In situ quantification of ribosome number by electron tomography

Area of Science:

• Cell Biology
• Molecular Biology
• Microscopy

Background:

• Ribosomes, crucial for protein synthesis, were discovered in 1955.
• While ribosome structure and function are well-studied, their total cellular number remains largely unquantified.
• Existing methods primarily focus on biochemical estimations rather than direct physical counts.

Purpose of the Study:

• To develop and validate a novel microscopic method for quantifying total ribosome numbers within cells.
• To determine ribosome counts in human cell lines (hTERT-RPE-1) and specific tissues of Caenorhabditis elegans.
• To compare imaging-based quantification with established biochemical approaches.

Main Methods:

• Utilized electron tomography on high-pressure frozen, freeze-substituted, and resin-embedded samples.
• Applied the method to quantify ribosomes in hTERT-RPE-1 cells and various tissues of C. elegans.
• Included controls with depleted RNA polymerase I A subunit (RPOA-1) in nematode studies.

Main Results:

• The microscopic method provided ribosome counts in hTERT-RPE-1 cells comparable to biochemical measurements.
• Ribosome numbers were successfully quantified in different tissues of C. elegans.
• RPOA-1-depleted worms showed significantly fewer ribosomes than control worms in two of three analyzed tissues.

Conclusions:

• The developed imaging-based approach offers a direct and complementary method for ribosome quantification.
• This technique enhances the understanding of ribosome localization and distribution in diverse cellular and tissue contexts.
• The study provides a valuable tool for future research in cell biology and molecular genetics across various model organisms.