Microtubule organization within mitotic spindles revealed by serial block face scanning EM and image analysis

Abstract

Serial block face scanning electron microscopy (SBF-SEM) is a powerful method to analyze cells in 3D. Here, working at the resolution limit of the method, we describe a correlative light-SBF-SEM workflow to resolve microtubules of the mitotic spindle in human cells. We present three examples of uses for this workflow which are not practical by light microscopy and/or TEM. First, distinguishing closely associated microtubules within K-fibers; second, resolving bridging fibers in the mitotic spindle; third, visualizing membranes in mitotic cells, relative to the spindle apparatus.Our workflow also includes new computational tools for exploring the spatial arrangement of MTs within the mitotic spindle. We use these tools to show that microtubule order in mitotic spindles is sensitive to the level of TACC3 on the spindle.

Congressing kinetochores progressively load Ska complexes to prevent force-dependent detachment

“…Kinetochore pairs of interest where tracked as described earlier, and once the congression event had occurred, cells were immediately chemically fixed in 3% glutaraldehyde, 0.5% paraformaldehyde, and 0.1% tannic acid in 0.05 M phosphate buffer, pH 7.4, for 1 h and processed for SBF-SEM. Samples were processed and images segmented as previously described ( Nixon et al, 2016 Preprint ). In brief, cells were postfixed in reduced osmium (2% osmium tetroxide, 1.5% potassium ferricyanide) for 1 h, incubated in 0.1% thiocarbohydrazide in dH 2 O for 20 min, and incubated in 2% osmium tetroxide in dH 2 O for 30 min before being incubated overnight in 1% uranyl acetate dH 2 O at 4°C.…”

Biomechanics of chromosome alignment at the spindle midplane

“…Indeed, recent findings show that sister k-fibers are physically linked with an antiparallel interpolar microtubule bundle, termed the bridging fiber 5,33 . These fibers have been observed also in electron microscopy images of human cells 34,98,99 . Electron tomography reconstructions of spindles in human RPE1 cells revealed that the minus ends of bridging microtubules are typically found near the wall of a kinetochore microtubule 34 .…”

Chromosome (mis)segregation is biased by kinetochore size

“…However, none of these established routes takes into account the intrinsic features of the kinetochore -the critical chromosomal interface with spindle microtubules. Kinetochore size and respective microtubule binding capacity varies between different animal and plant species [3][4][5][6][7][8][9][10], among different chromosomes from the same species (including humans) [3,[11][12][13][14][15], and in response to microtubule attachments throughout mitosis [16][17][18]. How kinetochore size impacts chromosome segregation remains unknown.…”

“…Additional size changes are due to an expandable module formed by CENP-C and outer kinetochore proteins involved in SAC signaling, as well as motor proteins, such as CENP-E and cytoplasmic dynein [12,17, 23]. In humans, the length of α-satellite DNA arrays at centromeres ranges from 200 kb on the Y chromosome, to >5 Mb on chromosome 18 [24], leading to an 3 fold variability in the amount of centromeric CENP-A and respective kinetochore size among different chromosomes [11][12][13][14][19][20][21]. To investigate the relevance of kinetochore size for chromosome congression and segregation during mitosis we took advantage from the unique cytological features of the Indian muntjac (IM), a small deer whose females have the lowest known chromosome number (n=3) in mammals [25].…”