Phosphoinositides are glycerol-based phospholipids, and they play essential roles in cellular signalling, membrane and cytoskeletal dynamics, cell movement, and the modulation of ion channels and transporters. Phosphoinositides are also associated with fundamental nuclear processes through their nuclear protein-binding partners, even though membranes do not exist inside of the nucleus. Phosphatidylinositol 4-phosphate (PI(4)P) is one of the most abundant cellular phosphoinositides; however, its functions in the nucleus are still poorly understood. In this study, we describe PI(4)P localisation in the cell nucleus by super-resolution light and electron microscopy, and employ immunoprecipitation with a specific anti-PI(4)P antibody and subsequent mass spectrometry analysis to determine PI(4)P's interaction partners. We show that PI(4)P is present at the nuclear envelope, in nuclear lamina, in nuclear speckles and in nucleoli and also forms multiple small foci in the nucleoplasm. Nuclear PI(4)P undergoes re-localisation to the cytoplasm during cell division; it does not localise to chromosomes, nucleolar organising regions or mitotic interchromatin granules. When PI(4)P and PI(4,5)P2 are compared, they have different nuclear localisations during interphase and mitosis, pointing to their functional differences in the cell nucleus. Mass spectrometry identified hundreds of proteins, including 12 potentially novel PI(4)P interactors, most of them functioning in vital nuclear processes such as pre-mRNA splicing, transcription or nuclear transport, thus extending the current knowledge of PI(4)P's interaction partners. Based on these data, we propose that PI(4)P also plays a role in essential nuclear processes as a part of protein-lipid complexes. Altogether, these observations provide a novel insight into the role of PI(4)P in nuclear functions and provide a direction for further investigation.

Department of Biology of the Cell Nucleus Institute of Molecular Genetics of the Czech Academy of Sciences Vídeňská 1083 142 20 Prague Czech Republic

Department of Epigenetics of the Cell Nucleus Institute of Molecular Genetics of the Czech Academy of Sciences division BIOCEV Průmyslová 595 252 20 Vestec Czech Republic

Microscopy Centre of the Institute of Molecular Genetics of the Czech Academy of Sciences Vídeňská 1083 142 20 Prague Czech Republic

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Viaud J., Mansour R., Antkowiak A., Mujalli A., Valet C., Chicanne G., Xuereb J.M., Terrisse A.D., Séverin S., Gratacap M.P., et al. Phosphoinositides: Important lipids in the coordination of cell dynamics. Biochimie. 2016;125:250–258. doi: 10.1016/j.biochi.2015.09.005. PubMed DOI

Cauvin C., Echard A. Phosphoinositides: Lipids with informative heads and mastermind functions in cell division. Biochim. Biophys. Acta. 2015;1851:832–843. doi: 10.1016/j.bbalip.2014.10.013. PubMed DOI

Balla T. Phosphoinositides: Tiny lipids with giant impact on cell regulation. Physiol. Rev. 2013;93:1019–1137. doi: 10.1152/physrev.00028.2012. PubMed DOI PMC

Ramos A.R., Elong Edimo W., Erneux C. Phosphoinositide 5-phosphatase activities control cell motility in glioblastoma: Two phosphoinositides PI(4,5)P2 and PI(3,4)P2 are involved. Adv. Biol. Regul. 2018;67:40–48. doi: 10.1016/j.jbior.2017.09.001. PubMed DOI

Vann L.R., Wooding F.B., Irvine R.F., Divecha N. Metabolism and possible compartmentalization of inositol lipids in isolated rat-liver nuclei. Biochem. J. 1997;327:569–576. doi: 10.1042/bj3270569. PubMed DOI PMC

Mazzotti G., Zini N., Rizzi E., Rizzoli R., Galanzi A., Ognibene A., Santi S., Matteucci A., Martelli A.M., Maraldi N.M. Immunocytochemical detection of phosphatidylinositol 4,5-bisphosphate localization sites within the nucleus. J. Histochem. Cytochem. 1995;43:181–191. doi: 10.1177/43.2.7822774. PubMed DOI

Boronenkov I.V., Loijens J.C., Umeda M., Anderson R.A. Phosphoinositide signaling pathways in nuclei are associated with nuclear speckles containing pre-mRNA processing factors. Mol. Biol. Cell. 1998;9:3547–3560. PubMed PMC

Gillooly D.J., Morrow I.C., Lindsay M., Gould R., Bryant N.J., Gaullier J.M., Parton R.G., Stenmark H. Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells. EMBO J. 2000;19:4577–4588. doi: 10.1093/emboj/19.17.4577. PubMed DOI PMC

