ABSTRACT Cellular cryo-electron tomography (cryo-ET) enables 3-dimensional reconstructions of organelles in their native cellular environment at subnanometer resolution. However, quantifying ultrastructural features of pleomorphic organelles in three dimensions is challenging, as is defining the significance of observed changes induced by specific cellular perturbations. To address this challenge, we established a semi-automated workflow to segment organellar membranes and reconstruct their underlying surface geometry in cryo-ET. To complement this workflow, we developed an open source suite of ultrastructural quantifications, integrated into a single pipeline called the surface morphometrics toolkit. This toolkit allows detailed mapping of spacing, curvature, and orientation onto reconstructed membrane meshes, highlighting subtle organellar features that are challenging to detect in three dimensions and allowing for statistical comparison across many organelles. To demonstrate the advantages of this approach, we combine cryo-ET with cryo-fluorescence microscopy to correlate bulk mitochondrial network morphology (i.e., elongated versus fragmented) with membrane ultrastructure of individual mitochondria in the presence and absence of endoplasmic reticulum (ER) stress. Using our toolkit, we demonstrate ER stress promotes adaptive remodeling of ultrastructural features of mitochondria including spacing between the inner and outer membranes, local curvature of the inner membrane, and spacing between mitochondrial cristae. We show that differences in membrane ultrastructure correlate to mitochondrial network morphologies, suggesting that these two remodeling events are coupled. Our toolkit offers opportunities for quantifying changes in organellar architecture on a single-cell level using cryo-ET, opening new opportunities to define changes in ultrastructural features induced by diverse types of cellular perturbations.

A surface morphometrics toolkit to quantify organellar membrane ultrastructure using cryo-electron tomography