Abstract
Cell polarization is important for multiple physiological processes. In motile cells, microtubules (MTs) are organized as a polarized array, which is to a large extent comprised of Golgi-derived MTs (GDMTs), which asymmetrically extend toward the cell front. We have recently found that GDMT asymmetry is based on a nonrandom positioning of spatially restricted nucleation hotspots, where MTs form in a cooperative manner. Here, we summarize methods used for GDMT identification including microtubule regrowth after complete drug-induced depolymerization and tracking of growing microtubules using fluorescent MT plus-end-tracking proteins (+TIPs) in living cells, and subsequent detection of those GDMTs that originate from the nucleation hotspots. These approaches can be used for quantification of the spatial distribution of MT nucleation events associated with the Golgi or another large structure.
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Acknowledgments
This work was supported by a National Institutes of Health (NIH) grant 5T32 CA 119925-10 and American Heart Association (AHA) POST33990271 (to R.J.T.), AHA 17POST32650000 (to A.A.W.M.S.), NIH R01GM078373, NIH R35-GM127098, and NIH R01-DK106228 (to I.K.).
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Thoppil, R.J., Sanders, A.A.W.M., Kaverina, I. (2020). Detection of Microtubule Nucleation Hotspots at the Golgi. In: Maiato, H. (eds) Cytoskeleton Dynamics. Methods in Molecular Biology, vol 2101. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0219-5_12
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DOI: https://doi.org/10.1007/978-1-0716-0219-5_12
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