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.

Keywords: PI(4)P; nucleus; phosphoinositides.

Figure 1

Detection of phosphatidylinositol 4-phosphate (PI(4)P) at the nuclear envelope and in the nucleoli. Super-resolution STED microscopy revealed the localisation of PI(4)P in the nuclear lamina, the nucleoli, nuclear speckles and nucleoplasmic foci. (a) Fluorescently labelled PI(4)P and lamin B1. (b) PI(4)P co-localises with lamin B1. (c) Intensities of pixels along the lines in b, PI(4)P (green) and lamin B1 (purple). Lines are drawn in a direction from the nucleoplasm to the cytoplasm (from bottom to top). Graphs were made using the FiJi software. (d) Localisation of PI(4)P and a protein C23, a marker of the nucleoli. (e) PI(4)P localises to the nucleoli, but the intensity of PI(4)P staining is much lower than in the nucleoplasm. Scale bars: a—5 μm; b—1 μm; d—5 μm; e—2 μm.

Figure 2

PI(4)P also localises to the nuclear speckles. Beside the nuclear membrane, the nuclear lamina, the nucleoli and nucleoplasmic foci, super-resolution STED microscopy revealed the localisation of PI(4)P in the nuclear speckles. (a) Co-localisation of PI(4)P with the Son protein, a marker of the nuclear speckles. PI(4)P is enriched inside and at the edges of nuclear speckles. Scale bar: 5 μm, insets 1 μm. (b) Percentage of PI(4)P signal in the nuclear speckles and the nucleoplasm per nucleus. Nearly 16% of the nuclear PI(4)P signal comes from the nuclear speckles. The graph was made in the GraphPad Prism program; data are shown as the mean with SD. NS—nuclear speckles, NP—nucleoplasm. (c) Pie chart representation of the results in b.

Figure 3

Nuclear PI(4)P localisation according to transmission electron microscopy. Electron microscopy revealed the localisation of PI(4)P: (A) to the cell nucleus (red dots). Black asterisk—fibrillar centre; DFC—dense fibrillar component; GC—granular component; NE—nuclear envelope; C—cytoplasm; black arrowheads delineate nuclear speckles. (B) In the nucleolus, PI(4)P is enriched in the DFC and also GC, but it does not localise to the FC. (C) In the nucleoplasm, PI(4)P is enriched on chromatin. (D) PI(4)P is also enriched at the nuclear speckles, and nuclear envelope (E,F) where it localises to nuclear lamina. (B–F) are high magnification insets from the same cell.

Figure 4

PI(4)P forms different foci than PI(4,5)P2. Electron microscopy showed the localisation of PI(4)P (red) and PI(4,5)P2 (black) are different in the nucleoplasm. Localisation of PI(4)P visualised with OSH1-PH domain and PI(4,5)P2 visualised with antibody (A) in the nucleoplasm and nuclear envelope, (B) in the nucleolus and (C) in the nuclear speckles. NP—nucleoplasm, C—cytoplasm, FC—fibrillar centre, DFC—dense fibrillar component, GC—granular component, NS—nuclear speckle. Scale bars: 500 nm.

Figure 5

Clustering patterns of (a) PI(4)P labelled with anti-PI(4)P antibody or (b) PI(4)P labelled with OSH1-PH domain. The foci formed by PI(4)P are up to 50 nm in size. (c) Clustering pattern of PI(4,5)P2 labelled with anti-PI(4,5)P2 antibody. The foci formed by PI(4,5)P2 are up to 100 nm in size (d) Co-localisation analysis of PI(4,5)P2 with OSH1-PH labelling (Figure 4). PI(4)P does not specifically co-localise with PI(4,5)P2. PCF—pair correlation function, PCCF—pair cross-correlation function. Dashed lines mark the value of functions equal to 1, which corresponds to a random distribution.

Figure 6

PI(4)P is dispersed in the cytoplasm during mitosis. PI(4)P and Son were labelled, and their localisation was followed across mitotic stages by super-resolution STED microscopy. PI(4)P does not localise to chromatin, nucleolar organising regions (NORs) or mitotic interchromatin granules (MIGs) during cell division. DNA was stained by DAPI. Scale bars: 10 μm.

Figure 7

PI(4)P forms lipid–protein complexes in the nucleus. (a) PI(4)P-binding partners were immunoprecipitated with anti-PI(4)P antibody, and the gel was stained with Coomassie Blue; MS—mass spectrometry. (b) The graph shows a number of proteins identified by mass spectrometry, divided into different sub-groups based on their function in the nucleus (uniprot.org). (c) Example of proteins immunoprecipitated with anti-PI(4)P and anti-PI(4,5)P2 antibody (IP) and pulled down with PI(4)P/PI(4,5)P2-coated agarose beads (PD). Ten percent of an input nuclear lysate was loaded on the gel in rows “in”. In—input; C—control mouse IgM antibody; PA—phosphatidic acid-coated agarose beads; hnRNP U—heterogeneous nuclear ribonucleoprotein U; NXF1—nuclear RNA export factor 1; NuMa—nuclear mitotic apparatus protein 1.