brech A, Raiborg C, Stenmark H. Spastin and ESCRT-III coordinate mitotic spindle disassembly and nuclear envelope sealing. Nature. 2015;522(7555):231–235. doi: 10.1038/nature14408. PubMed DOI

Maeshima K, Yahata K, Sasaki Y, Nakatomi R, Tachibana T, Hashikawa T, Imamoto F, Imamoto N. Cell-cycle-dependent dynamics of nuclear pores: pore-free islands and lamins. J Cell Sci. 2006;119(Pt 21):4442–4451. doi: 10.1242/jcs.03207. PubMed DOI

Hurt E, Beck M. Towards understanding nuclear pore complex architecture and dynamics in the age of integrative structural analysis. Curr Opin Cell Biol. 2015;34:31–38. doi: 10.1016/j.ceb.2015.04.009. PubMed DOI

Walther TC, Fornerod M, Pickersgill H, Goldberg M, Allen TD, Mattaj IW. The nucleoporin Nup153 is required for nuclear pore basket formation, nuclear pore complex anchoring and import of a subset of nuclear proteins. EMBO J. 2001;20(20):5703–5714. doi: 10.1093/emboj/20.20.5703. PubMed DOI PMC

Smythe C, Jenkins HE, Hutchison CJ. Incorporation of the nuclear pore basket protein nup153 into nuclear pore structures is dependent upon lamina assembly: evidence from cell-free extracts of Xenopus eggs. EMBO J. 2000;19(15):3918–3931. doi: 10.1093/emboj/19.15.3918. PubMed DOI PMC

Al-Haboubi T, Shumaker DK, Koser J, Wehnert M, Fahrenkrog B. Distinct association of the nuclear pore protein Nup153 with A- and B-type lamins. Nucleus. 2011;2(5):500–509. doi: 10.4161/nucl.2.5.17913. PubMed DOI

Pal K, Bandyopadhyay A, Zhou XE, Xu Q, Marciano DP, Brunzelle JS, Yerrum S, Griffin PR, Vande Woude G, Melcher K, Xu HE. Structural basis of TPR-mediated oligomerization and activation of oncogenic fusion kinases. Structure. 2017;25(6):867–877 e863. doi: 10.1016/j.str.2017.04.015. PubMed DOI PMC

Coyle JH, Bor YC, Rekosh D, Hammarskjold ML. The Tpr protein regulates export of mRNAs with retained introns that traffic through the Nxf1 pathway. RNA. 2011;17(7):1344–1356. doi: 10.1261/rna.2616111. PubMed DOI PMC

Fontoura BM, Dales S, Blobel G, Zhong H. The nucleoporin Nup98 associates with the intranuclear filamentous protein network of TPR. Proc Natl Acad Sci USA. 2001;98(6):3208–3213. doi: 10.1073/pnas.061014698. PubMed DOI PMC

Rajanala K, Sarkar A, Jhingan GD, Priyadarshini R, Jalan M, Sengupta S, Nandicoori VK. Phosphorylation of nucleoporin Tpr governs its differential localization and is required for its mitotic function. J Cell Sci. 2014;127(Pt 16):3505–3520. doi: 10.1242/jcs.149112. PubMed DOI PMC

Vomastek T, Iwanicki MP, Burack WR, Tiwari D, Kumar D, Parsons JT, Weber MJ, Nandicoori VK. Extracellular signal-regulated kinase 2 (ERK2) phosphorylation sites and docking domain on the nuclear pore complex protein Tpr cooperatively regulate ERK2–Tpr interaction. Mol Cell Biol. 2008;28(22):6954–6966. doi: 10.1128/MCB.00925-08. PubMed DOI PMC

Nakano H, Funasaka T, Hashizume C, Wong RW. Nucleoporin translocated promoter region (Tpr) associates with dynein complex, preventing chromosome lagging formation during mitosis. J Biol Chem. 2010;285(14):10841–10849. doi: 10.1074/jbc.M110.105890. PubMed DOI PMC

