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Detection of Microtubule Nucleation Hotspots at the Golgi


Thoppil RJ , Sanders AAWM , Kaverina I , . Methods in molecular biology (Clifton, N.J.). 2020 1 1; 2101(). 179-189


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.