High-throughput electron tomography identifies centriole over-elongation as an early event in plasma cell disorders.

Plasma cell disorders are clonal outgrowths of pre-malignant or malignant plasma cells, characterized by extensive chromosomal aberrations. Centrosome abnormalities are a major driver of chromosomal instability in cancer but their origin, incidence, and composition in primary tumor cells is poorly understood. Using cutting-edge, semi-automated high-throughput electron tomography, we characterized at nanoscale 1386 centrioles in CD138pos plasma cells from eight healthy donors and 21 patients with plasma cell disorders, and 722 centrioles from different control populations. In plasma cells from healthy individuals, over-elongated centrioles accumulated with age. In plasma cell disorders, centriole over-elongation was notably frequent in early, pre-malignant disease stages, became less pronounced in overt multiple myeloma, and almost entirely disappeared in aggressive plasma cell leukemia. Centrioles in other types of patient-derived B cell neoplasms showed no over-elongation. In contrast to current belief, centriole length appears to be highly variable in long-lived, healthy plasma cells, and over-elongation and structural aberrations are common in this cell type. Our data suggest that structural centrosome aberrations accumulate with age in healthy CD138pos plasma cells and may thus play an important role in early aneuploidization as an oncogenic driver in plasma cell disorders.

Protocol for high-throughput electron tomography exemplified on centrioles in primary human plasma cells.

Electron microscopy is the gold standard to characterize cellular ultrastructure. However, production of significant morphometrical data is highly limited by acquisition time. Here, we describe a semi-automated high-throughput strategy using single-axis serial section electron tomography to investigate and analyze centriole ultrastructure in bone-marrow-derived, primary human CD138pos plasma cells. The protocol comprises steps for electron microscopy sample preparation, semi-automated transmission electron microscopy screening, and screening evaluation for cells of interest. Thereafter, we detail tomography acquisition, data reconstruction, and joining. For complete details on the use and execution of this protocol, please refer to Dittrich et al.1.

A high-throughput electron tomography workflow reveals over-elongated centrioles in relapsed/refractory multiple myeloma.

Electron microscopy is the gold standard to characterize centrosomal ultrastructure. However, production of significant morphometrical data is highly limited by acquisition time. We therefore developed a generalizable, semi-automated high-throughput electron tomography strategy to study centrosome aberrations in sparse patient-derived cancer cells at nanoscale. As proof of principle, we present electron tomography data on 455 centrioles of CD138pos plasma cells from one patient with relapsed/refractory multiple myeloma and CD138neg bone marrow mononuclear cells from three healthy donors as a control. Plasma cells from the myeloma patient displayed 122 over-elongated centrioles (48.8%). Particularly mother centrioles also harbored gross structural abnormalities, including fragmentation and disturbed microtubule cylinder formation, while control centrioles were phenotypically unremarkable. These data demonstrate the feasibility of our scalable high-throughput electron tomography strategy to study structural centrosome aberrations in primary tumor cells. Moreover, our electron tomography workflow and data provide a resource for the characterization of cell organelles beyond centrosomes.

Integrative Imaging Reveals SARS-CoV-2-Induced Reshaping of Subcellular Morphologies.

Pathogenesis induced by SARS-CoV-2 is thought to result from both an inflammation-dominated cytokine response and virus-induced cell perturbation causing cell death. Here, we employ an integrative imaging analysis to determine morphological organelle alterations induced in SARS-CoV-2-infected human lung epithelial cells. We report 3D electron microscopy reconstructions of whole cells and subcellular compartments, revealing extensive fragmentation of the Golgi apparatus, alteration of the mitochondrial network and recruitment of peroxisomes to viral replication organelles formed by clusters of double-membrane vesicles (DMVs). These are tethered to the endoplasmic reticulum, providing insights into DMV biogenesis and spatial coordination of SARS-CoV-2 replication. Live cell imaging combined with an infection sensor reveals profound remodeling of cytoskeleton elements. Pharmacological inhibition of their dynamics suppresses SARS-CoV-2 replication. We thus report insights into virus-induced cytopathic effects and provide alongside a comprehensive publicly available repository of 3D datasets of SARS-CoV-2-infected cells for download and smooth online visualization.