Osborne S.L., Thomas C.L., Gschmeissner S., Schiavo G. Nuclear PtdIns(4,5)P2 assembles in a mitotically regulated particle involved in pre-mRNA splicing. J. Cell Sci. 2001;114:2501–2511. PubMed

Watt S.A., Kular G., Fleming I.N., Downes C.P., Lucocq J.M. Subcellular localization of phosphatidylinositol 4,5-bisphosphate using the pleckstrin homology domain of phospholipase C delta1. Biochem. J. 2002;363:657–666. doi: 10.1042/bj3630657. PubMed DOI PMC

Lindsay Y., McCoull D., Davidson L., Leslie N.R., Fairservice A., Gray A., Lucocq J., Downes C.P. Localization of agonist-sensitive PtdIns(3,4,5)P3 reveals a nuclear pool that is insensitive to PTEN expression. J. Cell Sci. 2006;119:5160–5168. doi: 10.1242/jcs.000133. PubMed DOI

Hammond G.R., Sim Y., Lagnado L., Irvine R.F. Reversible binding and rapid diffusion of proteins in complex with inositol lipids serves to coordinate free movement with spatial information. J. Cell Biol. 2009;184:297–308. doi: 10.1083/jcb.200809073. PubMed DOI PMC

Yildirim S., Castano E., Sobol M., Philimonenko V.V., Dzijak R., Venit T., Hozák P. Involvement of phosphatidylinositol 4,5-bisphosphate in RNA polymerase I transcription. J. Cell Sci. 2013;126:2730–2739. doi: 10.1242/jcs.123661. PubMed DOI

Castano E., Yildirim S., Faberova V., Krausova A., Ulicna L., Paprckova D., Sztacho M., Hozák P. Nuclear phosphoinositides-versatile regulators of genome functions. Cells. 2019;8:649. doi: 10.3390/cells8070649. PubMed DOI PMC

Jacobsen R.G., Mazloumi Gavgani F., Edson A.J., Goris M., Altankhuyag A., Lewis A.E. Polyphosphoinositides in the nucleus: Roadmap of their effectors and mechanisms of interaction. Adv. Biol. Regul. 2019;72:7–21. doi: 10.1016/j.jbior.2019.04.001. PubMed DOI

Blind R.D., Suzawa M., Ingraham H.A. Direct modification and activation of a nuclear receptor-PIP(2) complex by the inositol lipid kinase IPMK. Sci. Signal. 2012;5:ra44. doi: 10.1126/scisignal.2003111. PubMed DOI PMC

Toska E., Campbell H.A., Shandilya J., Goodfellow S.J., Shore P., Medler K.F., Roberts S.G. Repression of transcription by WT1-BASP1 requires the myristoylation of BASP1 and the PIP2-dependent recruitment of histone deacetylase. Cell Rep. 2012;2:462–469. doi: 10.1016/j.celrep.2012.08.005. PubMed DOI PMC

Sobol M., Yildirim S., Philimonenko V.V., Marasek P., Castano E., Hozak P. UBF complexes with phosphatidylinositol 4,5-bisphosphate in nucleolar organizer regions regardless of ongoing RNA polymerase I activity. Nucleus. 2013;4:478–486. doi: 10.4161/nucl.27154. PubMed DOI PMC

Sobol M., Krausova A., Yildirim S., Kalasova I., Faberova V., Vrkoslav V., Philimonenko V., Marášek P., Pastorek L., Čapek M., et al. Nuclear phosphatidylinositol 4,5-bisphosphate islets contribute to efficient RNA polymerase II-dependent transcription. J. Cell Sci. 2018;131 doi: 10.1242/jcs.211094. PubMed DOI

Stijf-Bultsma Y., Sommer L., Tauber M., Baalbaki M., Giardoglou P., Jones D.R., Gelato K.A., van Pelt J., Shah Z., Rahnamoun H., et al. The basal transcription complex component TAF3 transduces changes in nuclear phosphoinositides into transcriptional output. Mol. Cell. 2015;58:453–467. doi: 10.1016/j.molcel.2015.03.009. PubMed DOI PMC

Rando O.J., Zhao K., Janmey P., Crabtree G.R. Phosphatidylinositol-dependent actin filament binding by the SWI/SNF-like BAF chromatin remodeling complex. Proc. Natl. Acad. Sci. USA. 2002;99:2824–2829. doi: 10.1073/pnas.032662899. PubMed DOI PMC

