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.

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.

Cryo-electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for immature virus assembly.

The assembly of HIV-1 is mediated by oligomerization of the major structural polyprotein, Gag, into a hexameric protein lattice at the plasma membrane of the infected cell. This leads to budding and release of progeny immature virus particles. Subsequent proteolytic cleavage of Gag triggers rearrangement of the particles to form mature infectious virions. Obtaining a structural model of the assembled lattice of Gag within immature virus particles is necessary to understand the interactions that mediate assembly of HIV-1 particles in the infected cell, and to describe the substrate that is subsequently cleaved by the viral protease. An 8-Å resolution structure of an immature virus-like tubular array assembled from a Gag-derived protein of the related retrovirus Mason-Pfizer monkey virus (M-PMV) has previously been reported, and a model for the arrangement of the HIV-1 capsid (CA) domains has been generated based on homology to this structure. Here we have assembled tubular arrays of a HIV-1 Gag-derived protein with an immature-like arrangement of the C-terminal CA domains and have solved their structure by using hybrid cryo-EM and tomography analysis. The structure reveals the arrangement of the C-terminal domain of CA within an immature-like HIV-1 Gag lattice, and provides, to our knowledge, the first high-resolution view of the region immediately downstream of CA, which is essential for assembly, and is significantly different from the respective region in M-PMV. Our results reveal a hollow column of density for this region in HIV-1 that is compatible with the presence of a six-helix bundle at this position.

Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision.

Correlative electron and fluorescence microscopy has the potential to elucidate the ultrastructural details of dynamic and rare cellular events, but has been limited by low precision and sensitivity. Here we present a method for direct mapping of signals originating from ∼20 fluorescent protein molecules to 3D electron tomograms with a precision of less than 100 nm. We demonstrate that this method can be used to identify individual HIV particles bound to mammalian cell surfaces. We also apply the method to image microtubule end structures bound to mal3p in fission yeast, and demonstrate that growing microtubule plus-ends are flared in vivo. We localize Rvs167 to endocytic sites in budding yeast, and show that scission takes place halfway through a 10-s time period during which amphiphysins are bound to the vesicle neck. This new technique opens the door for direct correlation of fluorescence and electron microscopy to visualize cellular processes at the ultrastructural scale.