Super-Resolution Localisation of Nuclear PI(4)P and Identification of Its Interacting Proteome

Abstract

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.

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

“…PIPs are important functional regulators of mammalian nuclear processes [ 4 ]. PIPs are protein ligands with the ability to recruit and thus affect the localization, conformation, and activity of their protein effectors [ 4 , 9 , 23 ]. Therefore, it is of particular interest to identify the PIP2 nuclear effectors.…”

Nuclear Phosphatidylinositol 3,4,5-Trisphosphate Interactome Uncovers an Enrichment in Nucleolar Proteins

“…SHIP2 ( 107 ) or in its phosphorylated form on serine 132 ( 108 ) was found in nuclear speckles in different cells. Alternatively, the class II PI3K, PI3KC2α, known to produce PtdIns(3,4) P 2 by phosphorylating PtdIns4 P , was also reported to localize in nuclear foci ( 109 ) as well as its substrate PtdIns4 P ( 26 , 27 ), thus suggesting a potential synthesis route for PtdIns(3,4) P 2 in nuclear speckles. A key question is whether PtdIns(3,4) P 2 and PtdIns(3,4,5) P 3 bind to or even recruit PARP1 to their nuclear sites.…”

“…The concept of PPIn metabolism and signaling occurring in the nucleus independently of the cytoplasm was reported shortly after in several studies ( 10 , 11 , 12 ). Consequently, with the exception of phosphatidylinositol 3,5-bisphosphate, the remaining six PPIns have been detected and/or quantified in the nucleus ( 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ). The intranuclear biophysico-chemical state of PPIns is still unclear, but several possibilities are emerging to explain how the acyl chains can be shielded from the nuclear aqueous environment.…”

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

“…Nuclear lipids and phosphatidylinositol phosphates (PIPs) in particular, form small foci variably distributed in the eukaryotic cell nucleus (Fig. 3), localized to the specific sub-nuclear compartments and linked with the crucial nuclear processes [110][111][112][113][114][115][116][117][118]. So far, we started to paint with nanometer precision the static pictures of nuclear PIP distributions [119].…”