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

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.

PERK Signaling Promotes Mitochondrial Elongation By Remodeling Membrane Phosphatidic Acid

“…For example, recent cryo-electron tomography results indicate that mitochondrial elongation correlates with cristae remodeling in ER stressed cells, suggesting that these changes to bulk mitochondrial morphology and ultrastructure may be coordinated. 74 Further, PRELID1-dependent trafficking of PA to the IMM is a critical step in the biosynthesis of cardiolipins – a mitochondrial-specific phospholipid important for regulating membrane morphology and multiple mitochondrial functions including respiratory chain activity and apoptotic signaling. 48 While acute ER insults are unlikely to significantly influence cardiolipin levels owing to the higher stability of this phospholipid as compared to other membrane phospholipids 75 , chronic PERK-dependent translational attenuation, and subsequent sustained reductions in PRELID1, could lead to reductions in cardiolipin that impact mitochondrial energy metabolism and cell fate.…”

Quantitative Cryo-Electron Tomography

“…Furthermore, a quantitative analysis of cytoskeletal elements has provided insights into the macromolecular networks they form and their role in motility, trafficking, and cell cycle ( Rigort et al, 2012 ; Xu et al, 2015 ; Anderson et al, 2017 ; Dimchev et al, 2021 ). Membrane parameterization has been also essential to achieve a quantitative ultrastructural analysis of subcellular features ( Salfer et al, 2020 ; Barad et al, 2022 ). The consolidation of such analyses into integrative computational toolboxes highly facilitates the quantitative ultrastructural analysis of cryo-ET data in a time-efficient manner ( Dimchev et al, 2021 ; Barad et al, 2022 ), and appears as the fittest modality for future quantitative cryo-ET research.…”

“…Membrane parameterization has been also essential to achieve a quantitative ultrastructural analysis of subcellular features ( Salfer et al, 2020 ; Barad et al, 2022 ). The consolidation of such analyses into integrative computational toolboxes highly facilitates the quantitative ultrastructural analysis of cryo-ET data in a time-efficient manner ( Dimchev et al, 2021 ; Barad et al, 2022 ), and appears as the fittest modality for future quantitative cryo-ET research.…”

SEC62 and TMX4 control asymmetric autophagy of the nuclear envelope upon LINC complex disassembly

“…CET: ONM to INM distance measurements ONM and INM were manually segmented in Avizo software (Thermo Fisher Scientific) for each tomogram. The exported labels were submitted to surface morphometrics 49 to create a triangular mesh and measure the intra membrane distance along the surfaces. Results were plotted in Python using matplotlib.…”