Background

Microtubules are dynamically controlled by various modulatory proteins that act as stabilizers or destabilizers. Although some of these regulatory factors have been identified, additional microtubule regulators are yet to be found. Thus, it is pivotal to have a reliable method to detect microtubules and their stability.

Among antibodies or chemical reagents for labeling tubulin, SiR-Tub is an excellent tool for detecting microtubules due to its high specificity, cell permeability, and strong fluorescent signal. Since SiR-Tub was first introduced in 2014 (Lukinavičius et al., 2014), it has been utilized for visualizing microtubules in different organisms (Derivery et al., 2015). SiR-Tub has also been used to label microtubules in Drosophila larval tissues, including brain, gut, and body wall muscle (Lukinavičius et al., 2018). However, most of these applications of SiR-Tub labeling were limited to imaging of microtubules in various cells and tissues.

Recently, we have combined the SiR-Tub labeling method with genetic application for testing the role of a novel Drosophila protein named microtubule-associated N-terminal acetyltransferase 9 (Mnat9) (Mok and Choi, 2021). In this assay, we used the larval wing imaginal disc, the primordium for the adult wing, as an ex-vivo system. Because a wing disc consists of distinct anterior and posterior compartments, it is possible to drive Mnat9 overexpression specifically in one of the compartments by the Gal4-UAS system (Brand and Perrimon, 1993) while the other compartment is used as an internal control. Interestingly, although Mnat9 overexpression is insufficient to induce a noticeable change in normal microtubules, it strongly suppresses microtubule defects caused by a demecolcine treatment. This provides evidence for the role of Mnat9 in promoting microtubule stability. This result also suggests that SiR-Tub labeling under a sensitized microtubule condition can be an efficient way of testing the function of microtubule regulators that might not be identified by simple loss- or gain-of-function approaches.

Microtubules have been extensively studied, but genetic regulation of their function and stability is far from complete, and a number of microtubule regulators remain to be found. Hence, we introduce SiR-Tub labeling as an ex-vivo assay for identifying and testing microtubule regulatory proteins. This assay system has four experimental elements: (i) SiR-Tub is used for labeling microtubules in either unfixed or fixed tissues. (ii) Wing discs are used as ex-vivo organs (but other imaginal discs may also be used). (iii) Demecolcine is used to generate a sensitized background by partially impairing microtubules. However, such sensitized microtubule conditions can also be induced by silencing a known microtubule stabilizer, such as Tau (Bowne-Anderson et al., 2015; Barbier et al., 2019). (iv) An experimental gene of interest is down- or up-regulated in specific tissues by targeted expression. Compared with existing protocols for studying microtubule stability, our assay has technical advantages for sensitive detection of microtubules in developing organs. An additional merit of this protocol is its applicability to ex vivo assays and genetic screens for identifying regulators of microtubule stability. In the protocol below, step-by-step procedures from dissection to SiR-Tub labeling and imaging will be described in detail. Although this protocol is mainly designed for testing microtubule regulators in the wing disc of Drosophila, it may also be adapted for identifying or testing regulatory factors of microtubules in other invertebrates and vertebrate genetic model systems.