Visualizing Membrane Nanotube Dynamics in Drosophila Oocyte Using Live-Cell Imaging

Thin membrane protrusions in cells help them communicate, create traction forces during their movement, and coordinate complex development in multicellular organisms. These structures include cytonemes, tunneling nanotubes, and microtubule-based nanotubes (MT-nanotubes), each with a different cytoskeletal constitution and function. Actin-based cytonemes help deliver signaling molecules, while microtubule-based nanotubes assist with transporting vesicles and organelles. Despite their physiological role, we still do not fully understand how these thin membrane protrusions form and function. In this study, we introduce an improved live-cell imaging method to observe polar cell protrusions during micropyle morphogenesis in developing Drosophila eggs. This technique combines precise developmental staging, careful dissection, and optimized ex vivo culture conditions to maintain tissue health during extended imaging. We also fine-tuned the imaging settings to reduce phototoxicity and thermal stress. This allows for continuous, high-resolution tracking of protrusion dynamics in real time. Our protocol addresses major drawbacks of fixed-tissue methods by capturing the entire process of protrusion formation, extension, and remodeling in intact living tissue. Additionally, it works well with drugs, making it a useful tool for functional studies. Overall, this approach builds a strong foundation for exploring membrane protrusion biology. It can also be applied to investigate similar developmental processes in other systems, aiding our understanding of normal development and diseases. Key features • We optimized a live-imaging protocol ensuring accurate staging and fine dissection of Drosophila egg chambers for reproducible polar cell nanotube visualization. • The enhanced culture conditions maintain egg chamber viability for extended periods, enabling the continuous, real-time observation of dynamic membrane protrusion formation. • The optimized image acquisition settings minimize phototoxicity and sample heating, preventing imaging-induced artifacts and ensuring the acquisition of high-resolution, reproducible datasets while preserving tissue health. • This protocol supports pharmacological treatments for functional perturbation experiments and can be adapted to study similar developmental processes across various other insect systems.