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

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.

[1]  Kees Jalink,et al.  Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy , 2016, PloS one.

[2]  M. Capek,et al.  Nuclear phosphatidylinositol 4,5-bisphosphate islets contribute to efficient RNA polymerase II-dependent transcription , 2018, Journal of Cell Science.

[3]  Jonas Ries,et al.  A tessellation-based colocalization analysis approach for single-molecule localization microscopy , 2019, Nature Communications.

[4]  Guy M. Hagen,et al.  ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging , 2014, Bioinform..

[5]  M. Heilemann,et al.  Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. , 2008, Angewandte Chemie.

[6]  David Baddeley,et al.  Visualization of Localization Microscopy Data , 2010, Microscopy and Microanalysis.

[7]  D. W. Scott Averaged Shifted Histograms: Effective Nonparametric Density Estimators in Several Dimensions , 1985 .

[8]  M. Heilemann,et al.  Direct stochastic optical reconstruction microscopy with standard fluorescent probes , 2011, Nature Protocols.

[9]  M. C. Cardoso,et al.  Separation of replication and transcription domains in nucleoli. , 2014, Journal of structural biology.

[10]  P. Hozák,et al.  Nanoscale mapping of nuclear phosphatidylinositol phosphate landscape by dual-color dSTORM. , 2021, Biochimica et biophysica acta. Molecular and cell biology of lipids.

[11]  Kenneth W Dunn,et al.  A practical guide to evaluating colocalization in biological microscopy. , 2011, American journal of physiology. Cell physiology.

[12]  H. Vogel,et al.  A general method for the covalent labeling of fusion proteins with small molecules in vivo , 2003, Nature Biotechnology.

[13]  Michael D. Mason,et al.  Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. , 2006, Biophysical journal.

[14]  G. Schiavo,et al.  Nuclear PtdIns(4,5)P2 assembles in a mitotically regulated particle involved in pre-mRNA splicing. , 2001, Journal of cell science.

[15]  J. Lippincott-Schwartz,et al.  Imaging Intracellular Fluorescent Proteins at Nanometer Resolution , 2006, Science.

[16]  S. Hell,et al.  Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[17]  Gorjan Alagic,et al.  #p , 2019, Quantum information & computation.

[18]  P. Verveer,et al.  Coordinate-based colocalization analysis of single-molecule localization microscopy data , 2011, Histochemistry and Cell Biology.

[19]  M. Sauer,et al.  Super-resolution microscopy demystified , 2019, Nature Cell Biology.

[20]  M. Gustafsson Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy , 2000, Journal of microscopy.

[21]  R. Henriques,et al.  The cell biologist's guide to super-resolution microscopy , 2020, Journal of Cell Science.

[22]  Michael J Rust,et al.  Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) , 2006, Nature Methods.

[23]  N. Vitale,et al.  Fibrillarin Ribonuclease Activity is Dependent on the GAR Domain and Modulated by Phospholipids , 2020, Cells.

[24]  Barbora Šalovská,et al.  Limited Proteolysis-Coupled Mass Spectrometry Identifies Phosphatidylinositol 4,5-Bisphosphate Effectors in Human Nuclear Proteome , 2021, Cells.

[25]  Kevin W. Eliceiri,et al.  ImageJ2: ImageJ for the next generation of scientific image data , 2017, BMC Bioinformatics.