Gelato K.A., Tauber M., Ong M.S., Winter S., Hiragami-Hamada K., Sindlinger J., Lemak A., Bultsma Y., Houliston S., Schwarzer D., et al. Accessibility of different histone H3-binding domains of UHRF1 is allosterically regulated by phosphatidylinositol 5-phosphate. Mol. Cell. 2014;54:905–919. doi: 10.1016/j.molcel.2014.04.004. PubMed DOI

Ulicna L., Kalendova A., Kalasova I., Vacik T., Hozak P. PIP2 epigenetically represses rRNA genes transcription interacting with PHF8. Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2018;1863:266–275. doi: 10.1016/j.bbalip.2017.12.008. PubMed DOI

Ulicna L., Paprckova D., Faberova V., Hozak P. Phospholipids and inositol phosphates linked to the epigenome. Histochem. Cell Biol. 2018;150:245–253. doi: 10.1007/s00418-018-1690-9. PubMed DOI

Graham T.R., Burd C.G. Coordination of Golgi functions by phosphatidylinositol 4-kinases. Trends Cell Biol. 2011;21:113–121. doi: 10.1016/j.tcb.2010.10.002. PubMed DOI PMC

Hammond G.R., Schiavo G., Irvine R.F. Immunocytochemical techniques reveal multiple, distinct cellular pools of PtdIns4P and PtdIns(4,5)P(2) Biochem. J. 2009;422:23–35. doi: 10.1042/BJ20090428. PubMed DOI PMC

Kalasova I., Faberova V., Kalendova A., Yildirim S., Ulicna L., Venit T., Hozák P. Tools for visualization of phosphoinositides in the cell nucleus. Histochem. Cell Biol. 2016;145:485–496. doi: 10.1007/s00418-016-1409-8. PubMed DOI

Andersen J.S., Lyon C.E., Fox A.H., Leung A.K., Lam Y.W., Steen H., Mann M., Lamond A.I. Directed proteomic analysis of the human nucleolus. Curr. Biol. 2002;12:1–11. doi: 10.1016/S0960-9822(01)00650-9. PubMed DOI

Masuda T., Tomita M., Ishihama Y. Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J. Proteome Res. 2008;7:731–740. doi: 10.1021/pr700658q. PubMed DOI

Rappsilber J., Mann M., Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2007;2:1896–1906. doi: 10.1038/nprot.2007.261. PubMed DOI

Hebert A.S., Richards A.L., Bailey D.J., Ulbrich A., Coughlin E.E., Westphall M.S., Coon J.J. The one hour yeast proteome. Mol. Cell Proteom. 2014;13:339–347. doi: 10.1074/mcp.M113.034769. PubMed DOI PMC

Cox J., Hein M.Y., Luber C.A., Paron I., Nagaraj N., Mann M. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell Proteom. 2014;13:2513–2526. doi: 10.1074/mcp.M113.031591. PubMed DOI PMC

Stradalova V., Gaplovska-Kysela K., Hozak P. Ultrastructural and nuclear antigen preservation after high-pressure freezing/freeze-substitution and low-temperature LR White embedding of HeLa cells. Histochem. Cell Biol. 2008;130:1047–1052. doi: 10.1007/s00418-008-0504-x. PubMed DOI

Philimonenko A.A., Janacek J., Hozak P. Statistical evaluation of colocalization patterns in immunogold labeling experiments. J. Struct. Biol. 2000;132:201–210. doi: 10.1006/jsbi.2000.4326. PubMed DOI

Sharma A., Takata H., Shibahara K., Bubulya A., Bubulya P.A. Son is essential for nuclear speckle organization and cell cycle progression. Mol. Biol. Cell. 2010;21:650–663. doi: 10.1091/mbc.e09-02-0126. PubMed DOI PMC

Huen M.S., Sy S.M., Leung K.M., Ching Y.P., Tipoe G.L., Man C., Dong S., Chen J. SON is a spliceosome-associated factor required for mitotic progression. Cell Cycle. 2010;9:2679–2685. doi: 10.4161/cc.9.13.12151. PubMed DOI PMC

Spector D.L., Lamond A.I. Nuclear speckles. Cold Spring Harb Perspect Biol. 2011;3 doi: 10.1101/cshperspect.a000646. PubMed DOI PMC

Quinodoz S.A., Ollikainen N., Tabak B., Palla A., Schmidt J.M., Detmar E., Lai M.M., Shishkin A.A., Bhat P., Takei Y., et al. Higher-order inter-chromosomal hubs shape 3D genome organization in the nucleus. Cell. 2018;174:744–757. doi: 10.1016/j.cell.2018.05.024. PubMed DOI PMC

