Expansion Microscopy Provides Nanoscale Insight into Nucleolar Reorganization and Nuclear Foci Formation during...

Area of Science:

• Cell Biology
• Molecular Biology
• Microscopy Techniques

Background:

• The nucleolus is a vital membraneless organelle for ribosome biogenesis, possessing a complex nanoscale organization sensitive to cellular signals and disease.
• Understanding the nucleolus's structural dynamics is crucial for deciphering cellular responses and disease mechanisms.

Purpose of the Study:

• To explore the potential of Expansion Microscopy (ExM) for high-resolution imaging of the nucleolus and other membraneless organelles.
• To investigate the structural and functional changes in the nucleolus under induced nucleolar stress.
• To elucidate the early mechanisms of the nucleolar stress response and its molecular basis.

Main Methods:

• Application of dual protein Expansion Microscopy (dual-proExM) and click Expansion Microscopy (click-ExM) for nanoscale imaging.
• Induction of nucleolar stress using oxaliplatin, actinomycin D, and platinum-based compounds.
• Time-dependent analysis of nucleolar structural and functional alterations.

Main Results:

• Achieved unprecedented nucleolar resolution of approximately 45 ± 2 nm using ExM techniques.
• Identified novel stages in the nucleolar stress response preceding RNA Polymerase I sequestration.
• Visualized nanoscale protein rearrangements linked to RNA transcription and identified locations of active transcripts.
• Observed segregation of fibrillarin and NPM1 into nucleoplasmic foci under prolonged stress, imaged at nanometer resolution.

Conclusions:

• Expansion Microscopy (ExM) provides exceptional nanometer-scale precision for imaging membraneless organelles like the nucleolus.
• The study reveals new morphological details and sheds light on the early molecular mechanisms of the nucleolar stress response.
• ExM is a powerful tool for advancing the understanding of nucleolar structure, function, and response to cellular perturbations.