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Early diagnosis of acute myeloid leukemia (AML) in the pre-leukemic stage remains a clinical challenge, as pre-leukemic patients show no symptoms, lacking any known morphological or numerical abnormalities in blood cells. Here, we demonstrate that platelets with structurally abnormal mitochondria emerge at the pre-leukemic phase of AML, preceding detectable changes in blood cell counts or detection of leukemic blasts in blood. We visualized frozen-hydrated platelets from mice at different time points during AML development in situ using electron cryo-tomography (cryo-ET) and identified intracellular organelles through an unbiased semi-automatic process followed by quantitative measurement. A large proportion of platelets exhibited changes in the overall shape and depletion of organelles in AML. Notably, 23% of platelets in pre-leukemic cells exhibit abnormal, round mitochondria with unfolded cristae, accompanied by a significant drop in ATP levels and altered expression of metabolism-related gene signatures. Our study demonstrates that detectable structural changes in pre-leukemic platelets may serve as a biomarker for the early diagnosis of AML., Wang et al characterise platelets in a mouse model of acute myeloid leukemia (AML) using Cryo-electron tomography (Cryo-ET) and find that 23% of platelets in pre-leukemic cells exhibit abnormal, round mitochondria with unfolded cristae. They also detect reduced ATP levels and altered expression of metabolism-related genes, altogether suggesting that platelet alterations may serve as a biomarker for AML.

Lamellar bodies (LBs) are surfactant-rich organelles in alveolar cells. LBs disassemble into a lipid-protein network that reduces surface tension and facilitates gas exchange in the alveolar cavity. Current knowledge of LB architecture is predominantly based on electron microscopy studies using disruptive sample preparation methods. We established and validated a post-correlation on-lamella cryo-correlative light and electron microscopy approach for cryo-FIB milled cells to structurally characterize and validate the identity of LBs in their unperturbed state. Using deconvolution and 3D image registration, we were able to identify fluorescently labeled membrane structures analyzed by cryo-electron tomography. In situ cryo-electron tomography of A549 cells as well as primary Human Small Airway Epithelial Cells revealed that LBs are composed of membrane sheets frequently attached to the limiting membrane through “T”-junctions. We report a so far undescribed outer membrane dome protein complex (OMDP) on the limiting membrane of LBs. Our data suggest that LB biogenesis is driven by parallel membrane sheet import and by the curvature of the limiting membrane to maximize lipid storage capacity., Using the post-correlation on-lamella cryo-CLEM workflow, Klein, Wimmer et al. show that lamellar bodies (LBs) are composed of membrane sheets frequently attached to the limiting membrane through T-junctions in ABCA3 overexpressing cells and in primary human small airway epithelial cells. This study provides insights into LB biogenesis and membrane packing inside the LB.

Huntington disease (HD) is a neurodegenerative trinucleotide repeat disorder caused by an expanded poly-glutamine (polyQ) tract in the mutant huntingtin (mHTT) protein. The formation and topology of filamentous mHTT inclusions in the brain (hallmarks of HD implicated in neurotoxicity) remain elusive. Using cryo-electron tomography and subtomogram averaging, here we show that mHTT exon 1 and polyQ-only aggregates in vitro are structurally heterogenous and filamentous, similar to prior observations with other methods. Yet, we find filaments in both types of aggregates under ~2 nm in width, thinner than previously reported, and regions forming large sheets. In addition, our data show a prevalent subpopulation of filaments exhibiting a lumpy slab morphology in both aggregates, supportive of the polyQ core model. This provides a basis for future cryoET studies of various aggregated mHTT and polyQ constructs to improve their structure-based modeling as well as their identification in cells without fusion tags., Galaz-Montoya et al. report nanometer-resolution 3D cryo-electron tomography structures of mutant huntingtin (mHTT) and polyglutamine-only (polyQ) filaments in large aggregates free of stains, fixatives, tags, or dehydration artifacts. These results provide a framework for future structural studies of mHTT and polyQ aggregates, thereby improving our understanding of polyQ disorders such as Huntington disease.

Gag is the HIV structural precursor protein which is cleaved by viral protease to produce mature infectious viruses. Gag is a polyprotein composed of MA (matrix), CA (capsid), SP1, NC (nucleocapsid), SP2 and p6 domains. SP1, together with the last eight residues of CA, have been hypothesized to form a six-helix bundle responsible for the higher-order multimerization of Gag necessary for HIV particle assembly. However, the structure of the complete six-helix bundle has been elusive. Here, we determined the structures of both Gag in vitro assemblies and Gag viral-like particles (VLPs) to 4.2 Å and 4.5 Å resolutions using cryo-electron tomography and subtomogram averaging by emClarity. A single amino acid mutation (T8I) in SP1 stabilizes the six-helix bundle, allowing to discern the entire CA-SP1 helix connecting to the NC domain. These structures provide a blueprint for future development of small molecule inhibitors that can lock SP1 in a stable helical conformation, interfere with virus maturation, and thus block HIV-1 infection., Mendonça et al. determine the structures of both in vitro assemblies of the HIV structural precursor protein Gag and Gag viral-like particles (VLPs) to 4.2 Å and 4.5 Å resolutions, using cryo-electron tomography and subtomogram averaging. This study provides insights into the future development of small molecule inhibitors for HIV-1 infection.

The Z-disc forms a boundary between sarcomeres, which constitute structural and functional units of striated muscle tissue. Actin filaments from adjacent sarcomeres are cross-bridged by α-actinin in the Z-disc, allowing transmission of tension across the myofibril. Despite decades of studies, the 3D structure of Z-disc has remained elusive due to the limited resolution of conventional electron microscopy. Here, we observed porcine cardiac myofibrils using cryo-electron tomography and reconstructed the 3D structures of the actin-actinin cross-bridging complexes within the Z-discs in relaxed and activated states. We found that the α-actinin dimers showed contraction-dependent swinging and sliding motions in response to a global twist in the F-actin lattice. Our observation suggests that the actin-actinin complex constitutes a molecular lattice spring, which maintains the integrity of the Z-disc during the muscle contraction cycle., Oda and Yanagisawa report 3D cryo-electron tomography structures of Z-disc from porcine cardiac myofibrils in relaxed and activated states. They show that α-actinin dimers exhibit contraction dependent swinging and sliding motions in response to a global twist in the F-actin lattice. These findings provide insights into conformational changes of the Z-disc in the context of active myofibrils.