Mitotic spindles in mammalian cells
Mitotic spindles are three-dimensional molecular machines. They are assembled to segregate chromosomes to the forming daughter cells (Movie 1). Our current understanding of spindle architecture during mitosis is mainly based on a plethora of information derived from light microscopy. However, we have rather few ultrastructural data on spindle organization from 3D electron microscopy.
My approach is to apply serial-section electron tomography (ET) to obtain large-scale reconstructions of mitotic spindles. For this, we prepare HeLa cells by high-pressure freezing (HPF), freeze substitution, and block embedding. Serial-sections of the specimen are then imaged for dual-axis ET. We use a transmission electron microscope (TEM) operated at 300 kV to capture images every 1.0°. This approach allows us to reconstruct mitotic spindles in 3D with the resolution of about 2.3 nm.
Such large-scale reconstructions allow us to segment centrosomes, chromosomes and microtubules in 3D. This was recently achieved for the mitotic spindle in the early C. elegans embryo (Redemann et al., 2017). My intention is to apply this approach now for a quantitative analysis of mitotic spindle structure in mammalian cells. I will take advantage of this approach to analyze the number and the length distribution of microtubules of different classes at specific stages of mitosis.
Movie 1. Live-cell imaging of a Hela cell in mitosis. Microtubules are shown in green (GFP-α-tubulin), chromosomes in red (mCherry-histone). © R. Kiewisz/Müller-Reichert Lab.
© Stephan Wiegand
Doktorand
NameRobert Kiewisz
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