FIGURE 3.

Methods for studying the properties of stable microtubules. (A) Schematic showing an in vitro assay to study microtubule dynamics and how it is affected by different MAPs. A microtubule seed stabilized with GMPCPP is immobilized via antibodies/streptavidin on an otherwise passivated coverslip and soluble tubulin (and in this case DCX) are added with GTP, such that the tubulin can undergo cycles of nucleated growth and shrinkage. Below are kymographs of two microtubules with the microtubule seed visible in red, and DCX visible in green at two different concentrations: 10 nM (left) and 100 nM (right). Interestingly, DCX shows different behaviours at these two concentrations. Taken from (Bechstedt and Brouhard, 2012). (B) Image showing tyrosinated and acetylated tubulin stainings in DIV9 rat hippocampal neurons in control conditions (left) or after a 2.5 h treatment with 4 µM nocodazole. Note how without the nocodazole treatment, the overall levels of the tyrosinated and acetylated microtubules is similar, but the low dose nocodazole treatment preferentially depolymerizes dynamic (tyrosinated) microtubules such that very little tyrosinated microtubule signal is present on the right, while the level of acetylated microtubules is less affected. Taken from (Tas et al., 2017). (C) Cryo-EM maps of tubulin within the microtubule lattice (viewed from the lumen) with tubulin bound to GMPCPP (a GTP analog) (left), GDP (middle), or GDP + Taxol (right). Note that overall, the GMPCPP-bound tubulin and Taxol-stabilized tubulin look similar, having a longer lattice spacing (expanded conformation), while GDP-bound tubulin is more compacted. As indicated, both GMPCPP- and Taxol-bound microtubules are stable, while GDP-bound microtubules are not, suggesting that lattice expansion and microtubule stability are intricately connected. Taken from (Alushin et al., 2014). (D) A stage 3 (axon specified) rat hippocampal neuron expressing mRFP-fill (magenta) and photoactivatable-tubulin (green) with converted regions also indicated. The motion of the photoactivated tubulin can be clearly tracked for ∼10 min, as shown in the kymographs underneath for neurites in stage 2, 3, and 4 neurons. This reveals the predominantly retrograde motion of microtubules in neurites except for the one designated as an axon. Taken from (Burute et al., 2022). (E) Experimental set-up and image of mCherry-fill in a stage 2-to-3 mouse hippocampal neuron expressing mCherry-fill and mNeonGreen-CAMSAP3. CAMSAP3-labelled microtubules could be tracked for much longer periods of time (10–70 h) than photoactivated-tubulin, as evidenced by the kymographs showing the faster retrograde flow in all neurites except the axon once specified. Taken from (Schelski and Bradke, 2022). (F) U2OS cells expressing KIF5B-rigor tagged with 2xmNeonGreen fixed and stained for acetylated and total tubulin. Note how the KIF5B-rigor preferentially decorates the subset of microtubules that are acetylated, a commonly used marker for stable microtubules. Taken from (Jansen et al., 2023).