High-throughput screening of mitotic mammalian cells for electron microscopy using classic histological dyes

MCRS1 modulates the heterogeneity of microtubule minus-end morphologies in mitotic spindles

“…We used 3D reconstructions of whole mitotic spindles assembled in HeLa cells (data from Kiewisz et al 2021) to analyze the morphology of the MT ends ( Figure 1A ; Table 1 ). In these control cells, we defined two main classes of ends: closed ends that have an electron-dense “cap” ( Figure 1B , left panels; Figure 1-figure supplement 1A ) and open ends that typically have a flared morphology ( Figure 1B , right panels; Figure 1-figure supplement 1B ).…”

“…We first focused on the MTs forming the k-fibers (i.e., the KMTs), strictly defined as those directly associated through their plus ends to the kinetochores at the chromosome surface (Kiewisz et al, 2021). In agreement with the dynamic nature described for KMT plus ends (Cheeseman and Desai, 2008), the close examination of the KMT end morphology at the kinetochore showed that they were open (green circles) and flared ( Figure 1C ; Figure 1 -movie supplement 1-2; Table 2 ).…”

“…To determine whether there was any correlation between the KMT minus-end morphologies and their position along the half-spindle axis, we then looked at their distribution along this axis. About half of the KMT minus ends were located at the spindle poles, while the other half was distributed along the half-spindle (Kiewisz et al, 2021). We went on to determine the relative position of each KMT minus end on the half-spindle axis ( Figure 1D ).…”

“…A direct visualization of the MT minus ends at any given point may thus provide insightful clues about their actual dynamics. Here, we applied large-scale electron tomography (Redemann et al 2017; Kiewisz et al 2021) to directly visualize MT minus end morphologies at mitotic spindle poles in human cells in metaphase. We found that MT minus ends do not have homogeneous morphologies.…”

“…In order to screen for cells in metaphase, the resin blocks were observed from the top using an upright brightfield microscope (Zeiss, Germany). The two main features used to select cells in metaphase were a rounded shape and a distinguishable metaphase plate (Kiewisz et al, 2021). Serial semi-thick (300 nm) sections were cut using an EM UC6 ultramicrotome (Leica Microsystems, Austria) and collected on Formvar-coated slot grids.…”