Stimulated emission depletion (STED) super resolution imaging of RNA- and protein-containing domains in fixed cells

Gabrijela Dumbović; Xavier Sanjuan; Manuel Perucho; Sonia-V Forcales

doi:10.1016/j.ymeth.2020.04.009

Stimulated emission depletion (STED) super resolution imaging of RNA- and protein-containing domains in fixed cells

Methods. 2021 Mar:187:68-76. doi: 10.1016/j.ymeth.2020.04.009. Epub 2020 Apr 30.

Authors

Gabrijela Dumbović¹, Xavier Sanjuan², Manuel Perucho³, Sonia-V Forcales⁴

Affiliations

¹ BioFrontiers Institute, University of Colorado at Boulder, Boulder, CO, USA. Electronic address: gabrijela.dumbovic@gmail.com.
² Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain; Advanced Light Microscopy Unit, Center for Genomic Regulation, Barcelona, Spain.
³ Cancer Genetics and Epigenetics, Program of Predictive and Personalized Medicine of Cancer (PMPPC), Health Science Research Institute Germans Trias i Pujol (IGTP), Badalona, Spain; Tumor Initiation and Maintenance Program, Sanford Burnham Prebys (SBP) Medical Discovery Institute, La Jolla, CA, USA.
⁴ Department of Pathology and Experimental Therapeutics, Faculty of Medicine and Health Sciences, University of Barcelona, L' Hospitalet de Llobregat, Barcelona, Spain. Electronic address: sforcales@ub.edu.

PMID: 32360441
DOI: 10.1016/j.ymeth.2020.04.009

Abstract

Super resolution microscopy has changed our capability to visualize and understand spatial arrangements of RNA- and protein-containing domains in individual cells. In a previous study, we described a novel lncRNA, Tumor-associated NBL2 transcript (TNBL), which originates from a primate specific macrosatellite repeat. We aimed to describe several aspects of TNBL lncRNA, with one focus being pinpointing its precise location in the nucleus, as well as visualizing its interactions with proteins to deduce its functionality. Using a combination of STimulated Emission Depletion (STED) super resolution microscopy, single molecule RNA (smRNA) FISH against TNBL, and immunofluorescence against SAM68 perinucleolar body, we resolved the spatial complexity of the interaction between TNBL aggregates and SAM68 bodies at the perinucleolar region. Here, we describe protocols for a step-by-step optimized smRNA FISH/IF and STED imaging, detailing parameter settings, and three-dimensional data analysis of spatial positioning of subnuclear structures. These protocols can be employed for single-cell imaging of complex nuclear RNA-protein structures.

Keywords: Nuclear architecture; Nuclear domains; STED; Super resolution microscopy; lncRNA.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adaptor Proteins, Signal Transducing / genetics*
Cell Line, Tumor
Cell Nucleus / metabolism
DNA-Binding Proteins / genetics*
Epigenomics / methods*
Humans
In Situ Hybridization, Fluorescence / methods
Microscopy, Fluorescence / methods
RNA, Long Noncoding / analysis
RNA, Long Noncoding / metabolism*
RNA-Binding Proteins / genetics*
Single Molecule Imaging / methods*
Spatio-Temporal Analysis

Substances

Adaptor Proteins, Signal Transducing
DNA-Binding Proteins
KHDRBS1 protein, human
RNA, Long Noncoding
RNA-Binding Proteins