Lee SH, Sterling H, Burlingame A, McCormick F. Tpr directly binds to Mad1 and Mad2 and is important for the Mad1–Mad2-mediated mitotic spindle checkpoint. Genes Dev. 2008;22(21):2926–2931. doi: 10.1101/gad.1677208. PubMed DOI PMC

Schweizer N, Ferras C, Kern DM, Logarinho E, Cheeseman IM, Maiato H. Spindle assembly checkpoint robustness requires Tpr-mediated regulation of Mad1/Mad2 proteostasis. J Cell Biol. 2013;203(6):883–893. doi: 10.1083/jcb.201309076. PubMed DOI PMC

David-Watine B. Silencing nuclear pore protein Tpr elicits a senescent-like phenotype in cancer cells. PLoS One. 2011;6(7):e22423. doi: 10.1371/journal.pone.0022423. PubMed DOI PMC

Snow CJ, Paschal BM. Roles of the nucleoporin Tpr in cancer and aging. Adv Exp Med Biol. 2014;773:309–322. doi: 10.1007/978-1-4899-8032-8_14. PubMed DOI

Krull S, Dorries J, Boysen B, Reidenbach S, Magnius L, Norder H, Thyberg J, Cordes VC. Protein Tpr is required for establishing nuclear pore-associated zones of heterochromatin exclusion. EMBO J. 2010;29(10):1659–1673. doi: 10.1038/emboj.2010.54. PubMed DOI PMC

Vaquerizas JM, Suyama R, Kind J, Miura K, Luscombe NM, Akhtar A. Nuclear pore proteins nup153 and megator define transcriptionally active regions in the Drosophila genome. PLoS Genet. 2010;6(2):e1000846. doi: 10.1371/journal.pgen.1000846. PubMed DOI PMC

Funasaka T, Tsuka E, Wong RW. Regulation of autophagy by nucleoporin Tpr. Sci Rep. 2012;2:878. doi: 10.1038/srep00878. PubMed DOI PMC

McCloskey A, Ibarra A, Hetzer MW. Tpr regulates the total number of nuclear pore complexes per cell nucleus. Genes Dev. 2018;32(19–20):1321–1331. doi: 10.1101/gad.315523.118. PubMed DOI PMC

Shimi T, Pfleghaar K, Kojima S, Pack CG, Solovei I, Goldman AE, Adam SA, Shumaker DK, Kinjo M, Cremer T, Goldman RD. The A- and B-type nuclear lamin networks: microdomains involved in chromatin organization and transcription. Genes Dev. 2008;22(24):3409–3421. doi: 10.1101/gad.1735208. PubMed DOI PMC

Wu R, Terry AV, Singh PB, Gilbert DM. Differential subnuclear localization and replication timing of histone H3 lysine 9 methylation states. Mol Biol Cell. 2005;16(6):2872–2881. doi: 10.1091/mbc.e04-11-0997. PubMed DOI PMC

Vollmer B, Lorenz M, Moreno-Andres D, Bodenhofer M, De Magistris P, Astrinidis SA, Schooley A, Flotenmeyer M, Leptihn S, Antonin W. Nup153 recruits the Nup107–160 complex to the inner nuclear membrane for interphasic nuclear pore complex assembly. Dev Cell. 2015;33(6):717–728. doi: 10.1016/j.devcel.2015.04.027. PubMed DOI

Hase ME, Cordes VC. Direct interaction with nup153 mediates binding of Tpr to the periphery of the nuclear pore complex. Mol Biol Cell. 2003;14(5):1923–1940. doi: 10.1091/mbc.e02-09-0620. PubMed DOI PMC

Belgareh N, Rabut G, Bai SW, van Overbeek M, Beaudouin J, Daigle N, Zatsepina OV, Pasteau F, Labas V, Fromont-Racine M, Ellenberg J, Doye V. An evolutionarily conserved NPC subcomplex, which redistributes in part to kinetochores in mammalian cells. J Cell Biol. 2001;154(6):1147–1160. doi: 10.1083/jcb.200101081. PubMed DOI PMC