Kim J., Han K.Y., Khanna N., Ha T., Belmont A.S. Nuclear speckle fusion via long-range directional motion regulates speckle morphology after transcriptional inhibition. J. Cell Sci. 2019;132 doi: 10.1242/jcs.226563. PubMed DOI PMC

Yao R.W., Xu G., Wang Y., Shan L., Luan P.F., Wang Y., Wu M., Yang L.Z., Xing Y.H., Yang L., et al. Nascent Pre-rRNA Sorting via phase separation drives the assembly of dense fibrillar components in the human nucleolus. Mol. Cell. 2019;76:767–783. PubMed

Prasanth K.V., Sacco-Bubulya P.A., Prasanth S.G., Spector D.L. Sequential entry of components of the gene expression machinery into daughter nuclei. Mol. Biol. Cell. 2003;14:1043–1057. doi: 10.1091/mbc.e02-10-0669. PubMed DOI PMC

Gomes X.V., Wold M.S. Functional domains of the 70-kilodalton subunit of human replication protein A. Biochemistry. 1996;35:10558–10568. doi: 10.1021/bi9607517. PubMed DOI

Smith C.D., Wells W.W. Phosphorylation of rat liver nuclear envelopes. II. Characterization of in vitro lipid phosphorylation. J. Biol. Chem. 1983;258:9368–9373. PubMed

Cocco L., Gilmour R.S., Ognibene A., Letcher A.J., Manzoli F.A., Irvine R.F. Synthesis of polyphosphoinositides in nuclei of Friend cells. Evidence for polyphosphoinositide metabolism inside the nucleus which changes with cell differentiation. Biochem. J. 1987;248:765–770. doi: 10.1042/bj2480765. PubMed DOI PMC

Yang Y., Lee M., Fairn G.D. Phospholipid subcellular localization and dynamics. J. Biol. Chem. 2018;293:6230–6240. doi: 10.1074/jbc.R117.000582. PubMed DOI PMC

Vance J.E. Phospholipid synthesis and transport in mammalian cells. Traffic. 2015;16:1–18. doi: 10.1111/tra.12230. PubMed DOI

de Graaf P., Klapisz E.E., Schulz T.K., Cremers A.F., Verkleij A.J., van Bergen en Henegouwen P.M. Nuclear localization of phosphatidylinositol 4-kinase beta. J. Cell Sci. 2002;115:1769–1775. PubMed

Strahl T., Hama H., DeWald D.B., Thorner J. Yeast phosphatidylinositol 4-kinase, Pik1, has essential roles at the Golgi and in the nucleus. J. Cell Biol. 2005;171:967–979. doi: 10.1083/jcb.200504104. PubMed DOI PMC

Szivak I., Lamb N., Heilmeyer L.M. Subcellular localization and structural function of endogenous phosphorylated phosphatidylinositol 4-kinase (PI4K92) J. Biol. Chem. 2006;281:16740–16749. doi: 10.1074/jbc.M511645200. PubMed DOI

Kakuk A., Friedlander E., Vereb G., Jr., Kasa A., Balla A., Balla T., Heilmeyer L.M., Jr., Gergely P., Vereb G. Nucleolar localization of phosphatidylinositol 4-kinase PI4K230 in various mammalian cells. Cytometry A. 2006;69:1174–1183. doi: 10.1002/cyto.a.20347. PubMed DOI

Lachyankar M.B., Sultana N., Schonhoff C.M., Mitra P., Poluha W., Lambert S., Quesenberry P.J., Litofsky N.S., Recht L.D., Nabi R., et al. A role for nuclear PTEN in neuronal differentiation. J. Neurosci. 2000;20:1404–1413. doi: 10.1523/JNEUROSCI.20-04-01404.2000. PubMed DOI PMC

Gimm O., Perren A., Weng L.P., Marsh D.J., Yeh J.J., Ziebold U., Gil E., Hinze R., Delbridge L., Lees J.A., et al. Differential nuclear and cytoplasmic expression of PTEN in normal thyroid tissue, and benign and malignant epithelial thyroid tumors. Am. J. Pathol. 2000;156:1693–1700. doi: 10.1016/S0002-9440(10)65040-7. PubMed DOI PMC

