Dual-color dSTORM imaging and ThunderSTORM image reconstruction and analysis to study the spatial organization of the nuclear phosphatidylinositol phosphates

Abstract

Single molecule localization microscopy (SMLM) provided an unprecedented insight into the sub-nuclear organization of proteins and nucleic acids but apart from the nuclear envelope the role of the nuclear lipids in the functional organization of the cell nucleus was less studied. Nevertheless, nuclear lipids and specifically phosphatidylinositol phosphates (PIPs) play increasingly evident roles in gene expression. Therefore, here we provide the SMLM-based approach for the quantitative evaluation of the nuclear PIPs distribution while preserving the context of nuclear architecture. Specifically, on the example of phosphatidylinositol 4,5-bisphosphate (PIP2) we have: Implemented and optimized the dual-color dSTORM imaging of nuclear PIP2. Customized the Nearest Neighbor Distance analysis using ImageJ2 plug-in ThunderSTORM to quantitatively evaluate the spatial distribution of nuclear PIP2. Developed an ImageJ2 tool for the visualization of the Nearest Neighbor Distance analysis results in cellulo . Our customization of the dual-color dSTORM imaging and quantitative analysis provide a tool that is independent of but complementary to the biochemical and lipidomic analyses of the nuclear PIPs. Contrary to the biochemical and lipidomic analyses, the advantage of our analysis is that it preserves the spatial context of the nuclear PIP distribution.

PIP2-Effector Protein MPRIP Regulates RNA Polymerase II Condensation and Transcription

“…The Ser5-phosphorylated RNAPII pauses at the promoter-proximal region (+20 to +60 bp). PIP2-containing nuclear lipid islets (NLIs) associate with the initiation condensate [ 18 , 19 , 20 , 21 ]. Following the Tyr1 phosphorylation of the Rpb1 CTD, MPRIP mediates the interaction between the RNAPII complex and NLIs.…”

“…Phosphatidylinositol 4,5-bisphosphate (PIP2) is the most abundant phosphoinositide in the nucleus and it contributes to the nuclear architecture by regulating processes such as chromatin remodeling, DNA-damage response, and gene expression [ 17 ]. PIP2 localizes to nuclear speckles, nucleoli, and nucleoplasm, which is determined by electron, light, and super-resolution microscopy [ 18 , 19 , 20 , 21 , 22 , 23 ]. In nuclear speckles, PIP2 is associated with small nuclear ribonucleoproteins (U1-U6 snRNAs) and the hyper-phosphorylated form of RNAPII [ 24 ].…”

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 (Figure 3), localized to the specific sub-nuclear compartments, and linked with the crucial nuclear processes [115][116][117][118][119][120][121][122][123]. So far, we have started to paint with nanometer precision the static pictures of nuclear PIP distributions [124,125]. However, as we learned from the cytoplasmic membrane, intracellular membrane compartments, and nuclear envelope, lipids are highly mobile entities [52].…”

Comparative analysis of ChAdOx1 nCoV-19 and Ad26.COV2.S SARS-CoV-2 vector vaccines

“…Coverslips were mounted in Everspark dSTORM buffer (Idylle Labs, France) 16 and blinking sequences imaged on a Zeiss Elyra PS.1 super resolution system. Single-molecule localization microscopy data were processed in FIJI using NanoJ core 17 and Thunderstorm 18 and analyzed using custom FIJI 19 scripts.…”