Fiserova J, Efenberkova M, Sieger T, Maninova M, Uhlirova J, Hozak P. Chromatin organization at the nuclear periphery as revealed by image analysis of structured illumination microscopy data. J Cell Sci. 2017;130(12):2066–2077. doi: 10.1242/jcs.198424. PubMed DOI

Ball G, Demmerle J, Kaufmann R, Davis I, Dobbie IM, Schermelleh L. SIMcheck: a toolbox for successful super-resolution structured illumination microscopy. Sci Rep. 2015;5:15915. doi: 10.1038/srep15915. PubMed DOI PMC

Tsai W. Moment-preserving tresholding: a new approach. Comput Vis Graph Image Process. 1985;29:377–393. doi: 10.1016/0734-189X(85)90133-1. DOI

Arganda-Carreras I, Fernandez-Gonzalez R, Munoz-Barrutia A, Ortiz-De-Solorzano C. 3D reconstruction of histological sections: application to mammary gland tissue. Microsc Res Tech. 2010;73(11):1019–1029. doi: 10.1002/jemt.20829. PubMed DOI

Wee CY, Parmesran R. Measure of image sharpness using eigenvalues. Inf Sci. 2007;177(12):2533–2552. doi: 10.1016/j.ins.2006.12.023. DOI

Razafsky D, Ward C, Potter C, Zhu W, Xue Y, Kefalov VJ, Fong LG, Young SG, Hodzic D. Lamin B1 and lamin B2 are long-lived proteins with distinct functions in retinal development. Mol Biol Cell. 2016;27(12):1928–1937. doi: 10.1091/mbc.e16-03-0143. PubMed DOI PMC

Jacinto FV, Benner C, Hetzer MW. The nucleoporin Nup153 regulates embryonic stem cell pluripotency through gene silencing. Genes Dev. 2015;29(12):1224–1238. doi: 10.1101/gad.260919.115. PubMed DOI PMC

Nanni S, Re A, Ripoli C, Gowran A, Nigro P, D’Amario D, Amodeo A, Crea F, Grassi C, Pontecorvi A, Farsetti A, Colussi C. The nuclear pore protein Nup153 associates with chromatin and regulates cardiac gene expression in dystrophic mdx hearts. Cardiovasc Res. 2016;112(2):555–567. doi: 10.1093/cvr/cvw204. PubMed DOI

Toda T, Hsu JY, Linker SB, Hu L, Schafer ST, Mertens J, Jacinto FV, Hetzer MW, Gage FH. Nup153 interacts with Sox2 to enable bimodal gene regulation and maintenance of neural progenitor cells. Cell Stem Cell. 2017;21(5):618–634 e617. doi: 10.1016/j.stem.2017.08.012. PubMed DOI

Freund A, Laberge RM, Demaria M, Campisi J. Lamin B1 loss is a senescence-associated biomarker. Mol Biol Cell. 2012;23(11):2066–2075. doi: 10.1091/mbc.e11-10-0884. PubMed DOI PMC

Otsuka S, Bui KH, Schorb M, Hossain MJ, Politi AZ, Koch B, Eltsov M, Beck M, Ellenberg J. Nuclear pore assembly proceeds by an inside-out extrusion of the nuclear envelope. Elife. 2016;5:e19071. doi: 10.7554/eLife.19071. PubMed DOI PMC

Otsuka S, Steyer AM, Schorb M, Heriche JK, Hossain MJ, Sethi S, Kueblbeck M, Schwab Y, Beck M, Ellenberg J. Postmitotic nuclear pore assembly proceeds by radial dilation of small membrane openings. Nat Struct Mol Biol. 2018;25(1):21–28. doi: 10.1038/s41594-017-0001-9. PubMed DOI

Nucleoporin TPR Affects C2C12 Myogenic Differentiation via Regulation of Myh4 Expression