Deleris P., Bacqueville D., Gayral S., Carrez L., Salles J.P., Perret B., Breton-Douillon M. SHIP-2 and PTEN are expressed and active in vascular smooth muscle cell nuclei, but only SHIP-2 is associated with nuclear speckles. J. Biol. Chem. 2003;278:38884–38891. doi: 10.1074/jbc.M300816200. PubMed DOI

Elong Edimo W., Derua R., Janssens V., Nakamura T., Vanderwinden J.M., Waelkens E., Erneux C. Evidence of SHIP2 Ser132 phosphorylation, its nuclear localization and stability. Biochem. J. 2011;439:391–401. doi: 10.1042/BJ20110173. PubMed DOI

Nalaskowski M.M., Metzner A., Brehm M.A., Labiadh S., Brauer H., Grabinski N., Mayr G.W., Jücker M. The inositol 5-phosphatase SHIP1 is a nucleo-cytoplasmic shuttling protein and enzymatically active in cell nuclei. Cell Signal. 2012;24:621–628. doi: 10.1016/j.cellsig.2011.07.012. PubMed DOI

Ehm P., Nalaskowski M.M., Wundenberg T., Jucker M. The tumor suppressor SHIP1 colocalizes in nucleolar cavities with p53 and components of PML nuclear bodies. Nucleus. 2015;6:154–164. doi: 10.1080/19491034.2015.1022701. PubMed DOI PMC

Mellman D.L., Gonzales M.L., Song C., Barlow C.A., Wang P., Kendziorski C., Anderson R.A. A PtdIns4,5P2-regulated nuclear poly(A) polymerase controls expression of select mRNAs. Nature. 2008;451:1013–1017. doi: 10.1038/nature06666. PubMed DOI

Kakuk A., Friedlander E., Vereb G., Jr., Lisboa D., Bagossi P., Toth G., Gergely P., Vereb G. Nuclear and nucleolar localization signals and their targeting function in phosphatidylinositol 4-kinase PI4K230. Exp. Cell Res. 2008;314:2376–8238. doi: 10.1016/j.yexcr.2008.05.006. PubMed DOI

Balla A., Kim Y.J., Varnai P., Szentpetery Z., Knight Z., Shokat K.M., Balla T. Maintenance of hormone-sensitive phosphoinositide pools in the plasma membrane requires phosphatidylinositol 4-kinase IIIalpha. Mol. Biol. Cell. 2008;19:711–721. doi: 10.1091/mbc.e07-07-0713. PubMed DOI PMC

Harak C., Radujkovic D., Taveneau C., Reiss S., Klein R., Bressanelli S., Lohmann V. Mapping of functional domains of the lipid kinase phosphatidylinositol 4-kinase type III alpha involved in enzymatic activity and hepatitis C virus replication. J. Virol. 2014;88:9909–9926. doi: 10.1128/JVI.01063-14. PubMed DOI PMC

Izquierdo J.M., Majos N., Bonnal S., Martinez C., Castelo R., Guigo R., Bilbao D., Valcárcel J. Regulation of Fas alternative splicing by antagonistic effects of TIA-1 and PTB on exon definition. Mol. Cell. 2005;19:475–484. doi: 10.1016/j.molcel.2005.06.015. PubMed DOI

Kotak S., Busso C., Gonczy P. NuMA interacts with phosphoinositides and links the mitotic spindle with the plasma membrane. EMBO J. 2014;33:1815–1830. doi: 10.15252/embj.201488147. PubMed DOI PMC

Kukalev A., Nord Y., Palmberg C., Bergman T., Percipalle P. Actin and hnRNP U cooperate for productive transcription by RNA polymerase II. Nat. Struct. Mol. Biol. 2005;12:238–244. doi: 10.1038/nsmb904. PubMed DOI

Hautbergue G.M., Hung M.L., Golovanov A.P., Lian L.Y., Wilson S.A. Mutually exclusive interactions drive handover of mRNA from export adaptors to TAP. Proc. Natl. Acad. Sci. USA. 2008;105:5154–5159. doi: 10.1073/pnas.0709167105. PubMed DOI PMC

Sztacho M., Sobol M., Balaban C., Escudeiro Lopes S.E., Hozak P. NucSlear phosphoinositides and phase separation: Important players in nuclear compartmentalization. Adv. Biol. Regul. 2019;71:111–117. doi: 10.1016/j.jbior.2018.09.009. PubMed DOI

How Single-Molecule Localization Microscopy Expanded Our Mechanistic Understanding of RNA Polymerase II Transcription

Limited Proteolysis-Coupled Mass Spectrometry Identifies Phosphatidylinositol 4,5-Bisphosphate Effectors in Human Nuclear Proteome