Observing and tracking single small ribosomal subunits in vivo

Area of Science:

• Cell Biology
• Molecular Biology
• Biophysics

Background:

• Ribosomes, essential for protein synthesis, comprise small (SSU) and large (LSU) subunits.
• While ribosomal protein composition and synthesis are known, the dynamics and trafficking of ribosomal subunits are poorly understood.
• Existing knowledge gaps hinder a complete understanding of ribosome biogenesis and cellular localization.

Purpose of the Study:

• To investigate the molecular dynamics and intracellular trafficking kinetics of the SSU in vivo.
• To visualize and track single SSU within the nucleolus and nucleoplasm using advanced microscopy techniques.
• To quantify the mobility states of SSU in different cellular compartments.

Main Methods:

• Developed a stable cell line with an inducible SNAP-DIM2 fusion protein for SSU fluorescent labeling.
• Employed a dual-color fluorescent labeling strategy (green for bulk, red for sparse single-molecule visualization).
• Utilized single-molecule microscopy combined with jump-distance analysis and variational Bayes single-particle tracking for trajectory analysis.

Main Results:

• Successfully visualized and tracked individual SSU in the nucleolus and nucleoplasm.
• Identified three distinct SSU populations: mobile (2.3–2.8 µm²/s), retarded (0.18–0.31 µm²/s), and immobilized (0.04–0.05 µm²/s).
• Observed a significant fraction (33%) of immobile SSU in the nucleoplasm, contrasting with inert control molecules.

Conclusions:

• The study provides novel insights into the dynamic behavior of SSU within the cell.
• The presence of a substantial immobile SSU fraction in the nucleoplasm suggests regulatory mechanisms or specific functional states.
• These findings advance our understanding of ribosome biogenesis, intracellular transport, and potential regulatory processes.