Quantifying organellar ultrastructure in cryo-electron tomography using a surface morphometrics pipeline.

Area of Science:

• Cell Biology
• Structural Biology
• Biophysics

Background:

• Cellular cryo-electron tomography (cryo-ET) provides high-resolution 3D views of organelles in situ.
• Quantifying complex 3D organelle structures and perturbation-induced changes remains a challenge.

Purpose of the Study:

• To develop a semiautomated workflow and open-source pipeline for segmenting organellar membranes and quantifying their ultrastructural features from cryo-ET data.
• To investigate the impact of endoplasmic reticulum (ER) stress on mitochondrial ultrastructure and morphology.

Main Methods:

• A semiautomated workflow for segmenting organellar membranes and reconstructing surface geometry from cryo-ET data.
• Development of the open-source surface morphometrics pipeline for detailed ultrastructural quantification (e.g., spacing, curvedness, orientation).
• Integration of cryo-ET with cryo-fluorescence microscopy to correlate mitochondrial network morphology with individual mitochondrial ultrastructure.

Main Results:

• The surface morphometrics pipeline enables rapid modeling of complex membrane structures and quantitative analysis.
• ER stress induces adaptive remodeling of mitochondrial ultrastructure, including changes in inner/outer membrane spacing and inner membrane curvedness.
• Mitochondrial membrane ultrastructure differences correlate with observed mitochondrial network morphologies (elongated vs. fragmented).

Conclusions:

• The developed pipeline effectively quantifies subtle, three-dimensional organellar ultrastructural changes.
• ER stress and mitochondrial network morphology are coupled through adaptive remodeling of mitochondrial membrane ultrastructure.
• This approach facilitates single-cell level ultrastructural quantification, enabling the study of diverse cellular perturbations.