Experimental Tools and Techniques for Studying Microtubule Nucleation at the Centrosome and the Golgi Apparatus.

Microtubules are dynamic cytoskeletal filaments that organize intracellular space, facilitate vesicle and organelle transport, and are essential for cell division and polarity. They also contribute to signal transduction and the generation of mechanical forces. This wide range of functions depends on the ability of microtubules to form well-organized arrays. The spatial organization of these arrays is largely determined by specific subcellular sites-known as microtubule-organizing centers- where new microtubules are nucleated. The centrosome and the Golgi apparatus are the primary microtubule-organizing centers, and their microtubule nucleation activity is tightly regulated to fulfill cellular requirements. In this chapter, we present immunofluorescence techniques to study microtubule cytoskeleton, centrosome, and Golgi apparatus in both two- and three-dimensional cultures. We focus on fixation and pre-fixation extraction methods to enhance protein visualization and analysis. We also describe assays to investigate microtubule nucleation at the centrosome and the Golgi apparatus after microtubule depolymerization, along with methods to accurately quantify microtubules from each site. Finally, as a complementary live-cell imaging method, we explain how to visualize growing microtubules using fluorescent microtubule plus-end-binding proteins together with fluorescent markers for the centrosome or the Golgi apparatus. These approaches offer valuable tools to better understand microtubule organization and dynamics across different cellular contexts, providing insights into their roles in health and disease.