Mitotic spindles tend to align with the longest axis of the cell, resulting in a division plane that is perpendicular to the long axis59–61.

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From: Progress in Molecular Biology and Translational Science, 2014

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Ivana PonjavićKruno VukušićIva M. Tolić, in Methods in Cell Biology, 2021

2 Rationale

Mitotic spindle is a very dynamic and dense structure and it is often hard to capture the proper localization of proteins that are essential for the spindle, especially with live microscopy, and that is why we need to rely on fixed specimens and immunofluorescence to study the architecture of the spindle. Today, we can use ExM as an improved version of the immunofluorescence protocol (Chozinski et al., 2016) and get super-resolution images of essential proteins in the mitotic spindle. Tubulin is perhaps one of the most intriguing proteins to visualize in the spindle using ExM and the first example of this approach was published in a method paper using tubulin immunostaining of PTK1 cells (Chozinski et al., 2016). Tubulin in the mitotic spindles of zebrafish (Freifeld et al., 2017) and tubulin-GFP in early metaphase-I spindle of mouse oocytes (So et al., 2019) has also been expanded. Except anti-alpha tubulin and tubulin-GFP, kinetochore protein Hec1 (Chozinski et al., 2016), tetrameric kinesin KIF25 (Decarreau et al., 2017), GFP-Spindly and mCherry-tubulin (Sacristan et al., 2018) have been expanded recently as well in mammalian systems.

Here, we describe the optimized protocols for ExM of human mitotic spindles immunostained for tubulin, and centromere protein Hec1, as well as protocols for ExM of mitotic spindles labeled with endogenously tagged microtubule-associated protein PRC1, which required adaptations of the original protocol. One of the most useful parts of our ExM protocol is the protocol for tubulin immunostaining which, even when used without the expansion, gives plenty of information about the architecture of the spindle.

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However, we illustrate the greater power of ExM protocols used to study the mitotic spindle structure, when compared to immunofluorescence without expansion, which enable better structural characterization of defined structures within the mitotic spindle, such as bridging fibers which link two sister k-fibers (Kajtez et al., 2016). Moreover, applied protocols allow superior resolution of microtubule bundle distribution in different populations of microtubules, and provide a better insight into the localization of specific proteins within the spindle.